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Subproteome analysis of Listeria monocytogenes Medizinische

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					                                            Table of Contents
TABLE OF CONTENTS ......................................................................................................... 1


FINAL PROGRAMME .......................................................................................................... 11


INTERNATIONAL SCIENTIFIC BOARD ............................................................................................................ 11

NATIONAL BOARD ............................................................................................................................................ 11

LOCAL BOARD .................................................................................................................................................. 11

    Sunday, may 13, 2001 ............................................................................................................................................ 12
    Monday, may 14, 2001 ........................................................................................................................................... 13
    Tuesday, 15 may, 2001 .......................................................................................................................................... 14
    Wednesday, 16 may, 2001 ..................................................................................................................................... 15

HISTORY OF ISOPOL ........................................................................................................................................ 17
  Herbert K.H. Hof.................................................................................................................................................... 17


STRUCTURE, GENOMICS, PHYSIOLOGY ........................................................................ 19


SUBPROTEOME ANALYSIS OF LISTERIA MONOCYTOGENES ................................................................... 20
  M. Baumgärtner, O. Diekmann, L. Jänsch, J. Wehland ........................................................................................ 20

A GLUTAMATE DECARBOXYLASE SYSTEM PROTECTS LISTERIA MONOCYTOGENES AT LOW PH,
AND IN GASTRIC FLUID.................................................................................................................................... 21
  Paul D. Cotter1. Cormac G.M. Gahan1,2 , and Colin Hill1,2 .................................................................................... 21

DIFFERENT AUTOLYTIC BEHAVIOUR OF STRAINS OF LISTERIA MONOCYTOGENES ISOLATED FROM
PORTUGUESE CHEESE .................................................................................................................................... 22
  M.L. Faleiro1 , P. W. Andrew2 , D. Power1 ............................................................................................................ 22

PEPTIDE       TRANSPORT         IN         LISTERIA                 MONOCYTOGENES:                               PHYSICAL                   AND            GENETIC
CHARACTERISATION AND POTENTIAL APPLICATIONS. ............................................................................ 23
  H.-N. Tsai, David. A. Hodgson .............................................................................................................................. 23

IS P60 REALLY AN ESSENTIAL HOUSEKEEPING GENE OF LISTERIA MONOCYTOGENES ? ................ 24
   Pilgrim S., Kolb-Mäurer A., Bergmann B., *Geginat G., Gentschev l., Goebel W. .............................................. 24

COLD SHOCK PROTEINS IN LISTERIA: EVIDENCE FOR ROLE OF DNA BINDING PROTEINS IN LOW
TEMPERATURE INDUCTION ............................................................................................................................ 25
  Catherine E.D. Rees, Faye M. Barnard .................................................................................................................. 25

POST-GENOMIC ANALYSIS OF THE LISTERIA MONOCYTOGENES GENOME: ROLE OF SURFACE
PROTEINS........................................................................................................................................................... 26
  Didier Cabanes, O. Dussurget, P. Dehoux, L. Frangeul, P. Glaser, C Buchrieser, The Listeria Consortium, Pascale
  Cossart .................................................................................................................................................................... 26




                                                                                       1
THE INVASION PROTEIN INLB OF LISTERIA MONOCYTOGENES BINDS BY ITS C-TERMINUS
LIPOTEICHOIC ACID ......................................................................................................................................... 27
  Renaud Jonquières1, Hélène Bierne,1 Franz Fiedler,3 Pierre Gounon3 and Pascale Cossart1.1 ............................... 27

INTERNALINS FROM THE HUMAN PATHOGEN LISTERIA MONOCYTOGENES CONTAIN A FUSED
SUPERDOMAIN STRUCTURE........................................................................................................................... 28
  Dirk W. Heinz1, Wolf-Dieter Schubert1, Gero Göbel1, Meikel Diepholz1, Ayub Darji3, Daniel Kloer1, Torsten Hain3,
  Trinad Chakraborty3, Jürgen Wehland2 & Eugen Domann3................................................................................... 28

IDENTIFICATION OF NEW ACTA-BINDING PROTEINS ................................................................................. 29
  Michael Kuhn, Thilo Pfeuffer, Susanne Bauer, Werner Goebel ........................................................................... 29

CHARACTERISATION OF PARTIAL CYTOSINE DNA METHYLATION IN SEROTYPE 4B STRAINS OF
LISTERIA MONOCYTOGENES.......................................................................................................................... 30
  M. Akhtar, T. M. Perehinec, A. Nazli, P. J. Hill, C. E. D. Rees ............................................................................. 30

A RESPONSE REGULATOR OF LISTERIA MONOCYTOGENES L028 SHOWING HOMOLOGY TO KDPE IS
IMPORTANT FOR GROWTH AT LOW TEMPERATURE AND HIGH OSMOLARITY ..................................... 31
  Lone Brondsted1, Birgitte H. Kallipolitis2, Hanne Ingmer2, and Susanne Knöchel1 .............................................. 31

COMPARATIVE GENOMICS FOR DECIPHERING SEROVAR SPECIFIC MARKERS AND VIRULENCE
DETERMINANTS OF EPIDEMIC LISTERIA MONOCYTOGENES ................................................................... 32
  Buchrieser C, Simoes N1, Jacquet CH 3, Frangeul L1, Rocourt J3, Couve E1, Kunst F1, Cossart P2, and Glaser P132

ISOLATION OF NOVEL MUTATIONS THAT INFLUENCE THE REGULATION OF LISTERIA
MONOCYTOGENES INTRACELLULAR GENE EXPRESSION........................................................................ 33
  Nancy E. Freitag, Lynne Shetron-Rama................................................................................................................. 33

DEVELOPMENT OF OPTIMISED MARKER GENE EXPRESSION VECTORS FOR LISTERIA ..................... 34
  Philip J. Hill, Saara N.A. Qazi, Kenneth Mellits and Catherine E.D. Rees ............................................................ 34

DEVELOPMENT AND VALIDATION OF A NOVEL REPORTER GENE SYSTEM FOR THE IDENTIFICATION
OF INTRACELLULARLY ACTIVE PROMOTERS FROM L. MONOCYTOGENES .......................................... 35
  Sonja Otten and Trinad Chakraborty...................................................................................................................... 35

EVOLUTION OF VIRULENCE IN LISTERIA ...................................................................................................... 36
  Jürgen Kreft, Robert Lampidis *, Eva Ng , Werner Goebel................................................................................... 36

CHARACTERISATION OF THE SIGB AND LMU OPERONS OF LISTERIA MONOCYTOGENES ................ 37
  Sandra Wagner and Trinad Chakraborty ................................................................................................................ 37

IDENTIFICATION OF NEW PROTEINS ASSOCIATED TO THE CELL WALL OF LISTERIA
MONOCYTOGENES ........................................................................................................................................... 38
  Jessica Schaumburg, Uwe Kärst and Jürgen Wehland ........................................................................................... 38

IDENTIFICATION OF PUTATIVE VIRULENCE GENES IN LISTERIA MONOCYTOGENES BY USE OF A
GFP ENCODING PROMOTER PROBE ............................................................................................................. 39
  Meirion L Jones, David A Hodgson ....................................................................................................................... 39

POLYPORPHISM ANALYSIS OF GENES IMPLICATED IN LISTERIA MONOCYTOGENES VIRULENCE... 40
  KEROUANTON A.a, BRISABOIS A.a, MARAULT M.a, COSSART P.c and PICARD Bb................................. 40

GENE FRAGMENTS SPECIFICALLY PRESENT IN AN EPIDEMIC STRAIN OF L. MONOCYTOGENES
RAISE THE POSSIBILITY OF LATERAL GENE TRANSFER BETWEEN PATHOGENIC AND NON-
PATHOGENIC LISTERIA SPECIES ................................................................................................................... 41
  Christine Kocks ...................................................................................................................................................... 41

ACTA AND INTRACELLULAR MOTILITY......................................................................................................... 42
  Eugen Domann ....................................................................................................................................................... 42

                                                                                    2
BACTERIOPHAGES, BACTERIOCINS .............................................................................. 44


COMPARATIVE GENOMICS OF LISTERIA BACTERIOPHAGES ................................................................... 45
  Richard Calendar1, ROSS B. Inman2, Elke Sattelberger3, Peter Lauer1, Daniel Portnoy1, Nora Chow1, Markus Zimmer3,
  and Martin J. Loessner3 .......................................................................................................................................... 45

MEMBRANE DISRUPTION AND BACTERIAL CELL LYSIS BY LISTERIA BACTERIOPHAGES: THE
FUNCTION OF LYSINS AND HOLINS ............................................................................................................... 46
  Martin J. Loessner. Natasa Vukov, Susanne Gaeng, and Siegfried Scherer .......................................................... 46

PRACTICAL APPLICATIONS OF LISTERIA PHAGE ....................................................................................... 47
  Catherine E.D Rees ................................................................................................................................................ 47

USE OF GENERALISED TRANSDUCTION TO STUDY VIRULENCE MECHANISMS IN LISTERIA
MONOCYTOGENES ........................................................................................................................................... 48
  David A. Hodgson, Hsiang-Ning Tsai and Jonathan L. Telfer .............................................................................. 48

DETERMINATION AND IDENTIFICATION OF LISTERIA MONOCYTOGENES BY BACTERIOPHAGES IN
ICECREAM .......................................................................................................................................................... 49
  Ebtehaj Pishva ........................................................................................................................................................ 49

INTERACTION OF BACTERIOPHAGES WITH THE BACTERIAL CELL: THE ROLE OF SEROTYPE-
SPECIFIC SURFACE ANTIGENS. ..................................................................................................................... 50
  Nattawan Promadej1, Zheng Lan1, Franz Fiedler2, Xiang-He Lei1, Sophia Kathariou1,3 ....................................... 50

SYNERGISTIC EFFECT OF NISIN AND BACTERIOPHAGE ON LISTERIA MONOCYTOGENES IN BROTH
CULTURES ......................................................................................................................................................... 51
  Sandra M. Moorhead, Gary A. Dykes .................................................................................................................... 51

CHARACTERIZATION OF A NEW PEDIOCIN-LIKE SEC-DEPENDANT BACTERIOCIN PRODUCED BY
LISTERIA INNOCUA 743.................................................................................................................................... 52
  M.L. Kalmokoff1, T. Cyr2, and M.A. Hefford2. ..................................................................................................... 52

DETECTION OF LISTERIA MONOCYTOGENES IN PRESENCE OF LISTERIA INNOCUA .......................... 53
  Marie Cornu 1,Martin Kalmokoff 2. ....................................................................................................................... 53

INHIBITION OF LISTERIA MONOCYTOGENES IN CHICKEN COLD-CUTS BY ADDITION OF SAKACIN P
AND SAKACIN P PRODUCING LACTOBACILLUS SAKEI ............................................................................. 55
  Katia, T., Rosshaug, I., Axelsson, L. and K. Naterstad .......................................................................................... 55


VIRULENCE, PATHOGENICITY, FITNESS ........................................................................ 56


PATHOGENICITY OF LISTERIA MONOCYTOGENES ..................................................................................... 57
  W. Goebel .............................................................................................................................................................. 57

THE HLYA GENE EXPRESSION PATTERN IN RESPONSE TO ENVIRONMENTAL CONDITIONS IS NOT
CORRELATED WITH THE LISTERIA MONOCYTOGENES GENOTYPE ........................................................ 58
  Knut Rudi*, Hege Karin Nogva, Kristine Naterstad, Signe Marie Dromtorp, Sylvia Bredholt, and Askild Holck 58

THE USE OF LISTERIOLYSIN TO IDENTIFY AND CHARACTERISE IN VIVO INDUCED GENES IN
LISTERIA MONOCYTOGENES.......................................................................................................................... 59
  Cormac G.M. Gahan, Colin Hill* .......................................................................................................................... 59

DETECTION AND CHARACTERIZATION OF VIRULENCE ASSOCIATED GENES IN LISTERIA
MONOCYTOGENES STRAINS FROM INVASIVE AND NON-INVASIVE LISTERIOSIS. ................................ 60
  Paolo Aureli, Giovanna Franciosa, Christina Wedell-Neergaard, Antonella Maugliani. ....................................... 60

                                                                                     3
ACID TOLERANCE IN LISTERIA MONOCYTOGENES ENHANCES THE SURVIVAL IN MACROPHAGES 61
  M.P. Contea , G. Petronea, C. Longhia, M. Pentaa, A. M. Di Biasea, F. Supertib, L. Segantia ................................. 61

STRESS TOLERANCE AND VIRULENCE: TWO-COMPONENT SIGNAL TRANSDUCTION SYSTEMS IN
LISTERIA MONOCYTOGENES.......................................................................................................................... 68
  Birgitte H. Kallipolitis, Hanne Ingmer ................................................................................................................... 68

INVASIVENESS OF DIFFERENT PFGE-TYPES OF LISTERIA MONOCYTOGENES IN CACO-2 CELLS .... 69
  Charlotte Nexmann Larsen1. Birgit Norrung2, Helle Molgaard Sommer2 and Mogens Jakobsen3 ........................ 69

INVESTIGATION OF LISTERIA MONOCYTOGENES INTRACELLULAR GROWTH. .................................... 70
  Telfer. J.L.,The European Listeria Genome Consortium, Hodgson, D.A., ............................................................ 70

THE OSMOLYTE TRANSPORTER OPUC IS INVOLVED IN THE VIRULENCE OF LISTERIA
MONOCYTOGENES ........................................................................................................................................... 71
  J. A. Wouters1,R. D. Sleator2, C. G. M. Gahan2, C, Hill2 and T. Abee1 ................................................................. 71

IDENTIFICATION OF AN OSMOLYTE UPTAKE SYSTEM LINKED TO THE VIRULENCE OF LISTERIA
MONOCYTOGENES ........................................................................................................................................... 72
  Roy D. Sleator. Cormac G.M. Gahan, and Colin Hill* .......................................................................................... 72

IN VITRO AND IN VIVO VIRULENCE HETEROGENEITY IN CLINICAL LISTERIA MONOCYTOGENES
STRAINS ............................................................................................................................................................. 73
  Wagner M1, Vazquez - Boland JA4, Bubert A3, Goebel W2 .................................................................................. 73

INVASION OF CELLS AND TISSUES BY THE BACTERIAL PATHOGEN LISTERIA MONOCYTOGENES . 74
  Marc Lecuit, Laurence Braun. Helene Bierne, Javier Pizarro-Cerda, Shaynoor Dramsi, P. Glaser*. The European
  Consortium. Pascale Cossart. ................................................................................................................................. 74

INFLUENCE OF VIRULENCE GENES OF LISTERIA MONOCYTOGENES ON ITS SURVIVAL AND
MULTIPLICATION WITHIN PROTOZOA ........................................................................................................... 75
  Marion Walcher1, Werner Goebel2, Martin Wiedmann3, and Michael Wagner1* ................................................. 75

SURVIVAL AND PERSISTENCE OF LISTERIA MONOCYTOGENES IN ACANTHAMOEBA AND
PROTECTION AGAINST LISTERIOCIDAL AGENTS ....................................................................................... 76
  Simon Kilvington, Peter Andrew, Isabel Morgado & Amy Peasland. ................................................................... 76

THE ACTIVATION OF PROTEIN KINASE C ISOFORMS IN J774 CELLS BY LISTERIOLYSIN O AND
PHOSPHATIDYLINOSITOL -SPECIFIC PHOSPHOLIPASE C (PI-PC99) MEDIATES BACTERIAL
ASSOCIATION AND ENTRY .............................................................................................................................. 77
  Howard Goldfine and Sandra J. Wadsworth. ......................................................................................................... 77

ANALYSIS OF THE ROLE OF ENA/VASP PROTEIN DOMAINS IN THE ACTIN-BASED MOTILITY OF
LISTERIA MONOCYTOGENES.......................................................................................................................... 78
  Marcus Geese, Joseph J. Loureiro*, Frank B. Gertler*,Jürgen Wehland, Antonio S. Sechi. ................................. 78

SMCL FROM LISTERIA IVANOVII: UNDERSTANDING THE ROLE OF A BACTERIAL
SPHINGOMYELINASE IN BACTERIA-HOST CELL INTERACTION. .............................................................. 81
  J.A. Vázquez-Boland, B. González-Zorn ............................................................................................................... 81

INTERACTION OF LISTERIA MONOCYTOGENES WITH MICROVASCULAR ENDOTHELIAL CELLS ....... 82
  Michael Kuhn, Lars Greiffenberg, Tobias Hertzig, Werner Goebel ...................................................................... 82

CHARACTERISATION OF GROESL IN LISTERIA MONOCYTOGENES: EVIDENCE FOR IN VIVO
EXPRESSION ..................................................................................................................................................... 83
  Cormac G.M. Gahan, James O'Mahony, Colin Hill ............................................................................................... 83

ROLE OF THE AUTOLYSIN AMI IN THE ADHESION OF LISTERIA MONOCYTOGENES TO EUKARYOTIC
CELLS ................................................................................................................................................................. 84
  Eliane Milohanic, Renaud Jonquieres, Pascale Cossart, Patrick Berche and Jean-Louis Gaillard ......................... 84
                                                                                   4
INDUCTION OF COLD-SHOCK: PROTEINS IN LISTERIA MONOCYTOGENES UPON EXPOSURE TO
DIFFERENT STRESS CONDITIONS ................................................................................................................. 85
  H. H. Kamphuis. J. A. Wouters, T. Abee ............................................................................................................... 85

EFFECT OF DIFFERENT SALTING AND SMOKING PROCEDURES ON L. INNOCUA 2030C AND TVC
DURING CHILLED STORAGE OF VACUUM-PACKED COLD-SMOKED SALMON-TROUT ......................... 86
  Vaz-Velho, M., Silva, M., Ribeiro, J., Pessoa, J., Gay, M., Aalberts, C. ,Águas, M. and Gibbs, P. ..................... 86

IN VIVO STUDIES WITH MUTANTS OF LISTERIA MONOCYTOGENES ....................................................... 89
   Schlech, 'W., R.A. Garduno, and T, Gill, ............................................................................................................... 89


DISEASE OF MAN AND ANIMALS…………………………………………………………….. 90


PATHOLOGY OF L. MONOCYTOGENES INFECTION .................................................................................... 91
  Martina Deckert1, Maja Abram2, Dirk Schlüter3 .................................................................................................... 91

VETERINARY PERSPECTIVE. .......................................................................................................................... 92
  W. Donachie1 and J.C. Low2 .................................................................................................................................. 92

CURRENT SITUATION OF ANIMAL LISTERIOSIS IN SLOVENIA .................................................................. 93
  Zdovc Irena, Mehle Janez ...................................................................................................................................... 93

NEURAL ROUTE OF CEREBRAL LISTERIA MONOCYTOGENES MURINE INFECTION ............................. 94
  Yuxuan Jin1, Lone Dons2. Krister Kristensson1, and Martin Rottenberg3 .............................................................. 94

EXPERIMENTAL CONGENITAL LISTERIOSIS: A MURINE MODEL .............................................................. 95
  Maja Abram1, Darinka Vuckovic1, Miljenko Doric1, Branka Wraber2, Herbert Hof3, Dirk Schlüter3, Martina Deckert4
  ................................................................................................................................................................................ 95

SYSTEMIC AND INTRACEREBRAL INFECTIONS OF MICE WITH LISTERIA MONOCYTOGENES
SUCCESSFULY TREATED WITH LINEZOLID.................................................................................................. 96
  M. Callapina, M. Kretschmar, A. Dietz, C. Mosbach, H. Hof, T. Nichterlein ....................................................... 96


DEFENSE, IMMUNOLOGY AND ANTIBIOTICS ................................................................. 97


CYTOKINE RELEASE OF L. MONOCYTOGENES-INFECTED DENTRITIC CELLS ...................................... 98
  Kolb-Mäurer A., Kemmerer U., Gentschev I., Kämpgen E., Goebel W.CO ......................................................... 98

EARLY HOST-PATHOGEN INTERACTIONS AND THE INDUCTION OF T CELLS ....................................... 99
  M.E.A. Mielke,....................................................................................................................................................... 99

HIERARCHY AND DYNAMICS OF PEPTIDE-SPECIFIC CD4 T CELL POPULATIONS DURING L.
MONOCYTOGENES-INFECTION .................................................................................................................... 101
  Gernot Geginat ..................................................................................................................................................... 101

CROSS-TALK BETWEEN INNATE AND ADAPTIVE IMMUNE SYSTEMS: CD8+T CELL PRIMING AGAINST
NON-SECRETED LISTERIA MONOCYTOGENES ANTIGENS. ..................................................................... 102
  A.R. Tvinnereim, S. Hamiliton, J.T. Harty, ......................................................................................................... 102

ABUNDANCE OF NATURALLY PROCESSED ANTIGENIC PEPTIDES IN LISTERIA MONOCYTOGENES
INFECTED MICE ............................................................................................................................................... 103
  Mojca Skoberne1, Herbert Hof2 and Gernot Geginat2 .......................................................................................... 103

IN VIVO EFFECTS OF INTERNALIN RELATED PROTEIN A AND LISTERIOLYSIN ON THE MURINE
IMMUNE SYSTEM. ........................................................................................................................................... 104
  Siegfried Weiss, Susanne zur Lage, Trinad Chakraborty, Ayub Darji ................................................................. 104
                                                                                         5
EXPLOITING LISTERIA MONOCYTOGENES FOR THE DESIGN OF NOVEL STRATEGIES ..................... 105
  Stefan H.E. Kaufmann, Leander Grode, Jürgen Hess, Michael Rolph, Anna Marit Sponaas .............................. 105

PATHOGENESIS AND NEUROIMMUNOLOGY OF CEREBRAL LISTERIOSIS ........................................... 106
  D. Schlüter1, S. Reiter1, M. Montesinos-Rongen2, S. Lütjen1,2, L.-Y. Kwok1, M. Deckert2 ............................... 106

LISTERIA MONOCYTOGENES: ABSENCE OF SYNERGISM BETWEEN AMPICILLIN AND GENTAMICIN
USING E-TEST AND CHECKERBOARD MICRODILUTION TECHNIQUES .................................................. 107
  I. Davis, N. Campbell, M.Currie, W. F.Schlech ................................................................................................... 107

ANTIMICROBIAL THERAPY OF LISTERIOSIS .............................................................................................. 108
  Hof H., Nichterlein T., Lampidis R. .................................................................................................................... 108

MOLECULAR CHARACTERIZATION OF THE INTRINSIC RESISTANCE OF LISTERIA AGAINST
NALIDIXIC ACID ............................................................................................................................................... 111
  LAMPIDIS ROBERT; HOF HERBERT ............................................................................................................. 111


DETECTION, FOOD AND FOOD TECHNOLOGY ............................................................ 112


CONVENTIONAL ISOLATION METHODS FOR LISTERIA MONOCYTOGENES. ........................................ 113
  Catherine W. Donnelly, Ph.D.,............................................................................................................................. 113

GROWTH OF LISTERIA MONOCYTOGENES PREDICTED BY MATHEMATICAL MODELS AND
OBSERVED IN FOOD PRODUCTS ................................................................................................................. 123
  Pemilla Arinder, Eva Nerbrink, Elisabeth Borch. ................................................................................................ 123

ISO 11290-1, HORIZONTAL METHOD FOR THE DETECTION OF LISTERIA MONOCYTOGENES: CHANGE
OF ISOLATION MEDIA..................................................................................................................................... 124
  RR Beumer ........................................................................................................................................................... 124

COMPARISON OF THREE ENRICHMENT PRODEDURES FOR THE DETECTION OF STRESSED
LISTERIA MONOCYTOGENES IN SOFT CHEESE ........................................................................................ 125
  N. Rijpens, L. Herman ......................................................................................................................................... 125

IMPROVING RECOVERY OF LISTERIA MONOCYTOGENES INJURED BY ACIDIFICATION OR SALTING
 ........................................................................................................................................................................... 126
     Gnanou Besse N., Lafarge V. ............................................................................................................................... 126

METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN HUMAN FAECES ..................... 127
  Katharina Grif, Manfred P. Dierich, Franz Allerberger ....................................................................................... 127

VALIDATING DETECTION METHODS FOR FOODBORNE PATHOGENIC BACTERIA: LISTERIA
MONOCYTOGENES ......................................................................................................................................... 128
  D. Hitchins. .......................................................................................................................................................... 128

UTILIZATION OF PCR-SYSTEMS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN ROUTINE
DIAGNOSTIC OF ACID-CURD PRODUCTS ................................................................................................... 129
  Simon, Pamela;Barbara Brunner, M. Bülte, ......................................................................................................... 129

COLONY-BLOT ASSAY WITH ANTI-P60 ANTIBODIES AS A METHOD FOR QUICK IDENTIFICATION OF
LISTERIA IN FOOD .......................................................................................................................................... 130
  Barbara Rozalska1, Marzena Wieckowska-Szakiel1, Andreas Bubert2, Marek Rozalski3, Urszula Krajewska3, Wieslawa
  Rudnicka1 ............................................................................................................................................................. 130

DETECTION OF LISTERIA ANTIGENS ........................................................................................................... 131
  Bubert A.1, K. Kramer2, B. Rozalska3, W. Goebel4, and B.S. Yoon5 ................................................................... 131


                                                                                      6
PRESENCE OF LISTERIA MONOCYTOGENES IN SAMPLES OF COOKED SAUSAGES ......................... 132
  Becker, B.; Trierweiler, B.; Fechler, J. and Holzapfel, W.H. ............................................................................... 132

ADSORPTION, AND BIOFILM FORMATION AMONG ISOLATES OF LISTERIA MONOCYTOGENES ON
FOOD GRADE STAINLESS STEEL ................................................................................................................ 136
  J.M Farber, M.L. Kalmokoff, and J.W. Austin. .................................................................................................. 136

THE DIFFERENTIAL ADHERENCE OF LISTERIA MONOCYTOGENES STRAINS IN MONOCULTURE AND
MULTISPECIES BIOFILMS .............................................................................................................................. 137
  Norwood. D.E.1, Harvey, J.2, and Gilmour, A.1,2 ................................................................................................. 137

LISTERIA MONOCYTOGENES ATTACHES TO A WIDE VARIETY OF MATERIALS COMMONLY USED IN
THE FOOD INDUSTRY. .................................................................................................................................... 138
  Mark Beresford1,2 Peter Andrew1, Gilbert Shama2............................................................................................... 138

OCCURENCE OF LISTERIA MONOCYTOGENES IN SOUR MILK CURD AND SOUR MILK CHEESE...... 139
  B. Brunner , M. Bülte ........................................................................................................................................... 139

REDUCED LISTER1A MONOCYTOGENES OCCURRENCE IN FINNISH VACUUM-PACKED FISH
PRODUCTS ....................................................................................................................................................... 140
  Hatakka Maija1, Johansson Tuula2, Rantala Leila2, Pakkala Pekka1, Honkanen-Buzalski Tuula2 ....................... 140

MAILLARD REACTION DURING COOKING OF MEAT PRODUCTS CAUSES SUPPRESSION OF
VIRULENCE GENE EXPRESSION IN LISTERIA MONOCYTOGENES ......................................................... 141
  Mahmoud Sheikh-Zeinoddin, Tania M. Perehinec, Sandra E. Hill and Catherine E.D. Rees .............................. 141

DIVERSITY OF LISTERIA MONOCYTOGENES STRAINS ISOLATED IN MEAT PRODUCTS .................... 142
  T. Autio, J. Lunden, J. Björkroth, H. Korkeala. ................................................................................................... 142

LISTERIA MONOCYTOGENES CONTAMINATION ASSOCIATED WITH THE TRANSFER OF A DICER .. 143
  Lunden. J, Autio, T., Korkeala, H. ....................................................................................................................... 143

INDUSTRIAL APPLICATION OF AN ANTI LISTERIAL STRAIN OF LACTOBACILLUS SAKEI AS A
PROTECTIVE CULTURE AND ITS EFFECT ON THE SENSORY ACCEPTABILITY OF COOKED, SLICED,
VACUUM - PACKAGED MEATS...................................................................................................................... 144
  Sylvia Bredholta, Truls Nesbakkenb and Askild Holcka ....................................................................................... 144

COMPARATIVE STUDY FOR THE ENUMERATION OF LISTERIA MONOCYTOGENES FROM FRESH
FERMENTED SAUSAGES ............................................................................................................................... 145
  Albert, T.1, Hechelmann, H.1, Reissbrodt, R.2 und Gareis M.1............................................................................ 145


TYPING, EPIDEMIOLOGY ................................................................................................ 146


THE LISTERIA MONOCYTOGENES DATABASE IN PULSENET, THE NATIONAL MOLECULAR
SUBTYPING NETWORK FOR FOODBORNE DISEASE SURVEILLANCE IN THE UNITED STATES. ....... 147
  S. B. Hunter, N. Tucker, A. R. B, Ong, L. M. Graves, and B. Swaminathan, ...................................................... 147

REPORT ON THE PHASE III OF THE WHO SEROTYPING STUDY OF LISTERIA MONOCYTOGENES ... 148
  Lehmann1), Simone, A. Schönberg2) .................................................................................................................... 148

CHANGES IN SEROGROUP DISTRIBUTION OF HUMAN LISTERIA MONOCYTOGENES STRAINS IN
SWEDEN ........................................................................................................................................................... 149
  Tham W.1, E. Bannerman2, J. Bille2, H. Ericsson1, S. Helmersson1, B. Henriques3, Ch. Jacquet4, S. Loncarevic5, J.
  Rocourt4, I. Tjernberg6, H. Unnerstad1, M-L Danielsson-Tham1. ........................................................................ 149




                                                                                  7
STATUS REPORT ON WHO-SPONSORED INTERNATIONAL COLLABORATIVE STUDY OF SUBTYPING
METHODS FOR LISTERIA MONOCYTOGENES: PULSED-FIELD GEL ELECTROPHORESIS, PHASE III.
........................................................................................................................................................................... 150
    L. M. Gravesa, S. B. Huntera, N. Tuckera, M. Brettb, J. Harveyc, C. Jacquetd, A. Kerouanton-Le Galle, E. Lehnkeringf, B.
    Ojeniyig, M. Wagnerh, A. Brisaboise, A. Gilmourc, J. Rocourtd and B. Swaminathana. ....................................... 150

STRAINS IDENTIFICATION OF LISTERIA SPP. BY DNA FINGERPRINTING: ERIC-PCR. ......................... 151
  Laciar A., Vega A., Lopresti R., Centorbi ONP. .................................................................................................. 151

CHARACTERIZATION OF FOOD AND HUMAN ISOLATES OF LISTERIA MONOCYTOGENES BY USE OF
PCR-SSCP IN THE MPL AND PRF GENES .................................................................................................... 152
  Loncarevic S., Lehner A., Wagner M. ................................................................................................................. 152

COMPARISON OF MOLECULAR METHODS FOR TYPING RECURRENT AND SPORADIC LISTERIA
MONOCYTOGENES FOOD ISOLATES. ......................................................................................................... 153
  J.Harvey1, D.E. Norwood1, A. Gilmour1,2 ............................................................................................................ 153

COMPARISON OF EPIDEMIOLOGICALLY LINKED LISTERIA MONOCYTOGENES STRAINS
CHARACTERIZED BY TWO DISTINCT PHAGE-SENSITIVITY PHENOTYPES. ........................................... 154
  N. van der Mee-Marquet, L. Mereghetti, A. Audurier. ........................................................................................ 154

A NATIONAL ELECTRIC NETWORK FOR COMPARISON OF PFGE PROFILES OF LISTERIA
MONOCYTOGENES ......................................................................................................................................... 155
  Rantala Leila1, Lukinmaa Susanna2, Siitonen Anja2, Honkanen-Buzalski Tuula1 ............................................... 155

HIGH-RESOLUTION GENOTYPING OF LISTERIA MONOCYTOGENES BY FLUORESCENT AMPLIFIED-
FRAGMENT LENGTH POLYMORPHISM ANALYSIS .................................................................................... 156
  Birte Fonnesbech Vogel,1* and Peter Ahrens,2 ..................................................................................................... 156

PYROSEQUENCING AS A METHOD FOR GROUPING OF LISTERIA MONOCYTOGENES STRAINS
BASED ON NUCLEOTIDE POLYMORPHISMS IN THE INLB GENE. ........................................................... 157
  Helle Unnerstad1. Henrik Ericsson1, Anders Alderbom3, Wilhelm Tham1, Marie-Louise Danielsson-Tham1, Jens G
  Mattsson2 .............................................................................................................................................................. 157

CHARACTERISATION OF LISTERIA MONOCYTOGENES ISOLATED FROM EUROPEAN PRODUCTION
LINES OF FRESH AND COLD-SMOKED FISH............................................................................................... 158
  Gabriela Duarte1 and Paul Gibbs1,2 ...................................................................................................................... 158

DISSEMINATION OF L. MONOCYTOGENES IN A FROZEN CHICKEN NUGGET PROCESSING LINE .... 159
  Destro. M.T., Andrigheto, C.; Landgraf, M. ........................................................................................................ 159

PREVALENCE OF LISTERIA MONOCYTOGENES IN SOME FOODS AND FOOD PROCESSING PLANTS
IN ICELAND. ..................................................................................................................................................... 160
   Birna Gudbjörnsdottir, Helene L. Lauzon, Sigrun Gudmundsdottir .................................................................... 160

LISTERIOSIS IN DENMARK 1998 - 2000 CLINICAL AND EPIDEMIOLOGICAL ASPECTS ........................ 162
  P. Gerner-Smidt, B. Bruun, V. Fussing, J. Engberg, A.M. Petersen, J. Schiellerup, Sta-tens Serum Institut, Artillerivej
  5, Copenhagen, Denmark ..................................................................................................................................... 162

TRANSFORMING CONTAMINATION PREVALENCE DATA INTO CONCENTRATIONS IN FOODBORNE
MICROBIAL RISK ASSESSMENTS: LISTERIA MONOCYTOGENES DATA AS A PROTOTYPICAL CASE
STUDY ............................................................................................................................................................... 163
  A.D. Hitchins. ...................................................................................................................................................... 163

EPIDEMIOLOGICAL ANALYSIS OF LISTERIA MONOCYTOGENES RECOVERED FROM FOOD AND
ENVIRONMENT PROCESSING PLANT .......................................................................................................... 164
  KEROUANTON A.a, BRISABOIS.Aa., MARAULT M.a and PICARD B.b ....................................................... 164

EPIDEMIOLOGY OF LISTERIOSIS IN FINLAND ............................................................................................ 165
  Susanna Lukinmaa1. Outi Lyytikainen2 and Anja Siitonen1................................................................................. 165
                                                                                      8
EXPOSURE OF LISTERIA MONOCYTOGENES IN AN OUTBREAK CAUSED BY BUTTER ...................... 166
  Riitta Maijala1, Outi Lyytikäinen2 and Tuula Johansson3 .................................................................................... 166

VACUUM PACKED GRAVAD OR COLD-SMOKED RAINBOW TROUT/SALMON ONCE MORE THE CAUSE
OF AN OUTBREAK OF LISTERIOSIS? .......................................................................................................... 167
  Tham W., H. Ericsson, S. Helmersson, T. Netterby, H. Unnerstad, M-L Danielsson-Tham. .............................. 167

OCCURRENCE OF LISTERIA SPP. AND L. MONOCYTOGENES IN PORTUGUESE PRODUCTION LINES
OF FRESH AND COLD-SMOKED FISH .......................................................................................................... 168
  Manuela Vaz-Velho..1,3, Gabriela Duarte, G.1 Paul Gibbs1,2 ................................................................................ 168

LISTERIA MONOCYTOGENES ON A DAIRY FARM ...................................................................................... 169
  Elisabet Waaka, Wilhelm Thamb and Marie-Louise Danielsson-Thamb .............................................................. 169

TWO CONSECUTIVE NATIONWIDE OUTBREAKS OF LISTERIOSIS, FRANCE, OCTOBER 1999 -
FEBRUARY 2000 .............................................................................................................................................. 170
  Veronique Vaillant 1, Henriette de Valk 1, Jocelyne Rocourt 2, Frederic Stainer 3, Olivier Pierre 4, Veronique Goulet1.
  .............................................................................................................................................................................. 170

THE PREVALENCE AND THE BIODIVERSITY OF LISTERIA MONOCYTOGENES IN BIRD FECES IN
FINLAND ........................................................................................................................................................... 171
  S. Hellström, T. Autio, J. Lunden, H. Korkeala ................................................................................................... 171

A POINT-SOURCE OUTBREAK OF LISTERIA MONOCYTOGENES LINKED TO WHIPPING CREAM. .... 172
  J.M. Farber1, F.J. Pagotto1, E. Daley1, S. Kopil1, A. Hughes1, J. Drew2, J. Wylie2, S. Giercke2, D. Nowicki2, S. Harlos3,
  G. Hammond4, and J. Kettner5. ............................................................................................................................ 172

CRITICAL      POINTS        FOR       LISTERIA     MONOCYTOGENES                              CONTAMINATION                           IN        SALMON
SLAUGHTERHOUSES AND FILLETING PLANTS ......................................................................................... 173
  Liv Marit Rorvik 1 2, Heidi Kjos 2, Ragna Heggebo2 ........................................................................................... 173


LAW, REGULATIONS, PREVENTION .............................................................................. 174


U.S. FDA/FSIS DRAFT ASSESSMENT OF THE RELATIVE RISK TO PUBLIC HEALTH FROM
FOODBORNE LISTERIA MONOCYTOGENES AMONG SELECTED CATEGORIES OF READY-TO-EAT
FOODS. ............................................................................................................................................................. 175
  R. E. Brackett and R. L. Buchanan ...................................................................................................................... 175

RISK COMMUNICATION .................................................................................................................................. 176
  Helga Odden Reksnes, ......................................................................................................................................... 176

LISTERIA MONOCYTOGENES IN DOMESTIC ENVIRONMENTS: RECOMMENDATIONS OF THE
INTERNATIONAL FORUM ON HOME HYGIENE............................................................................................ 182
  RR Beumer1,2, SF Bloomfield1, M Exner, GM Fara1, KJ Nath1 and E Scott1. ..................................................... 182

CONVENTIONAL AND NOVEL STRATEGIES FOR INHIBITING LISTERIA IN FOODS .............................. 183
  Elliot T. Ryser, Department of Food Science and Human Nutrition, 2108 S. Anthony Hall, Michigan Slate University,
  East Lansing, MI 48824-1225, USA .................................................................................................................... 183

INACTIVATION OF LISTERIA MONOCYTOGENES CA AND OHIO2 IN ORANGE JUICE AND MILK BY
HIGH HYDROSTATIC PRESSURE (HHP) ....................................................................................................... 184
  Hami Alpas, Faruk Bozoglu ................................................................................................................................. 184

RESULTS SINCE THE INTRODUCTION OF LISTERIA SURVEILLANCE IN HUNGARY ............................ 185
  R. Kiss1, K. Krisztalovics2. M. Füzi3:................................................................................................................... 185

MICROBIOLOGICAL CRITERIA FOR LISTERIA MONOCYTOGENES IN FOODS. ..................................... 187
  Birgit Nørrung, DVM, PhD,................................................................................................................................. 187
                                                                                        9
EPIDEMIOLOGICAL INVESTIGATIONS AND TYPING OF LISTERIA MONOCYTOGENES (L.M.) ISOLATES
FROM PASTEURIZED CHEESE AND HUMAN LISTERIOSIS CASES DURING A SUSPECTED OUTBREAK
IN GERMANY .................................................................................................................................................... 189
   Stefan Brockmann1, A. Sabrowski2, R. Oehme1 E, Gutwein1, D, Maslo G. Ptaff1, P. Kimmig1 .......................... 189




                                                                               10
         Final programme
ISOPOL XIV
International Symposium on Problems of Listeriosis

May 13 – 16, 2001 Mannheim / FRG
Organizer: Hof H.
Universitätsklinikum
Institut für Med. Mikrobiologie und Hygiene
Theodor Kutzer Ufer 1-3
68167 Mannheim
Tel: 0621/3832224, Fax 0621/3833816
e-mail: herbert.hof@imh.ma.uni-heidelberg.de


INTERNATIONAL SCIENTIFIC BOARD                       sponsored by:
Pamer E/ New York/ USA                               Aventis, Becton-Dickinson
Rocourt J./ Paris/ France                            Bio-Mérieux, Grünenthal
Ryser E./ East Lansing/ USA                          Pfizer, Pharmacia,
Schlech W./ Halifax/ Canada                          Lederle, Merck
Swaminathan B./ Bethesda/ USA                        Oxoid,
Tham W./ Uppsala/ Sweden                             DFG

NATIONAL BOARD
Goebel W./Würzburg
Kreft J./ Würzburg
Scherer S./ Weihenstephan
Wehland J./ Braunschweig


LOCAL BOARD
Geginat G.
Lampidis R.
Nichterlein T.
Schlüter D.




                                            11
Sunday, may 13, 2001

Venue: Schloß Mannheim, Rittersaal

19.00 (7 p.m.) Opening address
               Historic remarks on Listeria and ISOPOL   Hof H.
               Come together




                                                   12
Monday, may 14, 2001

Venue: Congress Center (Rosengarten), Variohalle

(chair: Schlech W)
 8.30 - 9.15 Progress in understanding listeriosis and research needs            Swaminathan B.
              for the next decade
 9.15 - 10.00 Novel strategies for inhibiting Listeria in foods                  Ryser E.
10.00 - 10.15 Coffee break■Exhibition
10.15 - 11.00 Clinical manifestations                                            Schlech W.
11.00 - 11.45 Pathology                                                          Deckert M.
11.45 - 12.30 Therapeutic options                                                Hof H.

Lunch

A The risk (chair: Swaminathan)
13.30 - 13.50 Risk analysis                                                      Schlundt J
13.50 - 14.10 Assessment of the relative risk to public health from foodborne    Brackett E.
              L. monocytogenes among selected categories of ready-to eat foods
14.10 - 14.30 Risk communication                                                 Reksnes O.
14.30 - 14.50 Benefits of enhanced surveillance                                  Goulet V.
              Break■Poster session■Exhibition
16.00 - 16.30 Food processing and risk of contamination by L. monocytogenes      Colin P.
16.30 - 17.00 Microbiological criteria for L. monocytogenes in food              Norrung B.
17.00 - 17.20 Epidemiological analysis of L.monocytogenes recovered              Kerouanton A.
              from food and environment processing plant
17.20 - 17.40 Vacuum packed gravad or cold-smoked rainbow trout .                Tham W.
              the cause of an outbreak of listeriosis?
17.40 - 18.00 Poster session


B The diagnosis (chair: Beumer R)
13.30 - 14.00 Conventional isolation methods                                     Donnelly C.
14.00 - 14.30 ISO-11290-1, change of media                                       Beumer R.
14.30 - 14.50 Comparison of three enrichement procedures                         Rijpens N.
              Break■Poster session■Exhibition
16.00 - 16.20 Detection of Listeria in human faeces                              Allerberger F
16.20 - 16.50 Status report on WHO Phase III PFGE study                          Graves.
16.50 - 17.20 Report on the phase III of the WHO serotyping study                Lehmann S.
17.20 - 17.40 Detection of Listeria antigens                                     Bubert A.
17.40 - 18.00 Comparative genomics for deciphering serovar specific              Buchrieser C.
              markers and virulence determinants

C The disease (chair: Schlech W.)
13.30 - 14.00 Enteritis                                                          Schlech W.
14.00 - 14.30 Meningitis                                                         Schlüter D.
14.30 - 15.00 Neural route of cerebral murine infection                          Dons L.
              Break■Poster session■Exhibition
16.00 - 16.30 Veterinary perspective                                             Donachie W.
16.30 - 17.00 Congenital listeriosis: a murine model                             Abram M.
17.00 - 17.30 Animal models for antimicrobial therapy                            Nichterlein T.
              Poster session
19.30         Reception at the Landestechnikmuseum (bus transfer from CC)




                                                    13
Tuesday, 15 may, 2001
(chair: Kaufmann S.H.E.)
 8.30 - 9.15 Pathogenicity                                                                 Goebel W
 9.15 - 10.00 The response of the host cell                                                Wehland J.
10.00 - 10.15 Coffee break■Exhibition
10.15 - 11.00 Early host-pathogen interactions and induction of T cells                    Mielke M.
11.00 - 11.45 Specific T cell-mediated immunity                                            Pamer E.
11.45 - 12.30 Exploiting Listeria for the design of novel vaccination strategies           Kaufmann S.H.E.

Lunch

A Epidemiology and Prevention (chair: Ryser E.)
13.30 - 14.00 Listeriosis in USA, insights from recent outbreak investigations             Mead P.
14.00 - 14.30 Listeria in seafood                                                          Ben Embarek P.
14.30 - 15.00 Prevalence of L.monocytogenes in some foods and food processing              Gudbjornsdottir B.
              plants in Iceland
              Break■Poster session■Exhibition
16.00 - 16.30 Database in Pulsnet, subtyping network for foodborne disease                 Hunter S.B.
16.30 - 17.00 Recommendatiosn of the international forum on home hygiene                   Beumer R.
17.00 - 17.20 A point-source outbreak linked to whipping cream                             Farber J.
17.20 - 17.40 L. monocytogenes on a dairy farm                                             Waak E.
17.40 - 18.00 Effect of different salting and smoking procedures on L. innocua.            Vaz-Velho M.

B Bacteriology (chair: Cossart P.)
13.30 - 14.00 Regulation of intracellular gene expression                                  Freytag N
14.00 - 14.30 Act A                                                                        Domann E.
14.30 - 15.00 Smcl from L. ivanovii: understanding the role of                             Vazquez- Boland J.
              sphyngomyelinase in bacteria-host cell interaction
              Break■Poster session■Exhibition
16.00 - 16.30 Development of optimised marker gene expression vectors                      Hill P.J.
16.30 - 17.00 Cold shock proteins: evidence for role of DNA binding                        Rees C.E.D.
              proteins in low temperature induction
17.00 - 17.20 The use of listeriolysin to identify and characterise                        Gahan C.G.M.
              in vivo induced genes
17.20 - 17.40 Peptide transport: physical and genetic characterization                     Tsai H.N.
              and potential applications
              Poster session

C The defense (chair: Pamer E.)
13.30 - 14.00 Hierarchy and dynamics of peptide-specific CD4 T-cell                        Geginat G.
               populations during L. monocytogenes-infection
14.00 - 14.30 Crosstalk between innate and adaptive immune systems:                        Harty J
              CD8 TCells priming against non-secreted antigens
14.30 - 15.00 Ex vivo analysis of Listeria-specific T cells using MHC                      Busch D.
              multimer reagents
              Break■Poster session■Exhibition
       .
16.00 - 16.30 In vivo effects of internalin related protein A and listeriolysin
              on the murine immune system .                                                Weiß S.
16.30 - 17.00 Naturally processed antigenic peptides in infected mice                      Skoberne M.
17.00 - 17.30 The local defense mechanisms in the brain                                    Schlüter D.
17.00 - 18.00 Poster session

20.00 Uhr      Bus transfer to Heidelberg/ Banquet at the king`s hall in the castle
               Awarding of prizes (Seeliger Award and other prizes for active listeriologists)




                                                          14
Wednesday, 16 may, 2001

(chair: Goebel W.)

 8.30 - 9.15    Genomics of Listeria                                                          Glaser P.
 9.15 - 10.00   Comparative gene regulation in Listeria                                       Chakraborty T.
       .
10.00 - 10.15   Coffee break■Exhibition
10.15 - 11.00   Evolution                                                                     Kreft J.
11.00 - 11.45   The coming project REALIS                                                     Wehland J.
11.45 - 12.30   Structure and components                                                      Kathariou S.

Lunch

A. Fitness of Listeriae in vitro and in vivo (chair: Nichterlein T.)
13.30 - 13.50 Adsorption, and biofilm formation on food grade stainless steel                 Kalmokoff M.L.
       .
13.50 - 14.10 The differential adherence of L.monocytogenes strains                           Norwood D.E.
               in monocultutre and multispecies biofilms
14.10 - 14.30 Gene fragments distinguishing an epidemic L. monocytogenes strain               Kocks C.
               from the prototype strain EGD raise the possibility of lateral gene transfer
14.30 - 14.45 Induction of cold shock proteins upon exposure to stress                        Kamphuis H. H.
14.45 - 15.00 Autolytic behaviour of strains isolated from portuguese cheese                  Faleiro M.L.
               Break■Poster session■Exhibition
16.00 - 16.15 A glutamate decarboxylase system protects Listeria at low pH,                   Cotter P.D.
               and in gastric fluid
16.15 - 16.30 The osmolyte transporter opu C is involved in the virulence                     Wouters J.A.
16.30 - 16.45 Internalins from the human pathogenic L. monocytogenes                          Heinz D.W.
               contain a fused superdomain structure
16.45 - 17.00 The invasion protein inlB binds by its C-terminus lipoteichoic acid             Jonquières R.
17.00 - 17.15. Role of the autolysin ami in the adhesion of L. monocytogenes                  Milohanic E.
                to eucaryotic cells
17.15 - 17.30 In vitro and in vivo virulence heterogeneity in clinical strains                Wagner M
17.30 - 17.45 Polymorphism analysis of genes implicated in virulence                          Kerouanton A.
17.45 - 18.00 Influence of virulence genes on the survival and multiplication                 Walcher M.
                within protozoa

B The bacteriophages (chair: Scherer, S.)
13.30 - 14.00 Comparative genomics of bacteriophages                                          Calendar R.
14.00 - 14.30 Interaction of bacteriophages with the bacterial cell                           Kathariou S.
14.30 - 15.00 Membrane disruption and bacterial cell lysis: lysins and holins                 Loessner M.J
              Break■Poster session■Exhibition
16.00 - 16.30 Practical applications of Listeria phages                                       Rees C. E. D.
16.30 - 16.50 Use of generalised transduction to study virulence mechanisms                   Hodgson D.A.
16.50 - 17.10 Comparison of epidemiologically linked Listeria strains                         van der Meer-Marquet
N.
               characterized by two distinct phage-sensitivity phenotypes
17.10 - 17.30 Characterization of a new pediocin-like sec-dependant bacteriocin               Kalkomoff M.L.
               produced by L. innocua 743

C Listeria as a tool in cell biology (chair: Wehland, J.
       .
13.30 - 14.00. Invasion of mammalian cells by L. monocytogenes                                Cossart P.
       .
14.00 - 14.30 Activation of protein kinase C by listeriolysin and phospholipase               Goldfine H.
14.30 - 15.00 Identification of new actA-binding proteins                                     Kuhn M.
15.00 - 15.30 Molecular dynamics of cytoskeletal proteins involved in Listeria motility       Sechi A.
               Break■Poster session■Exhibition




                                                        15
16.00 - 17.30 Round table discussion:
              The future a) of Listeria research b) of ISOPOL   Hof H., Rocourt J
                                                                Vazquez-Boland J.-A.,
                                                                Swaminathan B.
                                                                Schlech W., Tham W
                                                                PamerE., Kreft J.

18.00   Closing

19.00   Trip to the Palatinat/Visit of a wine cellar
        (optional; extra charge)




                                                       16
HISTORY OF ISOPOL
Herbert K.H. Hof

Institute for Medical Microbiology and Hygiene, Fac. Clin. Medicine Mannheim, Univ. of
Heidelberg, 68167 Mannheim

The first description of Listeriae by E.G.D. Murray/Cambridge/England in 1924 did not trigger
a broad public interest. Only after the rediscovery of Listeriae by J. Potel/Halle/Germany in
1949 and somewhat later by H.P.R. Seeliger/Bonn/Germany the pathogenic role of these
bacteria for man and beast was appreciated by the scientific community. Quite a long time
passed by before the first International Symposium On Problems Of Listeriosis took place.
Then the interest in these particular bacteria grew steadily, so that meetings dedicated to this
subject were organized from time to time:

             I      Giessen / Germany                  1957
             II     Bozeman / USA                      1963
             III    Bilthoven / The Netherlands        1966
             IV     Leipzig / Germany                  1968
             V      Pecs / Hungary                     1972
             VI     Nottingham / UK                    1974
             VII    Varna / Bulgaria                   1977
             VIII   Madrid / Spain                     1981
             IX     Nantes / France                    1985
             X      Pecs / Hungary                     1988
             XI     Copenhagen / Denmark               1992
             XII    Perth / Australia                  1995
             XIII   Halifax / Canada                   1998
             XIV    Mannheim / Germany                 2001

It is a special character of this international conference that it will provide a forum for an
interdisciplinary discussion on quite various aspects of problems with listeriosis. Some of
these problems have been solved by the time but others still remains to be settled. And
researchers tend to trace new ones. Thus, further ISOPOL meetings will have to follow.

                                              17
18
structure, genomics, physiology




               19
SUBPROTEOME ANALYSIS OF LISTERIA MONOCYTOGENES
M. Baumgärtner, O. Diekmann, L. Jänsch, J. Wehland

GBF - German Research Centre for Biotechnology, Dept. of Cell Biology, Mascheroder Weg
1,
D-38124 Braunschweig, Germany

Problem: Our aim is to define the interactions between Listeria and host cells at the
molecular level. This includes studies on the known virulence factors and their cellular
interaction partners as well as a search for hitherto unknown listerial proteins involved in the
invasion process. In order to identify those proteins and to elucidate their role in this process
we focus on secreted and surface proteins of listeria.

Methods: We established protocols for the enrichment and identification of anchored and
non-covalently attached proteins derived from the listerial cell wall, associated and integral
membrane proteins as well as secretory proteins. Secretory and cell wall associated proteins
are mainly separated by conventional 2D-PAGE techniques, whereas 16-BAC/SDS-PAGE is
used for insoluble integral membrane proteins.
For large scale separation and especially for the pre-concentration of certain pH-ranges a
free-flow electrophoresis system is used to identify less abundant proteins under denaturing
as well as non-denaturing conditions.
As a functional proteomics approach Blue Native PAGE is employed, a method that allows
for the separation and identification of multi-protein complexes. This will give us insight into
the interplay of bacterial proteins as well as into pathogen-host interactions.

Results and discussion: The goal of all these efforts is to compare the relevant
subproteomes of the wildtype strain of Listeria monocytogenes to those of deletion mutants
lacking the wildtype invasiveness and of the apathogenic species Listeria inoccua to identify
and characterize proteins responsible for the virulence of listeria.
Along with proteins that are predicted to occur in extracellular subproteomes we detected
several “moonlighting” proteins which do not contain a known signal sequence to explain
their extracellular localization. Future tasks will be to distinguish between laboratory artefacts
and proteins that have an in vivo-relevance in these subproteomes as well as to determine
their function on the cell surface and to characterize their so far unknown secretion
pathways.




                                               20
A GLUTAMATE DECARBOXYLASE SYSTEM PROTECTS
LISTERIA MONOCYTOGENES AT LOW PH, AND IN GASTRIC
FLUID.
Paul D. Cotter1. Cormac G.M. Gahan1,2 , and Colin Hill1,2

                              1
Department of Microbiology        and National Food Biotechnology Centre 2, University
College Cork, Ireland.

Problem: The mechanisms used by Listeria monocytogenes to survive low pH, both in foods
and during gastric transit, have not been well characterised. The glutamate decarboxylase
(Gad) acid resistance system has been identified in a number of commensal and pathogenic
gastrointestinal genera. It has been mooted that the Gad system may play a role in survival
through the stomach. Our aim was to determine whether L. monocytogenes possesses a
Gad system, to characterise the genes and to determine its contribution to acid resistance.

Method: The survival of L. monocytogenes in gastric juice, in acidified broth, and in a variety
of mildly acidic foods in the presence and absence of additional glutamate was determined.
The presence of significant glutamate decarboxylase activity was verified using both rapid
colorimetric and enzymatic GABase assays. The relevant genes (gadABC) were identified
using degenerate PCR and non-polar mutants were created using the SOEing technique.

Results: Transcriptional assays revealed that the genes are up-regulated by an acidic pH
shift. Addition of glutamate dramatically enhances the acid survival of L. monocytogenes
L028. Enzymatic activity assays conducted on parent and mutant strains confirmed that this
phenomenon is due to the Gad system. Gad- mutants are exquisitely acid sensitive, with a 6-
log difference in levels of survival between wild-type and Gad-mutants in ex vivo porcine
gastric juice after 12 minutes. The mutants were also found to have a higher death rate in
acidified complex broth as well as all acidic foods tested. The extent to which different L.
monocytogenes strains are capable of glutamate decarboxylation varies and is directly
proportional to their resistance in glutamate-supplemented gastric fluid.

Conclusion: L. monocytogenes possesses a Gad system which plays a major role in its
ability to survive all low pH environments. That the ability to decarboxylate glutamate is
directly proportional to resistance to acid indicates Gad to be the important system in such
environments. This has implications for the virulence potential of individual strains.




                                              21
DIFFERENT AUTOLYTIC BEHAVIOUR OF STRAINS OF LISTERIA
MONOCYTOGENES ISOLATED FROM PORTUGUESE CHEESE
M.L. Faleiro1 , P. W. Andrew2 , D. Power1

1
 Universidade do Algarve- Faculdade de Engenharia de Recursos Naturais. 8000 Faro,
Portugal, Fax:351 289815927. 2University of Leicester, Department of Microbiology and
Immunology PO BOX 138 University Road Leicester LE 9HN UK, Fax: 44 116 2525030.

Problem: A different autolytic behavior was registered for four strains of L. monocytogenes
isolated from Portuguese cheese when cultivated in a defined medium at 30°C. No autolysis
was observed. This study was realised in order to elucidate this different autolytic behaviour.

Methods: The autolytic and non-autolytic strains were cultivated at 30°C and at 8°C in a
defined medium and a complex medium. The effect of NaCl and KCl on autolysis was
investigated using a microplate system. The existence of autolysins in all strains was
investigated by cultivating the strains in the presence of Micrococcus luteus cell walls and
the autolysin profile was determined by renaturing SDS-polyacrilamide gel electrophoresis
using cell walls of each strain. The presence and activity of the reported autolysin invasion
associated protein (p60) in L. monocytogenes was investigated by Polymerase Chain
Reaction (PCR), immunoblot and renaturing SDS-polyacrylamide gel electrophoresis. To
evaluate the potential pathogenic behaviour of the Portuguese cheese isolates in vivo
investigations in mice were done.

Results: Autolysis did not occur in any of the strains grown at low temperatures or in the
presence of NaCl or KCl. No differences on the autolytic profile of L. monocytogenes strains
were registered when M. luteus cell walls were incorporated into SDS-PAGE. However,
differences on the sensitivity of the cell walls of the different L. monocytogenes strains to
listerial autolysins were registered and variation on the lytic activity of the culture
supernatants of the strains was observed. Two autolysins a p60 a p34 were detected. The
results of PCR of the C-terminal region indicated a difference in the PCR-product size of
autolytic and non-autolytic strains. The cheese isolate C882 was the most virulent of all L.
monocytogenes strains tested (cheese, clinical and laboratory strain NCTC 7973) and
another cheese isolate, strain C681 was the least virulent of all L. monocytogenes strains
tested.

Conclusions: The different autolytic behaviour seems to be related with the reported
autolysin invasion associated protein (p60) and this can be related to the number of Thr-Asp
repeats in each protein. Apparently high autolytic activity is not associated directly with
virulence.




                                              22
PEPTIDE TRANSPORT IN LISTERIA MONOCYTOGENES:
PHYSICAL AND GENETIC CHARACTERISATION AND
POTENTIAL APPLICATIONS.
H.-N. Tsai, David. A. Hodgson

Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, U.K.
Problem: Many bacteria can use peptides as a source of amino acids. Peptide transport
provides an advantage when growing in an environment rich in proteins and peptides. In a
number of species, peptide transport also serves in cell signaling, chemotaxis and
osmoregulation. During intracellular growth, peptide transport provides a major source of
essential amino acids in L. monocytogenes. Amino acid biosynthesis in L. monocytogenes
was disrupted in an attempt to obtain attenuated strains. However, the auxotrophic mutants
were not attenuated in vivo. Possibly the auxotrophs could obtain amino acids in the form of
peptide from the host cell cytoplasm. Our goal is to characterize both the physical and
genetic aspects of peptide transport in L. monocytogenes. A long-term goal of the work is to
construct peptide transport deficient mutants in the hope of developing attenuated strains for
vaccine development.

Methods: Using toxic peptides we isolated mutants blocked in dipeptide transport,
oligopeptide transport and all forms of peptide transport. Using a variety of genetic
techniques, including transposon mutagenesis and transposon tagging we are analyzing the
genes encoding peptide transport.

Results: Physical characterization of the mutants indicates the presence of dipeptide,
tripeptide and oligopeptide permiases. No co-operation or overlapping transport was found
between the dipeptide and oligopeptide permiases. Mutant studies indicate that there might
be genes that control peptide transport at the global level. Mutants unable to transport any
peptides are highly attenuated in tissue culture.




                                             23
IS P60 REALLY AN ESSENTIAL HOUSEKEEPING GENE OF
LISTERIA MONOCYTOGENES ?
Pilgrim S., Kolb-Mäurer A., Bergmann B., *Geginat G., Gentschev l., Goebel W.

Lehrstuhl für Mikrobiologie, Biozentrum, Am Hubland, 97074 Würzburg;
*Institut für Medizinische Mikrobiologie und Hygiene, 68167 Mannheim, Germany.

The iap gene of Listeria monocytogenes encodes a 60 kDa protein termed p60, which is a
major extracellular protein present in all known Listeria species. Spontaneously occuring
rough mutants, which are impaired in the expression of p60 have been shown to be avirulent
in mice and they also have a reduced ability to invade non-phagocytic host cells such as 3T6
fibroblasts or Caco-2 human enterocyte-like cells. Purified p60 has been shown to possess
mureine hydrolase activity aquired for late step in cell division. Till now, it was thought that
p60 is an essential housekeeping protein and it is not possible to make a viable iap deletion
mutant. However, in this study, we are reporting for the first time construction of a L.
monocytogenes iap deletion mutant. This mutant (A/ap) expresses no p60. Our results
strongly indicate that the resulting phenotype is attributed to this single mutation, but a
complementation of iap has not yet been performed.
The effect of the iap deletion on the bacterial phenotype and on invasion of host cells are
described in this work.




                                              24
COLD SHOCK PROTEINS IN LISTERIA: EVIDENCE FOR ROLE
OF DNA BINDING PROTEINS IN LOW TEMPERATURE
INDUCTION
Catherine E.D. Rees, Faye M. Barnard

University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough,
Leicestershire, UK. LE12 5RD
One of the most notable features of Listeria monocytogenes as a human pathogen is its
psychrotrophic nature. Its ability to proliferate at low temperature is often central to its ability
to cause food poisoning, both by allowing growth on foods during cold storage and due to its
establishment in chilled food-processing plants leading to post-process contamination. Our
interest in this feature of the bacterium is to try and understand the cold adaptation process
and ultimately learn how to suppress its growth. To date we have identified 3 major cold-
shock gene homologues (cspA, cspB and cspC) in Listeria, although imminent release of the
completed genome sequence may allow us to identify further members of the gene family
from database searches. Analysis of patterns of expression of cspA indicate that it is induced
at low temperature, with high levels of mRNA being induced within 10 min of temperature
downshift. cspA mRNA remained detectable at low temperatures in cultures once growth
had resumed, thus showing a different pattern of gene expression to the transient induction
identified in mesophilic organisms. Analysis of promoter sequences showed strong
homology to those of Bacillus cspB and alignment of sequences identified a conserved
"Downstream Box" motif common to Gram-positive bacteria and distinct from that identified
in E. coli. EMSA ('gelshift') analysis provided evidence for protein binding to an IHF
consensus sequence found in the promoter region of cspA. However no IMF homologues
have yet been identified in Gram-positive organisms. The closest member of this family of
DNA binding proteins known is the HU homologue Hbsu, and we have identified a gene
homologue in Listeria termed Him. The cspA promoter cloned into a lux promoter probe
vector showed lower levels of activity in an ihf E. coli strain compared to the ihf parent.




                                                25
POST-GENOMIC ANALYSIS OF THE LISTERIA
MONOCYTOGENES GENOME: ROLE OF SURFACE PROTEINS.
Didier Cabanes, O. Dussurget, P. Dehoux, L. Frangeul, P. Glaser, C Buchrieser, The Listeria
Consortium, Pascale Cossart

Unité des Interactions Bactéries-Cellules, Institut Pasteur, 28 rue du Docteur Roux, 75015
Paris, France.

Problem: Listeria monocytogenes is an ubiquitously occurring gram-positive pathogen.
Listerial infections threaten the unborn, immunocompromised and elderly individuals. During
an acute infection, many tissues are infected, demonstrating the ability of these bacteria to
invade numerous eukaryotic cells in different tissues. Several bacterial proteins contributing
to the mechanisms used to enter cells, escape from phagocytic vacuole and spread from
one cell to another have been identified, including the invasion proteins InlA and InlB, the
secreted pore-forming toxin listeriolysin 0, and the surface protein ActA. InlA and InlB display
a region with a succession of leucine-rich repeats (LRRs). InlA, as a protein which is
targeted to and exposed on the bacterial surface, contains a signal peptide and a C-terminal
region containing a LPXTG peptide which allows covalent linkage to the peptidoglycan. InlB
does not contain the LPXTG motif, but a C-terminal region with GW repeats that is
necessary and sufficient to anchor InlB to the bacterial surface. Additional factors important
for survival in (he infected host are either the proteins which regulate synthesis of these
virulence factors like PrfA the regulator of nearly all the virulence genes identified to date. All
known PrfA-regulated genes possess a conserved symmetric sequence (PrfA box)
corresponding to the binding site of PrfA within the promoter region.

Results: The genome sequence of L. monocytogenes strain EGD-e Serovar 1/2a was
completed within the framework of an European consortium coordinated by P. Cossart and
the sequence of (the L. innocua (non-pathogenic listeria) genome was also recently
completed by P. Glaser et al at the Institut Pasteur. We look advantage of the availability of
these two genome sequences to search for new genes potentially implicated in host
specificity and virulence. We focused on «internalin like» proteins (proteins with signal
peptide, LRRs and LPXTG motif). LPXTG proteins (proteins with signal peptide and LPXTG
motif and GW proteins (proteins with signal peptide and GW repeats). Genes were
inactivated.
The general mutagenesis strategy used and the results obtained after in vitro analysis of the
mutants for adhesion, invasion and survival in different tissue culture cell, and in vivo
analysis after oral or intravenous infection for virulence, invasion and growth in the organs in
animal models, will be presented




                                                26
THE INVASION PROTEIN INLB OF LISTERIA MONOCYTOGENES
BINDS BY ITS C-TERMINUS LIPOTEICHOIC ACID
Renaud Jonquières1, Hélène Bierne,1 Franz Fiedler,3 Pierre Gounon3 and Pascale Cossart1.1

1
 Unite des Interactions Bactéries-Cellules, 3Station Centrale de Microscopic Electronique, 25
et 28 rue du Docteur Roux. Institut Pasteur. 75724 Paris Cedex 15. 2Institut für Genetik und
Mikrobiologie, Maria Wardstraße 1a, 80638 München, Germany.
Problem: InlB is a Listeria monocytogenes protein that is sufficient to promote entry in a
variety of mammalian cells. The last 232 amino acid domain (Csa) of InlB has been shown to
mediate attachment on the listerial surface, although its sequence docs not suggest any
known mechanism of association to the bacterial surface. InlB is present both on the
bacterial surface and in culture supernatants. As recently demonstrated, both forms of InlB,
soluble and surface-bound, can trigger signalling in host cells. To elucidate the specific role
of each of the two forms, it was important to understand bow InlB associates with the
bacterial surface.

Results: Using microscopy, we bring evidence that InlB is partially buried in the cell wall
layer, and by fractionation experiments we demonstrate that InlB associates with the
bacterial cytoplasmic membrane fraction. Moreover, using purified lipoteichoic acid (LTA)
and the three polypeptides InlB, Csa, or InlBΔCsa (InlB lacking the last 232 amino acids), we
demonstrate that LTA is the main bacterial ligand for the Csa domain of InlB.

Conclusion: These results provide the first evidence of an interaction between
lipoteichoic acids and a bacterial protein involved in adhesion and signalling.




                                              27
INTERNALINS FROM THE HUMAN PATHOGEN LISTERIA
MONOCYTOGENES CONTAIN A FUSED SUPERDOMAIN
STRUCTURE
Dirk W. Heinz1, Wolf-Dieter Schubert1, Gero Göbel1, Meikel Diepholz1, Ayub Darji3, Daniel
Kloer1, Torsten Hain3, Trinad Chakraborty3, Jürgen Wehland2 & Eugen Domann3

Departments of 1Structural Biology and 2Cell Biology, German Research Center for
Biotechnology (GBF), Mascheroder Weg l, D-38124 Braunschweig, Germany 3Institute for
Medical Microbiology, University of Giessen, Frankfurter Straße 107, D-35392 Giessen

Problem: Listeria monocytogenes is an opportunistic food borne human and animal
pathogen. Host cell invasion requires the action of the internalins A (InlA) and B (InlB), which
are members of a family of listerial cell-surface proteins. Common to these proteins are three
N-terminal domains that have been shown to direct host-cell specific invasion for InlA and
InlB.

Results: Here we present the high resolution crystal structures of these domains present in
InlB and InlH and show that they constitute a single fused "internalin superdomain". In this
superdomain, a central leucine rich repeat (LRR) domain is contiguously flanked by a
truncated EF-hand-like cap domain and an immunoglobulin (Ig)-like domain. The extended
ß-sheet, resulting from the distinctive fusion of the LRR and the Ig-like domain, constitutes
an adaptable concave interaction surface, which we propose is responsible for the specific
recognition of the host cellular binding partners during infection.




                                              28
IDENTIFICATION OF NEW ACTA-BINDING PROTEINS
Michael Kuhn, Thilo Pfeuffer, Susanne Bauer, Werner Goebel

Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften der Universität
Würzburg, Am Hubland, 97074 Würzburg, Germany

Problem: The Listeria monocytogenes surface protein ActA is an important virulence factor
required for listerial intracellular movement by inducing actin polymerisation. The only host
cell proteins known that directly interact with ActA are the phosphoprotein VASP which binds
to the central proline-rich repeat region of ActA and proteins of the Arp2/3 complex which
bind to the N-terminal region of ActA. To identify additional ActA-binding proteins we applied
the yeast two-hybrid system to search for mouse proteins that interact with ActA. Methods:
A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from
strain L. monocytogenes EGD as bait.

Results: Three different AIPs were identified one of which was identical to the human
protein LaXp180. Binding of LaXp180 to ActA was also demonstrated in vitro using
recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180
antibody and fluorescence microscopy, we showed that LaXp180 colocalizes with a subset
of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly
growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L.
monocytogenes was asymmetrical and mutually exclusive with F-actin polymerisation on the
bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in
signal transduction pathways and the regulation of microtubule dynamics. By
immunofluorescence we showed that stathmin colocalizes with intracellular ActA-expressing
L. monocytogenes.

Ref.:
Pfeuffer et al., 2000. Cell. Microbiol. 2:101-114.




                                               29
CHARACTERISATION OF PARTIAL CYTOSINE DNA
METHYLATION IN SEROTYPE 4B STRAINS OF LISTERIA
MONOCYTOGENES
M. Akhtar, T. M. Perehinec, A. Nazli, P. J. Hill, C. E. D. Rees

University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough,
Leicestershire, UK. LE12 5RD
We have recently identified variable patterns of DNA methylation sites in Listeria
monocytogenes. The degree of partial methylation is variable but is specific to the particular
isolate, and high level methylation was only found in serotype 4b strains. Controls were
carried out to ensure that this partial restriction was due to specific methylation and not due
to simple enzyme inhibition and the use of isoschizomers with differing methylation
sensitivities allowed us to identify this as cytosine-specific methylation occurring within Clal
restriction sites.
The gene responsible for this methylation was cloned and found to have homology to other
cytosine methyl-transferases but is not found associated with a Clal restriction endonuclease
on the chromosome.
To create these reproducible patterns of partial DNA methylation, the methylation event must
be controlled. To investigate this broth cultures were prepared by inoculation with single L.
monocytogenes cells, which can only be methylated at one of the susceptible sites. DNA
was recovered from each of these cultures and all cultures exhibited exactly the same
pattern of DNA methylation, indicating that these patterns are not inherited but methylation at
this locus must occur in a controlled manner.
Studying the location of Cytosine methylation sites within the prfA gene region does not
suggest a direct role in modulating gene expression, but in invasion assays a correlation
could be found between the degree of DNA methylation associated with an isolate and the
invasiveness of that strain. This suggests that the methylation may have an influence on
gene expression and that in these cultures with highly variable methylation patterns, some
cells may be pre-primed to infect.




                                               30
A RESPONSE REGULATOR OF LISTERIA MONOCYTOGENES
L028 SHOWING HOMOLOGY TO KDPE IS IMPORTANT FOR
GROWTH AT LOW TEMPERATURE AND HIGH OSMOLARITY
Lone Brondsted1, Birgitte H. Kallipolitis2, Hanne Ingmer2, and Susanne Knöchel1

Department of Dairy and Food Science and Department of Veterinary Microbiology, The
Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C,
Denmark, Phone: +45 35 28 32 71, Fax: +45 35 28 32 31, e-mail: lobr@kvl.dk

Problem: During growth at low temperature and high osmolarity Listeria monocytogenes
accumulates compatible solutes such as carnitine and glycine betaine. Regulation of uptake
of carnitine and glycine betaine involves the alternative sigma factor B. In bacteria such as
Bacillus subtilis and Escherichia coli adaptation to high osmolarity is dependent on an initial
uptake of potassium, and in E. coli this is regulated by a two-component response regulator
KdpE.

Method: We have constructed a L. monocytogenes L028 mutant in the two-component
response regulator showing homology to KdpE of Clostridium acetobutylicum and E. coli.
By the use of defined as well as complex media conditions we have investigated the
involvement of L. monocytogenes kdpE in growth at low temperature and high osmolarity.
Furthermore, the effect of the presence of osmo- and cryoprotectants has been tested during
defined media conditions.

Results: Our results show that kdpE of L. monocytogenes is important for growth at low
temperature and at high osmolarity, indicating that adaptation to growth at low temperature
involves uptake of potassium.




                                              31
COMPARATIVE GENOMICS FOR DECIPHERING SEROVAR
SPECIFIC MARKERS AND VIRULENCE DETERMINANTS OF
EPIDEMIC LISTERIA MONOCYTOGENES
Buchrieser C, Simoes N1, Jacquet CH 3, Frangeul L1, Rocourt J3, Couve E1, Kunst F1,
Cossart P2, and Glaser P1

1
 Laboratoire de Genomique des Microorganismes Pathogenes, 2Unite des Interactions
Bacteries-Cellules and 3Laboratoire de Listeria, Institut Pasteur, 25 Rue du Dr. Roux, 75724
Paris, France

Problem: Listeria monocytogenes, the causative agent of listeriosis, is an ubiquitous gram-
positive food-borne pathogen. Among the three serovars which are usually involved in
human listeriosis (1/2a; 1/2b;4b), out of 13 serovars identified within the species L.
monocytogenes, only one (4b) is mainly associated with outbreaks of invasive disease. This
suggests that enhancement of organism specific virulence factors may play a role in
epidemic disease, although all isolates of L. monocytogenes have the constitutive ability to
produce the virulence factors known to date, and that there may exist other virulence factors
yet undetermined and probably specific ones for L. monocytogenes strains causing
epidemics.

Methods: In order to identify those specific markers for epidemic strains and to decipher
probable Serovar specific virulence genes we undertook low coverage sequencing of L.
monocytogenes Serovar 4b (CLIP 80459), which caused an epidemic in France end 1999
and beginning 2000. We obtained 10000 shotgun sequences covering 1 times the genome.
The sequences were compared to the complete genome sequences of L. monocytogenes
strain EGD-e Serovar 1/2a and L. innocua (CLIP 11262) using an in silico approach.
Identified Serovar specific sequences were confirmed by comparative genomics using high
density membranes and additional strains of L. monocytogenes Serovar 4b.

Results: This approach allowed to identify about 100 regions specific to L. monocytogenes
Serovar 4b.

Conclusion: This understanding will allow the development of rapid, sensitive and accurate
diagnostic and typing tools for identification of L. monocytogenes in agriculture, food and
health care and to define characteristics of particularly successful clonal pathovariants in
causing disease.




                                             32
ISOLATION OF NOVEL MUTATIONS THAT INFLUENCE THE
REGULATION OF LISTERIA MONOCYTOGENES
INTRACELLULAR GENE EXPRESSION
Nancy E. Freitag, Lynne Shetron-Rama

Seattle Biomedical Research Institute, 4 Nickel-son St., Seattle WA.USA 98109

Problem: Listeria monocytogenes is capable of sensing the different host cell compartment
environments it encounters during the course of infection and of responding with the
regulated expression of bacterial virulence genes. One example of differential virulence gene
expression is the regulated expression of actA, whose gene product is required for actin-
based motility. actA is expressed at low to undetectable levels during bacterial growth in
standard broth culture, however, its expression increases over 200 fold once the bacteria
reach the host cell cytosol. The mechanisms that lead to intracellular induction of actA
expression are unknown.

Method: To identify bacterial components that contribute to regulation of actA expression, L.
monocytogenes containing actA-gus transcriptional reporter gene fusions were chemically
mutagenized and examined for increased actA expression following growth on standard
culture media.

Results: More than 40 L. monocytogenes mutants were isolated with actA expression levels
ranging from 20 to 500-fold greater than those observed for wild type strains. Many of the
mutants appeared to contain
mutations that resulted in global changes in virulence gene expression. Several of the actA
regulatory mutations exhibited linkage to prfA, a transcriptional regulator of L.
monocytogenes virulence genes. Many other regulatory mutants contained mutations that
mapped at least 40 kb outside of the prfA regulon.

Conclusion: The isolation of L. monocytogenes actA expression mutants with mutations
mapping outside the prfA regulon strongly supports the existence of regulatory factors, in
addition to PrfA, that contribute to the control of actA expression.




                                             33
DEVELOPMENT OF OPTIMISED MARKER GENE EXPRESSION
VECTORS FOR LISTERIA
Philip J. Hill, Saara N.A. Qazi, Kenneth Mellits and Catherine E.D. Rees

University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough,
Leicestershire UK LE12 5RD

Problem:The use of marker genes for studies of bacterial physiology and genetics has
become an essential tool for microbiologists. However many of the marker genes have been
developed first for use in E. coli and other Gram-negative bacterial species. When a native
red-shifted GFP (green fluorescent protein) variant (GFP3) was cloned in to a Gram-positive
expression vector containing the Pxyn promoter and transformed into Listeria
monocytogenes, only a small proportion of the population was seen to fluoresce when
examined by epifluorescence microscopy. The phenomenon was independent of both the
GFP gene variant and promoter used.

Results: Analysis of both mRNA and protein synthesis revealed that translation of all GFP
genes was limiting in L. monocytogenes. Modification of the translation initiation region of the
GFP3 gene resulted in a homogeneously fluorescent population of cells and this approach
was used to construct a variety of reporter plasmids for Listeria and other Gram-positive
bacteria. Translationally enhanced vectors were also developed using the unstable GFP3
variants which, although having a lower fluorescent output, retain their short-half life
characteristics in L. monocytogenes and therefore can be used as a sensitive monitor of
gene expression. High level expression of these reporter constructs in L. monocytogenes
was evaluated to determine if it had any detrimental biological effect during intracellular
infection of eukaryotic cell lines. The gfp+ Listeria cells were found to invade Caco2 cells
equally well as the wildtype cells, demonstrating that these expression systems can be used
to monitor the bacterium in complex environments at the single cell level. Introduction of a
second marker gene (luxABCDE operon) produced vectors that can be used both to monitor
cell viability and used for histopathological location of Listeria following infection.




                                              34
DEVELOPMENT AND VALIDATION OF A NOVEL REPORTER
GENE SYSTEM FOR THE IDENTIFICATION OF
INTRACELLULARLY ACTIVE PROMOTERS FROM L.
MONOCYTOGENES
Sonja Otten and Trinad Chakraborty

Institute for Medical Microbiology, University of Giessen, Frankfurter Str, 107, D-35392
Giessen, Germany
To identify listerial genes showing preferential expression within the eukaryotic host cell, we
developed a novel reporter gene system, consisting of a transcriptional fusion between the
promoterless bgaB gene (coding for a ß-galactosidase from Bacillus stearothermophilus)
and the promoterless cat gene (coding for a chloramphenicol acetyl-transferase from
Bacillus pumilus). Promoters of several known virulence genes from L, monocytogenes
revealed a clear discrimination between extracellularly and intracellularly active promoters.
We subsequently constructed a gene bank with fragments of chromosomal DNA from L,.
monocytogenes, preceding the transcriptional fusion between bgaB and cat. L.
monocytogenes EGD was transformed with the miscellaneous plasmid constructs and a first
screening of approximately 1000 clones was carried out.
This screening experiment yielded four plasmids with chromosomal fragments displaying
intracelluIar promoter activity and thereby expression of the bgaB gene, The corresponding
DNA inserts were sequenced and compared with the L, monocytogenes chromosome. They
all represented promoter regions of putative housekeeping genes, confirmed by the fact that
besides also extracellular promoter activity was observed.
The novel bgaB/cat reporter gene system promises to be a useful tool for the identification of
intracellularly active promoters. A continuation of the screening will lead to the identification
of further promoters and their respective genes preferentially expressed during the
intracellular state of L. monocytogenes.




                                               35
EVOLUTION OF VIRULENCE IN LISTERIA
Jürgen Kreft, Robert Lampidis *, Eva Ng , Werner Goebel

Lehrstuhl für Mikrobiologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074
Würzburg, Germany
* Institut f. Med. Mikrobiologie
und Hygiene, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany

The genus Listeria is particularly suited for investigations into the evolution of bacterial
virulence because it comprises pathogenic species of different virulence potential as well as
related, but nonpathogenic representatives. The pathogenic species L. monocytogenes and
L. ivanovii are characterized by their ability to invade different cell types of eukaryotic hosts,
to multiply therein and to spread directly from cell to cell.
Molecular genetic analyses revealed that most of the genes known to date as being involved
in the different steps of the infectious cycle are located within a few chromosomal gene
clusters. Invasion of non-phagocytic host cells by Listeria requires the products of the
internalin multigene family. All internalins have a varying number of leucine-rich repeats
(LRRs) in their N-terminal domain, the so-called large internalins are covalently bound to the
peptidoglycan via a C-terminal LPXTG motif. The small internalins are devoid of such a
membrane anchor, their precise function in virulence is largely unknown. LRR-proteins have
been described for a variety of other bacteria, as well as surface proteins with a LPXTG
motif, however, to date only in Listeria proteins have been found which combine both
features. The internalin gene clusters have evolved by duplications and rearrangements,
taking into account their repetitive nature, they show a striking structural stability.
Another characteristic of pathogenic Listeria is the presence of a large cluster of non-related
genes, the products of which are all required for bacterial multiplication within host cells and
for cell-to-cell spread. This PrfA-dependent virulence gene cluster (vcg / vcl) is found almost
identical in L. monocytogenes and L. ivanovii, but is also present in the nonpathogenic
species L. seeligeri, albeit in an inactive form. Similar to the internalin gene clusters, is has
some properties of a pathogenicity island, therefore it is now also called LIPI-1.
The evolutionary implications which derive from the structure of LIPI-1 in the different Listeria
species, will be discussed, as well as the evidence for horizontal gene transfer between
Listeria and other pathogenic bacteria.




                                               36
CHARACTERISATION OF THE SIGB AND LMU OPERONS OF
LISTERIA MONOCYTOGENES
Sandra Wagner and Trinad Chakraborty


Institute for Medical Microbiology, Frankfurter Str.107, 35392 Giessen, Germany


The lmu operon of L. monocytogenes was identified by the subtractive hybridistation
between Listeria innocua and L. monocytogenes genomes. A second operon sigB was also
analysed as this was also indicated in L. monocytogenes pathogenesis.
The use of PCR confirmed the sigB operon was found within both L. monocytogenes and L.
innocua however the lmu operon was only found in L. monocytogenes. To determine if
these operons are required as pathogenic factors, deletion mutants were constructed and
subjected to invasion and intracellular growth studies.
The sigB strain demonstrated lower invasion and intracellular growth in Vero cells
compared to the EGD wild-type. The lmuC strain possessed a greater defect in invasion
and intracellular growth compared to both sigB and EGD.
Our results suggest that the sigB and lmu genes play a role in host cell infection and
intracellular growth of this bacterium.




                                            37
IDENTIFICATION OF NEW PROTEINS ASSOCIATED TO THE
CELL WALL OF LISTERIA MONOCYTOGENES
Jessica Schaumburg, Uwe Kärst and Jürgen Wehland

German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig

Cell wall associated internalines play a significant role in adhesion and invasion of Listeria
monocytogenes, a facultative intracellular pathogen, into host cells. To find new proteins
involved in these processes, we perform serial extraction of surface proteins from L.
monocytogenes with different buffers, salts and detergents. We identified a 35 kD
lipoprotein. Its genomic sequence shows a signal peptide and a putative prfA binding site. To
reveal the function of this protein, we compared the EGD wildtype strain with a deletion
mutant in growth experiments at 37 °C and 25°C, and the infection of different eukaryotic cell
lines. In these experiments the mutant did not display any significant differences in growth
rate, infectioness and intracellular growth. We are also looking for other gene products
upregulated in the mutant to compensate the deleted function.
Since prfA is a positive regulation factor for virulence genes, we compared extracts from the
EGD wildtype with a prfA deletion mutant and a constitutive prfA overexpressing strain. After
separating these protein mixtures in one- or two-dimensional gels, we identified 33 protein
bands or spots by N-terminal sequencing and mass spectrometry. We identified 8 proteins
that appear to be prfA-dependent including the known virulence factors ActA, listeriolysin
and phospholipase C.




                                             38
IDENTIFICATION OF PUTATIVE VIRULENCE GENES IN
LISTERIA MONOCYTOGENES BY USE OF A GFP ENCODING
PROMOTER PROBE
Meirion L Jones, David A Hodgson

Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL UK

A promoter probe has been designed to detect promoters which are expressed whilst
Listeria monocytogenes is growing in tissue culture, in order to identify putative virulence
genes.
A plasmid has been produced containing a promoterless Operon consisting of genes
encoding green fluorescent protein (gfp) and chloramphenicol acetyl transferase. A multiple
restriction enzyme polycloning site (pcs) is located upstream of the Operon. An origin of
transfer allows conjugation of the plasmid into L. monocytogenes and a temperature
sensitive origin of replication allows the construct to be integrated into the L. monocytogenes
chromosome by homologous recombination. The conjugation system has proved to be a
very efficient method of transferring DNA into L. monocytogenes.
A 1.5-5kb L. monocytogenes DNA library has been inserted into the plasmid, conjugated
into L. monocytogenes and exconjugants used to infect mouse L cells in tissue culture.
Infected cells were screened for resistance to chloramphenicol and gfp production, to
identify constructs containing expressed promoters. Promoters have been identified
which are active only in tissue cultures and others, which are active both in tissue culture
and in broth. These are likely to represent genes involved in virulence and housekeeping
function




                                              39
POLYPORPHISM ANALYSIS OF GENES IMPLICATED IN
LISTERIA MONOCYTOGENES VIRULENCE
KEROUANTON A.a, BRISABOIS A.a, MARAULT M.a, COSSART P.c and PICARD Bb
a
  Unite d'Epidemiologie Bacterienne, Agence Francaise de Securite Sanitaire des Aliments,
39-41 rue du 11 novembre 1918, 94700 Maisons-Alfort, France. b Laboratoire de
Microbiologie, Faculte de Medecine de Brest, 21 av Camilles Desmoulins, 29200 Brest,
France c Unite des Interaction Bactéries-Cellules, Institut Pasteur, 25-28 rue du Docteur
Roux, 75015 Paris, France

Problem: Bacteria of the genus Listeria are common in the environment and also occur in
the intestinal tracts of healthy animals. Among the different species Listeria monocytogenes
is the only commonly pathogenic species for humans. Several molecular typing methods are
now available to differentiate L. monocytogenes strains implicated in listeriosis outbreak or to
trace L. monocytogenes strains in food industry. The objective of this study was to evaluate
the ability of genotypic methods to distinguish potentially virulent isolates among L.
monocytogenes from different origins.

Method: To this purpose, a panel of 43 L. monocytogenes was selected, they were isolated
from human infections, food and food industry environment following outbreaks, sporadic
cases or in epidemiological investigations and two strategies of genes analysis have been
applied. First, PCR-RFLP was applied on six genes (hly, actA, mpl, pIcB, plcA, prfA)
recognized to be implicated in the various stages of the bacterium intra-cellular invasion
process. Secondly, actA and plcA genes were also analyzed by southern-blot after DNA
macrorestriction and pulsed-field gel electrophoresis.

Results: Results showed that nearly all L. monocytogenes strains, including those isolated
in food industry possessed the 6 virulence genes studied and the two strategies converged
to indicate that the polymorphism of the genes implicated in virulence was not related to
epidemiological origin of strains but to their serotypes.

Conclusion: These data strengthen the clonal structure of L. monocytogenes previously
described. However, RFLP techniques indirectly reflects the genetic polymorphism and a
direct comparison by sequencing would represent a more precise approach.




                                              40
GENE FRAGMENTS SPECIFICALLY PRESENT IN AN EPIDEMIC
STRAIN OF L. MONOCYTOGENES RAISE THE POSSIBILITY OF
LATERAL GENE TRANSFER BETWEEN PATHOGENIC AND
NON-PATHOGENIC LISTERIA SPECIES
Christine Kocks

Institute of Genetics, University of Cologne, Zuelpicher Str. 47, D-50674 Köln, Germany

Problem: Most major food-borne outbreaks of listeriosis in Europe and in the United States
were caused by genetically closely related L. monocytogenes strains of serotype 4b. It is
unknown to which extent the genomes of these strains differ from the virulent prototype
strain EGD and whether genomic loci exist that could underlie the epidemic phenotype.

Results: We genomicly subtracted the recently sequenced virulent prototype strain L.
monocytogenes EGD from a prototype epidemic strain. We estimate that about 5% of the
genome of the epidemic strain is not present in EGD. We have isolated and sequenced 39
gene fragments corresponding to 20% of the differential genome material. For half of these
fragments no function could be predicted on the basis of homology. Of the other half 83%
had homologies to bacterial surface proteins, some of which have been implicated in
virulence. This distribution was in marked contrast to fragments randomly drawn from the
genome. We have used Southern hybridization of arrays to assess the occurrence of these
epidemic strain-specific fragments in other bacterial genomes. Surprisingly, some of the
fragments hybridized in a mosaic-like fashion to the genomes of the animal pathogen L.
ivanovii and to the non-pathogenic L. innocua.

Conclusion: Our results have implications for Listeria strain typing because they raise the
possibility that pathogenic and non-pathogenic Listeria species exchange genetic surface
determinants by lateral gene transfer. Moreover, these results provide a starting point for the
isolation of epidemic strain-associated genes.




                                              41
ACTA AND INTRACELLULAR MOTILITY
Eugen Domann
Institute for Medical Microbiology, Frankfurter Strasse 107, D-35392 Giessen

INTRODUCTION: L. monocytogenes is a bacterium which employs compartments of the
host cell machinery for its pathogenesis. The life cycle of this pathogen starts with the
bacterium-induced uptake (internalization) where the bacterial surface molecules internalin A
and B interact with the host cell receptors E-cadherin and met-receptor tyrosine kinase,
respectively. Immediately after internalization the intruder egresses from the endosom by
support of the secreted molecules listeriolysin (LLO) and a phosphatidylinositol-specific
phospholipase C (PlcA) and enter the cytoplasm. The otherwise non-motile bacteria
accumulate actin filaments, generate polar actin tails and move intracellularly like „comets“.
This sophisticated process is enabled by the ActA polypeptide located on the surface of the
bacteria and is described below. Upon contact with the plasma membrane of the host cell, L.
monocytogenes induces pseudopod-like protrusions and enters adjacent cells. The main
factors involved in lysis of the double-membrane vacuole within the new host are a
phosphatidylcholine-specific phospholipase C, PlcB, and listeriolysin and another infection
cycle begins.
Intracellular motility of L. monocytogenes is a key event in listerial pathogenicity and an
excellent example for molecular mimicry. ActA is the sole factor to recruit actin filaments and
to generate actin tails for movement, harnessing host cell proteins by mimicking domains of
cell proteins normally engaged in the host cell cytoskeleton and eukaryotic movement. L.
monocytogenes is one member of a group of microorganisms able to move actin-based: the
enteric bacteria Shigella ssp., the obligat intracellular parasites Rickettsia, and Vaccinia
virus.

RESULTS AND DISCUSSION: The essential role of the actA gene was in part described by
the observation that an actA-deficient mutant strain was still able to enter host cells but was
unable to move intracellularly in infected tissue culture cells. This isogenic mutant strain was
devoid of actin filaments and and the offspring accumulated intracellularly as microcolonies.
In addition, this actA mutant strain was highly attenuated for virulence when assessed for
this property in the mouse model of infection.
The actA gene encodes a protein with 639 amino acids, whereas the mature protein
comprises 610 aa. The N-terminal part harbors a 29 amino acid long signal peptide
necessary for transport of this molecule across the bacterial membrane. The C-terminus has
a membrane anchor region to tether the protein to the bacterial cell wall. One region of this
molecule is rich in positively charged amino acid residues, 129 - 153, and is essential for
actin assembly. Amino acid residue region 97-121 is responsible for the continuity of the
assembly process and region 116-122 for tail formation. Another important region is a
fourfold presence of the centrally located proline-rich domains with high sequence
homologies to host cell proteins zyxin, vinculin, and ankyrin.
The proline-rich regions recruit VASP (vasodilator-stimulated phosphoprotein) and Mena
(mouse Enabled) proteins. Both belong to the Ena-VASP family of proteins which share
homology with the Drosophila Enabled (Ena) protein, known to be involved in neuronal
development. This type of protein is organized in three domains, an Ena-VASP homology 1
domain (EVH1), a proline-rich central domain, and an EVH2 C-terminal domain. In vitro
studies revealed that both VASP and Mena bound directly to the ActA molecule. Binding was
mediated by the EVH1 domain of these proteins and the FPPPP motif in ActA. VASP/Mena
                                                42
establish a direct connection between intracellular Listeria and the cytoskeletal components
of the host cell by recruiting profilin providing polymerization-competent actin monomers.
Hence, the polyproline-rich motif of ActA represents a binding motif for EVH1 domains and
the interaction of the Ena/VASP proteins with this domain mimics the interaction with
endogenous substrates present within the primary sequence of the focal adhesion proteins
zyxin, vinculin, and ankyrin.
The N-terminal part of ActA initiate actin assembly by recruiting the Arp2/3 complex, firstly
identified in Acantamoeba as a seven-polypeptide-complex which contains the proteins Arp2
and Arp3. This complex alone is inactive but can nucleate actin and induces actin
polymerization when incubated with ActA or proteins of the WASp (Wiskott-Aldrich
Syndrome proteins) family of proteins. The 146-KKRRK-150 motif of the ActA protein is
responsible for this interaction and mutations in the arginine residues abolish intracellular
motility by interfering with the recruitment of the Arp2/3 complex. The acidic stretch region of
ActA located C-terminal of the signal peptide is probably involved in the efficiency of
bacterial movement and is equivalent to the acidic-stretch region present in WASp-family
proteins.
The concentrated functions of all these host cell proteins accumulated on the surface of the
bacteria via ActA result in intracellular bacterial movement. The average velocity is about
0.02-0.3 µm/s, but as fast as 1.5 µm/s in some cells, and the speed is roughly proportional to
the rate of actin polymerization. The tail comprises two different types of polymerized actin:
closer to the bacterial surface relatively short (0.2-0.3 µm) disordered filaments are found
whereas very long linear actin tails (up to 30 µm) emanating from the bacterium are found
throughout the tail.

CONCLUSIONS: Overall, ActA evolved as a molecule which mimics domains of host cell
proteins involved in actin polymerization of eukaryotes and allows Listeria the perfect
exploitation of components of the infected eukaryotic host cell. The specific interaction of
several listerial virulence factors with numerous key components of the host cell contributed
to the rise of a new discipline, called „cellular microbiology“.

Literature:
1. Chakraborty, T. 1999. Molecular and cell biological aspects of infection by Listeria
    monocytogenes. Immunobiol. 201:155-163.
2. Cossart, P., and H. Bierne. 2001. The use of host cell machinery in the pathogenesis of
    Listeria monocytogenes.Curr. Opin. Immunol. 13:96-103.
3. Domann, E., J. Wehland, M. Rohde, S. Pistor, M. Hartl, W. Goebel, M. Leimeister-
    Wächter, M. Wuenscher, and Chakraborty T. 1992. A novel bacterial virulence gene in
    Listeria monocytogenes required for host cell microfilament interaction with homology to
    the proline-rich region of vinculin. EMBO J. 11:1981-1990.
4. Reinhard, M. T. Jarchau, and U. Walter. 2001. Actin-based motility: stop and go with
    Ena/VASP proteins. Trends Biochem. Sci. 26:243-249.




                                              43
bacteriophages, bacteriocins




             44
COMPARATIVE GENOMICS OF LISTERIA BACTERIOPHAGES

Richard Calendar1, ROSS B. Inman2, Elke Sattelberger3, Peter Lauer1, Daniel Portnoy1, Nora
Chow1, Markus Zimmer3, and Martin J. Loessner3

1
 Department of Molecular & Cell Biology, University of California, Berkeley, 401 Barker Hall,
Berkeley, CA 94720-3202, USA; 2Institute for Molecular Virology, University of Wisconsin at
Madison, 1525 Linden Drive, Madison, Wl 53706, USA; 3Institute for Microbiology, FML
Weihenstephan, Technical University of Munich, Weihenstephaner Berg 3, 85350 Freising,
Germany.

We have sequenced the genomes of different temperate bacteriophages infecting Listeria
monocytogenes, and compared their properties by bioinformatic analyses and physico-
molecular characterization. Phage A118 grows on serotype 1/2 strains and causes general
transduction.
Its genome is a collection of linear molecules that are circularly permuted and terminally
redundant. Its integration protein is of the invertase/resolvase class. Its attachment site is in
a gene that is homologous to comK, which is B. subtilis is the activator for genes involved in
competence in DNA transformation.
We have constructed an integration vector consisting of a plasmid that replicates in E. coli
and can be mated into L monocytogenes, using the RP4 transfer system. This plasmid does
not replicate in L. monocytogenes, but integrates into comK. The non-transducing phage
PSA is released by Scott A, a serotype 4b strain.
Its genome is unique and has 3' hydroxyl terminated cohesive ends. The integrase is also of
the invertase/resolvase class, and the attB attachment site is located within a Listeria
arginine tRNA gene. It is evident that Listeria phages carry many genes with high similarity to
genes from phages infecting various low G+C host bacteria, such as lactic acid bacteria,
Bacillus species, and gram-positive cocci.

This implies a relatively recent exchange of genetic information between these viruses.




                                               45
MEMBRANE DISRUPTION AND BACTERIAL CELL LYSIS BY
LISTERIA BACTERIOPHAGES: THE FUNCTION OF LYSINS AND
HOLINS
Martin J. Loessner. Natasa Vukov, Susanne Gaeng, and Siegfried Scherer

Institute for Microbiology, FML Weihenstephan,            Technical   University   of   Munich,
Weihenstephaner Berg 3, 85350 Freising, Germany.

Endolysins are cell wall lytic enzymes enabling the release of progeny virions. Listens phage
enzymes represent L-alanine-D-glutamate peptidases or N-acetylmuramoyl-L-alanine
amidases. They are composed of distinct functional domains: the N-terminus harbours the
enzymatic activity, and the C-terminal cell wall binding domains (CBD) mediate tight and
specific, but non-covalent, binding of the enzymes to their cell wall-associated ligands.
Genetic fusions between several CBDs and GFP demonstrated the highly specific
recognition and binding properties. Binding kinetics were determined by surface plasmon
resonance analysis, which revealed an affinity constant (K) in the range of matured
antibodies. CBDs also enabled the development of affinity assays, based on binding of
bacterial target cells to surface-immobilized CBDs. Recombinant endolysins enzymes
enabled rapid and highly specific lysis of Listeria cells. Expression of ply and quantitative
secretion of functional enzymes in the dairy starter organism Lactococcus lactis was
achieved by genetic fusion with a signal peptide sequence of a Lactobacillus S-layer protein;
processing of the propeptide did not influence enzyme activity.
The holin proteins associated with Listeria phage endolysins are small hydrophobic proteins
which cause non-specific membrane lesions and enable the Ply enzymes to reach their
peptidoglycan substrate. They show three potential transmembrane domains and a dual start
motif similar to λ S. This feature was evaluated using a newly developed           S-thf system,
which allows testing of the ability to support λ gpR-mediated lysis in E. coll.
In contrast to λ S, the longer L. monocytogenes phage Hol500-96 protein revealed a better
lytic activity than the shorter Hol500-93 product. We also studied several single amino acid
mutants that were shown to affect lysis timing.




                                              46
PRACTICAL APPLICATIONS OF LISTERIA PHAGE
Catherine E.D Rees

University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough,
Leicestershire, UK. LE12 5RD

The aim of this talk will be to review the range of applications which have developed from
studies of Listeria phage. The subjects to be covered will range from developments in phage
typing, the construction and application of lux phage and the detection of Listeria using the
"Phage Amplification" technique. The use of molecular biology has lead to the
characterisation of various phage components which have also been applied to the detection
of Listeria in rapid ATP tests, the eradication of Listeria in cell invasion assays and now
provides the potential for development of integration vectors for stable gene cloning. Looking
to the future we foresee further developments, perhaps in phage therapy or the development
of antisense phage to prevent virulence gene expression in environmental populations of
Listeria.




                                             47
USE OF GENERALISED TRANSDUCTION TO STUDY
VIRULENCE MECHANISMS IN LISTERIA MONOCYTOGENES
David A. Hodgson, Hsiang-Ning Tsai and Jonathan L. Telfer

Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL UK

We recently reported a large number of bacteriophage that can promote genetic exchange
between Listeria monocytogenes strains (Hodgson, D. A. (2000); Generalized transduction
of serotype 1/2 and serotype 4b strains of Listeria monocytogenes; Molecular Microbiology
35:312-323). We have used these generalised transducing bacteriophages for the analysis
of mutants of L. monocytogenes 1/2a strains 10403, EGD and L028. One of the simplest
uses is to demonstrate whether or not a transposon insertion is the cause of a specific
phenotype. This is a particular problem when using transposons to clone specific genes.
Transposons Tn916 and Tn1545 can cause multiple insertion and so it is essential to identify
which transposon insertion is causing the change in phenotype Generalised transduction
allows cloning individual transposon insertions. Another problem is that point mutations may
arise in transposon insertion strains that are responsible for a change in phenotype. We will
describe a strategy that allows transposon insertion mutations to be unequivocally selected.
Using generalised transduction it is possible to transposon-tag genes containing point
mutations and by cloning the transposon insertion site chromosome walk to the mutated
gene. The principle of transposon-tagging will be explained and a number of examples
given.




                                             48
DETERMINATION AND IDENTIFICATION OF LISTERIA
MONOCYTOGENES BY BACTERIOPHAGES IN ICECREAM
Ebtehaj Pishva

Dept. Bacteriol., Isfahan Medical Sciences            University,   Isfahan,   IRAN,    E-mail:
pishva@yahoo.com Tel: +98311687683

Problem: According to the instruction of the Institute of Standards and Industrial Research of
Iran (ISIR) packed food must be without Staphylococcus aureus, Salmonella and Shigella
spp, only small amounts of gramnegative bacteria like E. coli in food is allowed. Listeria
monocytogenes is a grampositive bacillus. This organism is facultative intracellular; it can
live in cells in pasteurized milk. It causes disease in immunosuppressed patients especially
in the elderly, during pregnancy, in cancer patients and in individuals receiving steroid drugs.
Identification L. monocytogenes is difficult for some reasons, changing of bacterial
morphology and colony during the culture, isolation methods and biochemical reactions
tests, furthermore the serologic tests is not valid.

Method: We used specific bacteriophages to identify Listeria monocytogenes and to
differentiate them from other diphtheroids that usually exist in diary products. This method is
suitable to diagnose L. monocytogenes in clinical samples and food items. 30 ice cream
samples taken from the Institute of Standard and Industrial Research of Iran (ISIR) were
studied. According to those instructions bacterial culture was used to identify the
contaminating bacterial agent. In initial surveys gramnegative organisms like Salmonella
were suspected to occur. Then the suspected organisms isolated from ice cream samples
and under special conditions (pH 6,~37°C) were exposed to Listeria bacteriophages. They
produced lytic or lysogenic forms, respectively when tested under different conditions .

Results : From all isolated diphtheroids of surveyed ice cream about 1/3 of them were
changed to lytic form in acidic pH and ~ 10°C. The organisms in higher temperature
produced lysogenic form and changed morphology and colonies. During the culture if the
colonies changes from smooth to rough, it won’t be influenced by bacteriophages any more.

Discussion : The morphology changes and variation of biochemical tests for identifying of L.
monocytogenes makes it difficult to diagnose .Thus using phagetyping in suitable conditions
is the most explicit way to identify this organisms in clinical and food samples.




                                              49
INTERACTION OF BACTERIOPHAGES WITH THE BACTERIAL
CELL: THE ROLE OF SEROTYPE-SPECIFIC SURFACE
ANTIGENS.

Nattawan Promadej1, Zheng Lan1, Franz Fiedler2, Xiang-He Lei1, Sophia Kathariou1,3

1
 University of Hawaii, Department of Microbiology, 2538 The Mali, Honolulu, Hawaii, USA;
2
 Institute for Genetics and Microbiology, University of Munich, Maria-Ward Str. la, 80638
Munich, Germany 3North Carolina State University, Department of Food Science, 339
Schaub hall, Raleigh NC 27695, USA.

Problem: In Listeria monocytogenes, teichoic acid (TA) composition varies structurally and
antigenically among different serotypes. In strains of serotype 4b, N-acetylglucosamine is as
integral component in the TA chains and bears galactose and glucose substituents.

Results: We identified two serotype-specific loci essential for glycosylation of serotype 4b
TA. Mutations in one locus (gtcA) resulted in absence of galactose and marked reduction in
glucose, whereas gltA or gltB mutants lacked glucose but had normal levels of galactose.
Galactose, but not glucose, was essential for adsorption of 2671 and other serotype 4b-
specific phage, and gtcA mutants were phage-resistant. In contrast, mutants in either gltA or
gltB remained phage-sensitive. Characterization of the gtcA genomic region in a strain of
serotype 1/2a revealed the presence of a divergent gtcA gene preceded by a novel gene
(mtrA) which is absent from serotype 4b but present in all screened serogroup 1/2 strains.
Insertional inactivation of the novel gene mtrA resulted in absence of N-acetylglucosamine
from the teichoic acid of serotype 1/2a bacteria, and resistance to the serotype-specific
phage A118.

Conclusion: It appears that in the course of its evolution L. monocytogenes has acquired
serotype-specific genes which are involved in expression of TA substituents that may also be
essential for adsorption of phage




                                             50
SYNERGISTIC EFFECT OF NISIN AND BACTERIOPHAGE ON
LISTERIA MONOCYTOGENES IN BROTH CULTURES
Sandra M. Moorhead, Gary A. Dykes

Food Safety Group, AgResearch, Private Bag 1323, Hamilton, New Zealand

Problem: The ability of Listeria monocytogenes to survive cold storage is of concern to the
food industry in particular with ready to eat meats and vegetables. As a means of control or
elimination of L. monocytogenes in these food products, the use of antibacterial agents that
can be used as a natural preservative is finding widespread appeal. The antilisterial effect of
nisin has been documented and is used as a natural preservative in various food processes,
albeit with limited success when used alone. Combinations of nisin and other antibacterial
agents have succeeded in significant reductions of L. monocytogenes over the use of nisin
alone, reiterating hurdle technology as an effective means of control or elimination of
pathogenic bacteria.

Method: This study investigated possible synergistic activity in the combination of nisin and
Listeria specific bacteriophage on the reduction of lag, log and stationary phase cultures of
both a clinically isolated and a meat isolated strain of L. monocytogenes.

Results: A synergistic effect giving enhanced reduction of L. monocytogenes during chill
storage in broth solution has been observed. Further experimental work is to be undertaken
on meat substrates during chill storage and with various concentrations of both agents.

Conclusion: The use of natural antibacterial agents such as nisin in conjunction with
aggressive specific bacteriophage presents an exciting possibility for the control or possible
elimination of L. monocytogenes on ready to eat products.




                                              51
CHARACTERIZATION OF A NEW PEDIOCIN-LIKE SEC-
DEPENDANT BACTERIOCIN PRODUCED BY LISTERIA
INNOCUA 743.

 M.L. Kalmokoff1, T. Cyr2, and M.A. Hefford2.

1
 Microbiology Research Division, Bureau of Microbial Hazards, 2Research Services
Division, Bureau of Biologics and Radiopharmaceuticals, Health Canada, Banting
Research Centre, Tunney’s pasture, P.L.# 2204A2, Ottawa, Canada, K1A-0L2.

Problem: Listeria innocua 743 was previously shown to produce a bacteriocin-like
inhibitory activity. Here, we report on the identification and genetic characterization of a
new bacteriocin produced by this isolate.

Method: Transposon insertion mutagenesis was used to produce mutants of L. innocua
743 which demonstrated either an impairment or loss in the ability to produce a bacteriocin-
like inhibitory activity. The resulting mutants were characterized through cloning,
sequencing and expression of the interrupted sequences.

Results: Gel-electrophoretic analysis of spent cultures indicated that this strain produced
two distinct inhibitory activities. Insertion of Tn917 into a low copy 2.8 Kb cryptic plasmid
(pHC743) generated mutants with either an impaired ability, or the loss in ability to produce
one of the inhibitors. Sequence analysis revealed the presence of two continuous open
reading frames , the first encoding a new pediocin-like bacteriocin (lisA), and the second
encoding a predicted protein having homology with genes involved in immunity towards
other bacteriocins (lisB). Translation of the bacteriocin gene initiated from a non-canonical
start codon, and encoded a 71 amino acid prebacteriocin lacking the double glycine leader
peptidase processing site common to other type IIa bacteriocins. Alignment of the predicted
sequence with the processed N-termini of related bacteriocins suggests that the mature
bacteriocin consists of 44 amino acids, with a predicted m.w. of 4555 Da. Mutants with
insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single
operon, and that lisB represents the immunity protein. Cloning of an amplicon containing
the lisAB operon into Escherichia coli, resulted in production and export of the bacteriocin,
confirming that the phenotype is dependant on only these two genes, and that export of this
bacteriocin is sec-dependant.

Conclusions: This represents the first confirmation of bacteriocin production in a Listeria
spp. The new bacteriocin (listeriocin 743A) is related to other pediocin-like class II
bacteriocins, which are well known as effective inhibitors of L. monocytogenes.




                                                52
DETECTION OF LISTERIA MONOCYTOGENES IN PRESENCE OF
LISTERIA INNOCUA

Marie Cornu 1,Martin Kalmokoff 2.

1
 AFSSA LERAC, 22 rue Pierre Curie - BP332 - 94709 Maisons Alfort Cedex, France.
2
 Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health Canada,
Sir Frederick G. Banting Research centre, Tunney's Pasture, P.L. 2204A2, Ottawa, ON,
Canada K1A OL2.

Problem: In the mid-1990s, three reference publications demonstrated that L. innocua can
mask L. monocytogenes after the enrichment procedure and explained this by the higher
growth rate of L. innocua in selective media. This overgrowth of L. innocua engenders a high
risk of false-negative results, as there is little chance to detect L. monocytogenes when the
product also contains L. innocua.

Methods: The protocol was based on the recent ISO 11290-1 method: a primary enrichment
in Half Fraser Broth at 30°C during 24h and a secondary enrichment in Fraser Broth at 35°C.
Characterisation of turbidimetric kinetics: Thirteen Listeria strains were cultured in 5 media to
compare within- and between-species variability. Mixed cultures of L. monocytogenes and
L. innocua: Growth of each species in mixed cultures was monitored along the enrichment
procedure. Risk assessment: A Monte Carlo procedure was applied to assess the risk of
false negatives.

Experimental results: Selective advantage: No significant differences were found in growth
characteristics between either species in the various enrichment media. Nevertheless, we
proved that a rapid L. innocua isolate may overgrow a slow L. monocytogenes isolate, with a
final proportion over 99%. Bacteriocin-like inhibition: Kinetic analysis of mixed cultures
demonstrated that some L. innocua isolates may produce bacteriocin-like compounds active
against L. monocytogenes and then suppress it.

Simulation results: The Monte Carlo procedure was based on hypotheses derived from this
experimental work and from published results. In absence of inhibition, the probability to
detect L. monocytogenes when the product contains both species was estimated to 74%.
These 26% of false-negative results are only due to the selective advantage of certain L.
innocua strains, with a final L. innocua proportion a the end of the primary (resp. secondary)
enrichment over 95% in 22% (resp. 29%) of the cases. In the hypothesis that 6% of L.
innocua strains supress L. monocytogenes, this sensitivity was estimated to 70%.

Conclusion: With plating media on which presumptive isolates have to be characterized, the
overgrowth of L. innocua strongly impacts the recovery rate of L. monocytogenes. However,
even on newer plating media in which it is possible to distinguish L. monocytogenes colonies
from L. innocua colonies, false negatives remain a possibility for enrichments where the final
L. innocua level exceeds 99%. Moreover, the impact of inhibitory effects may not be reduced
with these new media.
Despite current limitations (necessary improvement of the growth model with better
description of the lag phases and decelerations, lack of knowledge about the growth

                                               53
parameters and the proportion of inhibitory L. innocua strains), this modelling approach may
be useful to predict real situations. This application of predictive microbiology and
quantitative risk assessment has, to our knowledge, never been illustrated before.




                                            54
INHIBITION OF LISTERIA MONOCYTOGENES IN CHICKEN
COLD-CUTS BY ADDITION OF SAKACIN P AND SAKACIN P
PRODUCING LACTOBACILLUS SAKEI
Katia, T., Rosshaug, I., Axelsson, L. and K. Naterstad


MATFORSK, Norwegian Food Research Institute, Osloveien 1, 1430 As, NORWAY

Listeria (L.) monocytogenes is the causative agent of food related listeriosis and is potentially
pathogenic to immunocompromised patients. L. monocytogenes has the ability to grow in
vacuum packaged food at chilling temperatures, and is also relatively tolerant to salt and low
pH, thus it is difficult to control its growth in food, Listeria (L.) monocytogenes, the bacteriocin
sakacin P or a strain of the lactic acid bacteria Lactobacillus (Lb.) sakei were added to
vacuum packed chicken cold cuts, and incubated at 4 and 10°C for four weeks. Addition of
sakacin P had an inhibiting effect on growth of L. monocytogenes. A high dosage had a
pronounced effect, while a Sow dosage gave an initial inhibition of growth; Addition of a
sakacin P producing and a non-producing Lb. sakei both had an inhibiting effect on growth of
L. monocytogenes. The effects were similar at both temperatures used. When a less sakacin
P sensitive L. monocytogenes strain was used, the high dosage of sakacin P used had an
inhibiting effect.




                                                55
virulence, pathogenicity, fitness




                56
PATHOGENICITY OF LISTERIA MONOCYTOGENES
W. Goebel

Biozentrum der Universität Würzburg (Mikrobiologie), Würzburg, Germany

Listeria monocytogenes and Listeria ivanovii represent the two pathogenic members in the
genus Listeria. These gram-positive, facultative intracellular bacteria have developed
strategies in evolution to invade normally non-phagocytic mammalian cells, to survive and
efficiently multiply in the cytosol of these host cells as well as in phagocytes
(monocytes/macrophages and dendritic cells) and to spread between homologous and
heterologous neighboring cells. Many genes involved in these and other processes related to
the pathogenesis of a listeriosis have been identified and studied in great detail, mainly in L,
monocytogenes. The regulated expression of most of these virulence genes which occurs in
a complex differential manner outside and inside host cells, involves the central
transcriptional activator PrfA. The ordered regulation of the virulence genes is crucial for a
successful infection by L, monocytogenes. In this contribution I shall concentrate on some
recent aspects concerning these basic virulence properties of Listeria monocytogenes.




                                              57
THE HLYA GENE EXPRESSION PATTERN IN RESPONSE TO
ENVIRONMENTAL CONDITIONS IS NOT CORRELATED WITH
THE LISTERIA MONOCYTOGENES GENOTYPE
Knut Rudi*, Hege Karin Nogva, Kristine Naterstad, Signe Marie Dromtorp, Sylvia Bredholt,
and Askild Holck

MATFORSK, Norwegian Food Research Institute

Listeria monocytogenes is a Gram-positive, facultative intracllular bacterium that causes
invasive, often fatal, disease in susceptible hosts. Over the past decade, the organism has
caused several epidemics. The virulence of L monoctogenes is relatively ill defined, although
several virulence genes have been described in detail. The gene hlyA, encoding
listeriolysion 0, is the best characterized virulence gene. Listeriolysin 0 was initially identified
because of the hemolytic activity of this gene product. The expression of this gene is
environmentally regulated, and is important for the pathogenicity of L. monocytogenes. We
have addressed the questions of the effect on gene expression of growth medium and
temperature for six different strains, including the two type strains NTNC 7973 and DSMZ
20600. The gene expression level was determined by quantitative reverse transcription PCR
through the application of 5'-nuclease PCR, using 23S rRNA as an internal reference- The
expression patterns were subsequently correlated with the genotypes, as determined by
multilocus sequencing and amplified fragment length polymorphism (AFLP). Our results
show quantitatively that the expression pattern of hiyA is diverse, ranging from a strain
DSMZ 20600 in which hlyA apparently is highly expressed at 0°C and slightly repressed at
42°C, to the strains SIK and 2A/24 m which hlyA is repressed at 0°C, and expressed at
42°C, Furthermore, we did not find correlation between the expression patterns and the
genotype clustering. This knowledge has implications for identifying strains with virulence
properties.




                                                58
THE USE OF LISTERIOLYSIN TO IDENTIFY AND
CHARACTERISE IN VIVO INDUCED GENES IN LISTERIA
MONOCYTOGENES
Cormac G.M. Gahan, Colin Hill*

Department of Microbiology and National Food Biotechnology Centre University College
Cork, Ireland.

Problem: Prior to escape from the macrophage phagosome, Listeria monocytogenes
encounters low pH conditions which are capable of inducing the synthesis of a specific
subset of proteins. In order to identify the genes encoding these proteins, we have
developed a system which is analogous to in vivo expression technology (IVET), but which
utilises the haemolysin gene of L. monocytogenes, hly as both a reporter of gene expression
and as a means of selection of promoter elements expressed in vivo.

Method: A bank was created by fusing a promoterless copy of hly to random promoter
elements in a listeriolysin negative IVET host. Sequential inoculations of mice with this bank
resulted in the isolation of clones with increased survival potential in the mouse model
relative to a negative control, but which remained haemolysin negative on blood agar plates.
The system was successfully tested using the promoter of the inducible virulence gene plcA
and by creating a fusion strain expressing hly from the promoter region of groESL.

Results: A number of in vivo induced loci were identified including genes encoding a DNA
topoisomerase III, a cellobiose transporter and a fumarase. Two isolates represented fusions
to proteins of unknown function and three isolates contained no significant homologues in
the database. A mutant in the fumarase gene demonstrated reduced virulence for mice and
an inability to grow in cultured mouse macrophages.

Conclusion: We have adopted a novel approach to IVET for use in L. monocytogenes
which can identify genes expressed at a specific stage of pathogenesis. Furthermore,
placing synthesis of haemolysin under the influence of known promoters, such as groESL
and plcA, can be used as an effective in vivo reporter of gene expression. We envisage that
this reporter system will be of use in the functional evaluation of genetic loci in L.
monocytogenes.




                                             59
DETECTION AND CHARACTERIZATION OF VIRULENCE
ASSOCIATED GENES IN LISTERIA MONOCYTOGENES STRAINS
FROM INVASIVE AND NON-INVASIVE LISTERIOSIS.
Paolo Aureli, Giovanna Franciosa, Christina Wedell-Neergaard, Antonella Maugliani.

Istituto Superiore della Sanitä, Viale Regina Elena 299, 00161, Rome, Italy.

Problem: The mechanism of pathogenesis of L. monocytogenes involves multiple virulence
factors that affect host cell invasion, intracellular multiplication and cell-to-cell spread. Recent
data have shown that L. monocytogenes can cause either invasive or non-invasive
listeriosis, potentially suggesting for the existence of different pathogenic pathways. Here,
we tested L. monocytogenes human isolates from invasive and non-invasive listeriosis and
rare non-pathogenic (non-human) L. monocytogenes strains for the presence of virulence
associated genes; the genetic polymorphisms of these genes were also assessed. The aim
of this study was to provide an insight into the genetic determinants of virulence of different
pathogenic L. monocytogenes strains. The non-invasive listeriosis isolates were from four
distinct outbreaks occurred in Italy (2), Denmark and the United States, respectively.
Specifically, the virulence genes considered in this study were: inlA, iap, hly, actA, mpl, prfA,
plcA, pIcB and flaA.

Methods: All were amplified by PCR, by using specific sets of primers. The amplified genes
were then subjected to the restriction fragment length polymorphisms (RFLP) analysis, each
with 3 or 4 different endonucleases.

Results: All the virulence genes were amplified from the human isolates, while some of
them were persistently not detected by PCR in the non pathogenic strains.
The non pathogenic strains showed most of the genetic polymorphisms in the positive
amplification products.
Different restriction profiles were also identified in a few virulence genes from the human
isolates: in general, the polymorphic virulence genes of some non-invasive Listeria strains
showed either unique patterns or profiles comparable to those produced by some invasive or
some non pathogenic strains, respectively.

Conclusion:The distribution of the virulence genes subtypes among the different pathogenic
L. monocytogenes strains might contribute to a deeper understanding of the pathogenesis of
this microorganism




                                                60
ACID TOLERANCE IN LISTERIA MONOCYTOGENES ENHANCES
THE SURVIVAL IN MACROPHAGES
M.P. Contea , G. Petronea, C. Longhia, M. Pentaa, A. M. Di Biasea, F. Supertib, L. Segantia

a
  Microbiology Institute, University “La Sapienza”;b Ultrastructure Laboratory, Istituto
Superiore di Sanità; Rome; Italy

Problem: Listeria monocytogenes is a widespread gram-positive facultative intracellular
food-borne bacterium associated in humans with listeriosis (Mc Lauchlin, 96). After
entering the host through the intestinal tract, bacteria spread to various target tissues,
including liver, brain and placenta by the lymph and the blood strain.
This bacterium is able to survive and multiply within macrophages and also actively
internalised and incorporated into phagolisosomes of different nonphagocytic cell types,
(Gaillard et al, 87) (Dramsi et al., 96). Adherent infected mononuclear phagocytes can in
turn infect neurons, indicating that macrophages play a relevant role in the invasion of
CNS by cell to cell spread (Dramsi et al., 98; Drevets 99). The mode of entry and
intracellular survival of L.monocytogenes has been well studied in different cell types,
including the J774 murine macrophage-like cell line (de Chastellier & Berche, 94) and the
THP-1 human myelomonocytic cell line (Scorneaux et al., 96). Each step of the invasive
process is genetically controlled and regulated by the pleiotropic activator prfA (Kuhn and
Goebel 95).
The capability to adapt to low pH in external environments and infected host systems, in
which bacteria must overcome the acidic habitat of the stomach and cell phagosomes
represents a pivotal step in the infection strategy. It has been shown that acid adaptation
at mild acidic conditions can enhance the survival of bacterial cells when exposed to
acidic lethal conditions (Kroll & Patchett, 92) and is correlated to the synthesis of acid-
induced proteins (Sokolovic et al., 93). Acid adaptation of bacteria also induces increased
virulence in murine models (O’Driscoll et. al., 96; Saklani-Jusforgues et al., 00).
However, even than there are increasing evidences that response to acid stress can
regulate the virulence of L.monocytogenes, the molecular pathways involved in this event
are not yet well known. In a previous report we demonstrated that the entry efficiency of L.
monocytogenes in enterocyte-like cells was significantly enhanced in acid adapted
bacteria (Conte et al., 00). In the present research, to add further information on the
mechanism involved in the increased pathogenicity of acid tolerant bacteria we
investigated the fate of L. monocytogenes cells, pre-exposed to mild acidic conditions, in
murine (J774 cells) and human (THP-1 cells) macrophage-like cell lines.

Bacterial strains and culture conditions. The bacterial strains used in this study were L.
monocytogenes LM1, LM2, isolated in our laboratory from liquor of patients affected by
listeriosis and L. monocytogenes LMm4, a constitutively acid resistant LM1-derived
mutant, isolated following prolonged exposure to low pH (Conte et al., 00). The strains
were grown in Brain Heart Infusion Broth (BHI, Oxoid) and subcultured onto Tryptone Soy
Agar (TSA, Oxoid). Adaptation of L. monocytogenes strains to acid and measurement of
acid tolerance response were performed as previously described (Conte et al., 00). In
some experiments, prior the infection of macrophages, bacterial cells were incubated with
5% fresh pool of human serum as source of opsonin for 30 min at 37°C. Then bacteria
were pelleted by centrifugation at 3.000 rpm for 20 min and resuspended in RPMI.

                                              61
Cells. J774.A1 macrophage-like cells were cultured and activated with 0.1 g of LPS
(Sigma; E.coli O55:B5) per ml as previously described (Conte et al., 00). THP-1 cells, a
myelomonocytic cell line, were mantained in RPMI 1640 medium supplemented with 10%
foetal calf serum and 2 mM glutamine in an atmosphere of 95% air-5% CO2. Cells which
grow spontaneously in loose suspension under these conditions were subcultured every
third day. THP-1 cells (106 cell/ml) were differentiated by incubation with phorbol miristate
acetate (0.16 M) for 24 h at 37 °C in 24 well plates following the procedure described by
Scorneaux et al, (96). Adherent cells maintained in phorbol miristate acetate were activated
by the exposure to IFN- (100 U/ml; specific activity 2 x 107 U/mg of protein) for 24 h at 37
°C.

Invasion assays. The infection of murine macrophages was performed in LPS-activated
J774.A1 cells (Conte et al. 00) and in IFN--activated adherent THP-1 cells, with bacterial
suspensions in cell culture medium of non adapted and acid adapted strains, for 15 min or 1
h at 37°C at a MOI of approximately 100 bacteria per cell for J774 and 10 bacteria per cell
for THP-1 cells. Then the macrophages were five-fold washed and incubated with
Dulbecco’s modified Eagle’s MEM containing 10 g/ml of gentamicin. At various time
intervals after addition of bacterial inoculum (15 min, 4 and 8 h for J774 cells and 1, 3, 5 h
for THP-1 cells) the macrophages were lysed and plated to determine viable intracellular
bacteria by cfu counts.
Transmission electron microscopy. Murine and human macrophages infected with 100
bacteria per cell of L. monocytogenes acid adapted and non adapted strains. Then they were
fixed at 4, 5 and 8 h post addition of gentamicin containing medium and treated for electron
microscopy observations, as previously described (Conte et al., 00).

Transcriptional analysis of virulence genes in L. monocytogenes acid adapted and
non acid adapted strains. Total cellular RNA was extracted (Conte et al., 96) from acid
adapted and non acid adapted cells of L. monocytogenes LM2 and transcriptional analysis of
prfA, inlA, hly, plcB, actA and cplC genes was performed as previously described (Conte et
al., 96) and monitored using intragenic probes made by PCR.

Chemical determination of intracellular superoxidedismutase (SOD) and catalase. SOD
activity was measured by the pyrogallol method (1 unit of activity corresponded to the
amount of the enzyme necessary to achieve 50% inhibition of pyrogallol autoxidation).
Catalase activity was determined by measuring the amount of substrate (moles)
transformed min-1 mg protein-1.

Results and discussion

Survival behaviour of Listeria towards acidic pH
The growth of L.monocytogenes strains was hindered by the acidity of the medium.
Exponentially grown bacteria were rapidly killed when the pH was reduced to 3.5. If before
submitting bacterial cells to acid stress, they were treated at an intermediary non-lethal pH
(pH 5.1) for 1 h at 37°C, their capacity of resistance to a subsequent lethal acid stress
increased. After 120 min at pH 3.5, the following rates of survival were observed for acid
adapted cultures: 54 %, 70% and 60% of survival for L. monocytogenes LM1, LM1m4 and
LM2, respectively.

Survival  and   intracellular growth  of   acid-adapted               and     nonadapted
L.monocytogenes in murine and human macrophages
                                             62
The ability of L. monocytogenes strains to entry and proliferate in phagocytic cells was
tested in the J774.A1 and THP-1 macrophage-like cell lines. Fig.1 shows entry and
growth of acid-adapted and nonadapted L. monocytogenes wild type LM1 and L.
monocytogenes mutant LM1m4 in LPS-activated phagocytic cells. The percentage of
bacterial inoculum that invaded macrophages 15 min post infection was similar
independently of the acid tolerance property of bacteria. Differently after 4 h or 8 h
incubation in gentamicin containing medium, the number of intracytoplasmic bacterial cells
of both strains pre-exposed at pH 5.1 and of the nonadapted acid tolerant mutant resulted
about 4 or 5-fold higher than that observed in the nonadapted LM1 wild type strain. The
highest values of viable intracellular bacteria were reached after 8 h incubation, thus
indicating a noticeable increased survival and growth of these bacterial cells in J774.A1
macrophages.
In table 2 are reported entry and growth of L. monocytogenes LM2 in THP-1 cells. Acid
adaptation of bacterial cells did not significantly influence the entry in macrophages, with
the exception of opsonised bacteria that are phagocytosed at a lesser extent. However, in
all experimental conditions tested, the rate of multiplication of acid adapted bacteria
resulted higher than that observed for untreated bacteria. When human macrophages
were activated with IFN non acid adapted bacteria were unable to grow, whereas when
they were exposed to mild acidic conditions prior the infection, they were able to multiply,
being the percentage of viable intracellular bacteria 5 h post infection about 5-fold higher
as compared with that of non adapted bacteria.

Transmission electron microscopy of infected macrophages
After incubation of J774.1 and THP-1 infected cells in gentamicin containing medium,
some differences were observed between acid adapted and non adapted listerial cells.
Only the latter were observed partially digested into the phagosomes. After further 4 h or 5
h incubation the differences between acid-adapted and nonadapted strain were more
evident. In J774.1 cells, infected with non adapted LM1 strain, numerous bacteria
appeared to be enclosed into the phagosomes, a number of them showing extensive
structural damage. Macrophages infected with acid-adapted L. monocytogenes contained
a greater number of bacteria, often intact and almost all free in the cytoplasm. Acid-
adapted LM1 cells frequently appeared to be surrounded by a network of actin filaments
and bacteria with actin tails were frequently observed. Similar results were observed in
THP-1 cells 5 h after infection with acid adapted and non adapted bacteria.

Transcriptional analysis of L.monocytogenes virulence genes
To investigate whether acid adaptation affected L. monocytogenes, by regulating at
transcriptional level the expression of genes correlated to the invasive pathway and of the
clpC gene, codifying for a general stress protein, CLPC-ATPase, slot-blot analyses were
performed with total cellular RNA extracted from acid adapted and non adapted LM2 L.
monocytogenes. Hybridisation of RNA extracted from iron deficient bacteria with the prfA,
inlA, hly, plcB, actA and cplC genes resulted similar in bacterial cells pre-exposed to
sublethal pH and in control untreated cells. as detected by a Betascope model 603 blot
analyzer (Betagen).

Intracellular levels of L.monocytogenes catalase and superoxidedismutase
The SOD activity in acid adapted and non adapted bacterial lisates was 14 U and 10.4 U,
respectively, whereas catalase activity was similar (38.5 M/mg versus 39.1 M /mg) in
the two different experimental conditions.

                                              63
        Our earlier studies demonstrated that the expression of virulence determinants of
L. monocytogenes can be influenced by low temperature and iron deficiency (Conte et al.,
94, 96, 00). Moreover, acid-tolerant strains of L. monocytogenes showed increased entry
efficiency in enterocyte-like cells (Conte et al., 00). In this report we have evaluated the
influence of acid tolerance on the invasive behaviour of L.monocytogenes in
macrophages.
        In our experiments the entry efficiency of nonadapted and acid-adapted L.
monocytogenes strains in activated phagocytic cells (15 min post infection for murine
macrophages or 1 h for human macrophages) did not exhibit any significant difference,
with the exception of opsonised acid adapted bacteria which were phagocytosed by THP-
1 cells at a minor extent.. The decreased ability of acid-adapted bacteria to entry human
macrophages cells could be attributed to some interaction between of surface proteins
induced in acid tolerant bacteria ( Sokolovic et al., 93) and human serum components,
affecting the recognition between bacteria and macrophages.
        Concerning the survival in macrophages, it has been reported that L
.monocytogenes can easily escape from phagosomes and multiply in phagocytic cells
whereas when these cells are LPS- or IFN--activated, listerial growth is greatly inhibited ,
and bacteria are rapidly killed (de Chastellier e Berche 94, Ouadrhiri et al., 00). We
demonstrated that acid-adapted L. monocytogenes wild type strains are efficiently
phagocytosed and able to survive and proliferate intracellularly in LPS-treated murine
J774.1 cells, at a significant increased extent by respect of the non adapted wild type
strain. Results obtained in IFN--activated adherent THP-1 cells were consistent with
those obtained in J774 cells, demonstrating an overall increased intracellular survival of L.
monocytogenes acid tolerant bacteria.        The different fate of L. monocytogenes after
phagocytosis of acid exposed bacterial cells was also confirmed at a morphological level
by electron microscopy, as electron micrographs evidenced a higher ability of acid-
adapted bacteria to escape from phagosomes.
        In our research the enhanced ability to survive intracellularly of acid tolerant cells
did not appear correlated to a modulation of the expression of the prfA-regulated virulence
genes and of the clpC gene, codifying for a general stress protein. This result is in
agreement with findings of Hanaka et al. (95), demonstrating that L. monocytogenes can
grow in macrophages without the aid of proteins induced by environmental stresses of
heat and hydrogen peroxide. Chemical determinations of intracellular cytoplasmic SOD
indicated in acid adapted bacterial lysates a slight increased synthesis of SOD whereas
the levels of catalase appeared unaltered.
        Taken together these results add new information about the influence of the acid
tolerance response on L. monocytogenes virulence. The fate of this food-borne pathogen
in macrophages, i.e. intracellular survival and growth, which allow the spread of bacteria
in the host, could be highly promoted, following the exposure to acid stress. However, the
molecular mechanisms, used by L.monocytogenes acid tolerant strains and involved in
the increased survival ability, remain to be elucidated.


Acknowledgements
This work was supported by MURST cofinanziamento 2000, ENEA and Faculty grants.

References

Brehm K, Kreft J, Ripio MT, Vazquez-Boland JA. Microbiologica SEM 1996; 12: 219-236.

                                               64
Conte MP, Longhi C, Polidoro M, Petrone G, Buonfiglio V, Di Santo S, Papi E, Seganti L,
Visca P, Valenti P. Infect Immun 1996; 64(9): 3925-3929.
1. Conte MP, Longhi C, Petrone G, Polidoro M, Valenti P, Seganti L. J Med Microbiol (in
   press).
2. Conte MP, Longhi C, Petrone G, Polidoro M, Valenti P, Seganti L. Res Microbiol 1994;
   145: 677-682.
3. Conte MP, Petrone G, Di Biase AM, Ammendolia MG, Superti F, Seganti L. Microb
   Pathog 2000;29(3):137-44
4. de Chastellier C, Berche P. Infect Immun 1994, 62(2):543-53
5. Dramsi S, Lebrun M, Cossart P. Curr. Top. Microbiol, Immunol 1996, 209, 61-68,.
6. Dramsi S., Levi S., Triller A., Cossart P. Infect Immun. 1998. 66(9):4461-44688.
7. Drevets D A . Infect. Immun. 1999, 67, 3512-3517.
8. Gaillard J.L., Berche P., Mounier J., Richard S., Sansonetti P. Infect Immun. 1987,
   55(11):2822-2829.
9. Hanawa T., Yamamoto T., Kamiya S. Infect Immun. 1995, 63(12):4595-4599.
10. Inoue S, Itagaki SI, Amano F. Infect Immun 1995; 63: 1876-1886.
11. Kroll RG, Patchett RA. Lett Appl Microbiol 1992; 14:224-227.
12. Kuhn M., Goebel W. 1995, Genet Eng (N Y). 1995;17:31-51.
13. Mc Lauchlin J. Food Control 1996; 7(4/5): 187-193.
14. O’Driscoll B, Cormac G, Gahan M, Hill C. Appl Env Microbiol 1996; 62(5):1693-1698.
15. Ouadrhiri eY, Scorneaux B., Sibille Y., Tulkens P.M. Ant Ag Chemother, 1999, 43,
    1242- 1262.
16. Saklani-Jusforgues H., Fontan E., Goossens P.L. FEMS Microbiology letters 2000,
    193, 155-159.
17. Scorneaux B, Ouadrhiri Y, Anzalone G, Tulkens PM Antimicrob Agents Chemother
    1996, 40(5):1225-30.
18. Sokolovic Z, Riedel J, Wuenscher M, Goebel W.. Mol Microbiol 1993, 8: 219-227.




                                            65
              100



              80
 % Inoculum




              60



              40



              20



               0
                    nonadapted   acid-adapted   nonadapted   acid-adapted
                    LM1          LM1            LMm4         LMm4

FIG.1 Growth of L. monocytogenes wild-type LM1 and
 L. monocytogenes mutant LMm4 in J774.A1 cells.
Intracellular growth values are expressed as the percentage
of the initial inoculum of bacteria that are gentamicin resistant.
Viable intracellular bacteria 15 min post infection □; viable
intracellular bacteria 4h post gentamicin addition ; viable
intracellular bacteria 8h post gentamicin addition . Errors
bars represent the SD for triplicate experiments.




                                                                            66
Table 1. Viable intracellular acid adapted (pH 5) and non acid adapted (pH 7) L.
monocytogenes LM2 in non activated or IFN- activated (100 U/ml) THP-1 macrophages, in
presence or in absence of human serum. Intracellular growth values are expressed as the
percentage of the initial inoculum of bacteria that are gentamicin resistant, 1 h and 5 h post
post infection. Data are presented as means  standard deviation of at least three
experiments.

                                 Time      after Non       acid Acid adapted
                                 infection       adapted        bacteria
Non     activated          THP-1                 bacteria
macrophages                                      % of inoculum

                                          1h             102.1             81.9

                                          5h             333.1            323.2

                                   Time after        Non acid      Acid adapted
                                   infection         adapted       bacteria
IFN    activated          THP-1                     bacteria
macrophages                                          % of inoculum

                                          1h             6.01.5           6.51.6

                                          5h             7.82.0           312.7


                                 Time      after Non       acid Acid adapted
IFN     activated         THP-1 infection       adapted        bacteria
macrophages                                      bacteria
and opsonised bacteria                           % of inoculum

                                          1h             282.5           18.61.9

                                          5h             714.0            813.5




                                               67
STRESS TOLERANCE AND VIRULENCE: TWO-COMPONENT
SIGNAL TRANSDUCTION SYSTEMS IN LISTERIA
MONOCYTOGENES
Birgitte H. Kallipolitis, Hanne Ingmer

Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, KVL,
Stigbojien 4, DK-1870 Frederiksberg C, Denmark. Email: ingmer@biobase.dk

Problem: Listeria monocytogenes is able to survive under adverse conditions such as high
and low temperature, starvation, low pH or high osmolarity, which can be present in both
food products and within host tissues. The molecular mechanisms by which L.
monocytogenes adapt and survive during periods of changing environmental conditions are
largely unknown. In order to understand more about how L. monocytogenes sense and
respond to changes in the environment, we have commenced a search for two-component
signal transduction systems. These highly conserved systems, most typically consisting of a
sensor histidine kinase (HK) and a response regulatory (RR) protein, enable bacteria to
adapt to stressing conditions by changing their pattern of gene expression.

Methods: In our search for two-component systems, we applied a method based on PCR
amplification using degenerate oligo-nucleotides with sequences derived from the conserved
regions in the RR family of proteins (Morel-Deville et al., 1997).

Results: By this approach, seven RR genes of L. monocytogenes L028 was isolated. To
assess the role of these transcription factors for stress tolerance and virulence, we disrupted
each of the RR genes by insertional mutagenesis. One of the RR genes appeared to be
essential to viability and could not be inactivated. All viable mutant strains with disrupted
response regulator genes were further characterized with regard to growth in vitro under a
variety of conditions (high osmolarity, heat, cold, H2O2, acid, ethanol). Furthermore, the role
of the transcriptional regulators in pathogenesis was assessed by testing the survival of the
RR mutants in a mouse infection model.

Conclusion: The results suggest that several of the two-component signal transduction
systems play important roles in stress tolerance and pathogenicity of L. monocytogenes.

References: Morel-Deville, F., Ehrlich, S. D. and Morel, P. (1997). Identification by PCR of
genes encoding multiple response regulators. Microbiology 143, 1513-1520.




                                              68
INVASIVENESS OF DIFFERENT PFGE-TYPES OF LISTERIA
MONOCYTOGENES IN CACO-2 CELLS
Charlotte Nexmann Larsen1. Birgit Norrung2, Helle Molgaard Sommer2 and Mogens
Jakobsen3

1
  Royal Veterinary and Agricultural University, Dept. Of Veterinary Microbiology, Stigb0Jlen
4, 1870 Frederiksberg C, Denmark.2 Danish Veterinary and Food Administration, Inst. Of
Food Safety and Toxicology, Morkhoj Bygade 19, 2860 S0borg, Denmark.3 Royal Veterinary
and Agricultural University, Dept. of Dairy and Food Science, Rolighedsvej 30, 1958
Frederiksberg C, Denmark

Problem: Several studies of Listeria monocytogenes have shown that strains belonging to
serogroup 1/2 are more often recovered from foods than strains of serogroup 4. In addition a
significant difference has been found in the distribution of pulsed field gel electrophoresis
types (PFGE) among food isolates compared to human isolates. The reason why certain
serotypes and PFGE types seems to be more commonly associated with human listeriosis
than others is unknown. Differences in virulence or ability to survive and grow in certain
processed food items or their production lines might be possible explanations for this
phenomenon.

Method: The virulence of 26 previously PFGE-typed isolates of L. monocytogenes was
examined testing the ability to invade intestinal epithelial Caco-2 cells as a model. The
design of the experiment was build up as a three-way variance analysis examining the effect
of the origin (human or food) and of the PFGE type. The analyses were performed in 6 runs
(days) giving the third effect, due to random variations between the runs.

Results: Strains belonging to seven different PFGE types and originating from both foods
and humans were examined. No significant difference in invasiveness could be seen
between strains derived from humans compared to strains derived from foods. Strains
belonging to PFGE-type 1 express significantly lower ability to invade the cells than strains
belonging to other PFGE-types. Although strains of PFGE type 2 also seem to invade at a
low level this was not significant. PFGE type 1 and 2 are more frequently found in foods
compared to four of the five other PFGE-types examined.

Conclusion: Certain PFGE-types commonly found in foods are less invasive in Caco-2 cells
than other PFGE types.




                                             69
INVESTIGATION OF LISTERIA MONOCYTOGENES
INTRACELLULAR GROWTH.
Telfer. J.L.,The European Listeria Genome Consortium, Hodgson, D.A.,

Department of Biological Sciences, University of Warwick, Coventry, UK. CV4 7AL.

Problem: Although the overall mechanism of pathogenesis of infections with Listeria
monocytogenes is well characterised, we still have a lot to learn about the mechanisms of
intracellular growth and survival. By studying L. monocytogenes strains that abort infection
following invasion in a tissue culture model we can elucidate other genes involved in the
maintenance of the bacterium in vivo.

Method: In this work a transposon insertion library was used to transduce mutant strains
and a tagged gene of interest was indicated by a change of phenotype. Transduction
mapping was then used to give the distance between the mutant gene and the transposon.
The transposon insertion site was sequenced and using the genome sequence candidate
genes identified. The candidate genes were then directly sequenced in parallel with a cloning
approach looking for wild type sequences that restored the virulence of the mutants. A wild
type cosmid library was also constructed and used to investigate mutants not amenable to
other approaches.

Results: This work describes genes that when mutated cause significant attenuation in a
tissue culture system.

Conclusion: Gaining knowledge of the less apparent virulence factors may provide us with
a window into other intracellular pathogens and new candidate oral vaccine strains.




                                             70
THE OSMOLYTE TRANSPORTER OPUC IS INVOLVED IN THE
VIRULENCE OF LISTERIA MONOCYTOGENES
J. A. Wouters1,R. D. Sleator2, C. G. M. Gahan2, C, Hill2 and T. Abee1

1
  Laboratory of Food Microbiology, Wageningen University and Wageningen Centre for Food
Science (WCFS), Bomenweg 2, 6703 HD Wageningen, The Netherlands. 2 Department of
Microbiology and National Food Biotechnology Centre, University College Cork, Cork,
Ireland.
Problem: Survival of the food-borne pathogen Listeria monocytogenes at high osmolarity is
attributed mainly to the accumulation of the organic osmolytes glycine betaine and carnitine.
Osmolyte accumulation may also be of relevance to the virulence of this bacterium. In this
respect it is significant to characterize the relevant L. monocytogenes osmolyte transporters.
Here we report the identification and disruption of a novel compatible solute transporter in L.
monocytogenes, designated OpuC, and the analysis of its role in osmolyte transport and
virulence.

Results: The L. monocytogenes OpuC-system was identified using the lactococcal pWVOl-
derived Ori+RepA-vector pORI19 to generate random chromosomal mutations. Analysis of a
mutant unable to grow at high Nad-concentrations in the presence of carnitine revealed the
presence of genes with significant homology (up to 60% identity) to the Bacillus subtilis
OpuC multicomponent osmolyte uptake system.
Radiolabelled osmolyte transport studies conducted with an opuC-deletion mutant revealed
decreased uptake rates of both carnitine and glycine betaine in L. monocytogenes.
Interestingly, mutating OpuC resulted in a significant reduction in the ability of L.
monocytogenes to colonize the upper small intestine and cause subsequent systemic
infection following peroral inoculation.

Conclusion: The multicomponent OpuC transport system represents the newest addition to
the arsenal of transporters used by L. monocytogenes for osmolyte acquisition during salt
stress and survival in hosts.




                                              71
IDENTIFICATION OF AN OSMOLYTE UPTAKE SYSTEM LINKED
TO THE VIRULENCE OF LISTERIA MONOCYTOGENES
Roy D. Sleator. Cormac G.M. Gahan, and Colin Hill*

Department of Microbiology' and National Food Biotechnology Centre University College
Cork, Ireland.

Problem: The success of Listeria monocytogenes as a food borne pathogen owes much to
its ability to survive a variety of stresses, both in the external environment prior to Ingestion
and subsequently within the animal host. Growth at high salt concentrations and low
temperatures is attributed mainly to the accumulation of organic solutes such as glycine
betaine and carnitine.

Method: We utilized a novel system for generating chromosomal mutations (based on a
lactococcal pWV0l-derived Ori+ repA- vector, pOR1119) to identify the listerial OpuC
homologue; a multi-component membrane bound system dedicated to the uptake and
accumulation of both glycine betaine and carnitine. Radiolaballed osmolyte uptake studies,
together with growth experiments in defined media, were used to determine the specificity of
the transporter and asses the importance of the system in contributing to listerial
osmotolerance. We also investigated the role of OpuC in contributing to the growth and
survival of Listeria in an animal (murine) model of infection.

Results: Mutating the Operon in two distinct strains of L. monocytogenes revealed
significant strain variation in the observed activity of OpuC. Disrupting the transporter
reduced the ability of Listeria to colonise the upper small intestine and cause subsequent
systemic infection following peroral inoculation.

Conclusion: The multi-component OpuC transport system which represents yet another
addition to the arsenal of transporters used by Listeria for osmolyte acquisition during salt
stress, contributes significantly to both the osmotolerance and virulence potential of this
ubiquitous food.




                                               72
IN VITRO AND IN VIVO VIRULENCE HETEROGENEITY IN
CLINICAL LISTERIA MONOCYTOGENES STRAINS
Wagner M1, Vazquez - Boland JA4, Bubert A3, Goebel W2
1
 Institute for Milk Hygiene, Milk Technology and Food Science, Veterinärplatz 1, Vienna,
2
 Institute for Microbiology, Am Hubland, Würzburg, 3Merck KGaA, Frankfurterstrasse
250, Darmstadt, 4Patologia Animal I, Facultad de Veterinaria, Universidad Complutense,
Madrid.

Problem: It was the goal of the study to investigate the behaviour of 4 Listeria
monocytogenes strains (1 outbreak/encephalitis associated, sv 4b, 1 sporadic/gastroenteritis
associated, sv 4b, 1 outbreak/gastroeneteritis associated, sv 1/2b, Listeria monocytogenes
EGD, reference, sv 1/2a) for their virulence properties in in vitro and in vivo assays.

Methods: Virulence was assayed using a GFP-reporter analysis and plating in a cell culture
model (cell lines: Caco-2, TIB 73 and P388-D1). Virulence testing in vivo was done in a
mouse assay investigating organ kinetics.

Results: In comparison to EGD, adherence assays clearly demonstrated a lower capability
of the clinical strains to adhere to Caco - 2 and TIB 73 cells. The invasion rate was
comparable among the strains and cell lines tested except for the outbreak gastroenteritis
associated isolate where the invasion rate was lower. Generally sv 4b-strains were most
efficient in spreading in Caco-2 cells whereas in phagocytes, the sv 1/2 strains were most
efficient. No significant difference in spreading was observed in TIB 73 cells. However, in
absolute numbers, lowest recovery from the assayed TIB 73 cells was seen using the
outbreak/gastroenteritis associated isolate. This was concluded to occur from the lower
invasion capability. In a BALB C mouse model, this isolate showed a significantly reduced
colonization of the liver and spleen.

Conclusion: With respect to the virulence properties of clinical L. monocytogenes, these
findings suggest a high flexibility to spread within the different compartments in the host.




                                            73
INVASION OF CELLS AND TISSUES BY THE BACTERIAL
PATHOGEN LISTERIA MONOCYTOGENES
Marc Lecuit, Laurence Braun. Helene Bierne, Javier Pizarro-Cerda, Shaynoor Dramsi, P.
Glaser*. The European Consortium. Pascale Cossart.

Unité des Interactions Bactéries-Cellules, * Laboratoire de Génomique des Pathogènes.
Institut Pasteur, Paris, France.

Problem: -L monocytogenes is a food pathogen characterized by its capacity to cross the
intestinal barrier, the hematoencephalic barrier and the foeto-placental barrier resulting in
meningitis, abortions and as demonstrated recently gastroenteritis. Entry of L.
monocytogenes into mammalian cells is a key feature of a Listeria infection. We are
investigating 1) how this bacterium induces its internalization into cells in vitro; and 2) the in
vivo relevance of the in vitro data. Two surface proteins involved in entry into cultured cells
were identified. Internalin mediates entry in epithelial cells. InlB mediates entry into most cell
types. Entry via each of these two proteins can be studied independently as beads coated
with either protein can enter into cells. The receptor for internalin is E-cadherin, a
transmembrane cell adhesion protein which is connected to the cytoskeleton via catenins
which appear critical for the entry process. Internalin displays a stringent specificity of
interaction. It interacts with human but not mouse F-cadherin. These observations have
allowed us to design a strategy to demonstrate its role in vivo.

Results: Two ligands for InlB have been recently identified: the first is gClqR, an ubiquitous
molecule whose role and location are controversial. The second identifed by Shen et al is
Met, the receptor for the hepatocyte growth factor. Whether these ligands interact will be
discussed. Interactions of InlB with cells trigger signaling events which are involved in
internalisation (PI 3- kinase activation) or post internalization events (phospholipase C γl and
NF-κB activation). Interestingly, internalin and InlB are part of a multigene family
characterized by leucine rich repeats. The complete genome sequence has revealed that
this family is larger than expected and that Listeria has an exceptionnally high number of
surface proteins which may help this bacterium to interact with diverse environments,
including several types of mammalian cells.




                                               74
INFLUENCE OF VIRULENCE GENES OF LISTERIA
MONOCYTOGENES ON ITS SURVIVAL AND MULTIPLICATION
WITHIN PROTOZOA
Marion Walcher1, Werner Goebel2, Martin Wiedmann3, and Michael Wagner1*
1
 Lehrstuhl für Mikrobiologie, Am Hochanger 4, 85350 Freising, Germany, 2 Lehrstuhl für
Mikrobiologie, Am Hubland, 97070 Würzburg, Germany, 3 Department of Food Science,
Cornell University, 412 Stocking Hall, Ithaca, NY 14853, USA
Listeria monocytogenes is an important food-borne pathogen of humans which can multiply
within animal and human cells. The structure and function of the virulence genes of L.
monocytogenes were intensively investigated during the last years leading to novel insights
into interactions of this bacterium with its host cells. In addition to this well-characterized life
style of L monocytogenes past research indicated that this species might also be capable to
successfully infect protozoa. However, this phenomenon has never been investigated in
detail.
!n this study we established an improved protozoa infection model for members of the genus
Listeria. Acanthamoeba were selected as host organisms since (i) they can be cultivated in
axenic media and (ii) they have been described to serve as multiplication vehicles for various
bacterial pathogens. Acanthamoeba were infected with different strains of L. monocytogenes
representing the three evolutionary lineages within this species as well as with a set of well-
characterized L. monocytogenes mutants. Amoebal cell numbers and the number of
intracellular viable Listeriae were recorded over a period of 42 days. Results confirmed that
L. monocytogenes multiplies within Acanthamoeba and demonstrated that ActA, PrfA, P60
(iap gene product), and the internalins InIA and InIB are not required for L. monocytogenes
to infect and thrive within Acanthamoeba. Currently we are investigating different strains of L.
seeligen - a non-pathogenic species which does nevertheless possess an intact virulence
gene cluster - with the Acanthamoeba infection model. Furthermore electron microscopic
analysis are performed to precisely determine the intracellular location of the listeriai cells
within the amoeba.
In conclusion our results demonstrate that protozoa might represent an important ecological
niche for the survival of L. monocytogenes in the environment. Interestingly, many virulence
factors absolutely required for the successful infection of mammalian cells are obviously not
of importance for survival within free living amoeba.




                                                75
SURVIVAL AND PERSISTENCE OF LISTERIA
MONOCYTOGENES IN ACANTHAMOEBA AND PROTECTION
AGAINST LISTERIOCIDAL AGENTS
Simon Kilvington, Peter Andrew, Isabel Morgado & Amy Peasland.
Department of Microbiology and Immunology, University of Leicester, Medical Sciences
Building PO Box 138, University Road Leicester LEI 9HN. United Kingdom.

Problem. The co-cultivation of Listeria monocytogenes with the common environmental
amoeba Acanthamoeba was investigated to establish whether the bacterium could infect and
survive within amoebal trophozoites and cysts.

Method. A. polyphaga trophozoites were co-cultured with L. monocytogenes in 1/4 strength
Ringer's solution and viable bacteria counted over 0-5 days. After 24 hrs co-culture,
trophozoite encystment was induced and the cysts treated with 2% HCl to kill trophozoites
and L. monocytogenes but retain mature cyst viability and bacteria within. Infected cysts
were exposed to 100 µg /ml gentamicin or 50 ppm chlorine (levels shown to be lethal to L.
monocytogenes) for 24 hrs, washed and inoculated into fresh culture medium to induce
trophozoite excystment.

Results. Co-culture of L. monocytogenes with A. polyphaga resulted in an initial slight
increase in bacterial numbers that persisted during the course of the experiments. L.
monocytogenes in 1/4 Ringer's alone showed a rapid decline (3 log reduction after 24 hrs).
Electron microscopy demonstrated bacteria within membrane bound vesicles in the
trophozoite cytoplasm. L. monocytogenes inside cysts survived exposure to HCl, gentamicin
and chlorine.

Conclusion. L. monocytogenes can infect and persist within Acanthamoeba trophozoites.
Survival within cysts affords protection from antimicrobial agents and disinfection. This may
provide an important ecological niche for L. monocytogenes and have implications for the
presence and persistence of the bacterium in the environment.




                                             76
THE ACTIVATION OF PROTEIN KINASE C ISOFORMS IN J774
CELLS BY LISTERIOLYSIN O AND PHOSPHATIDYLINOSITOL -
SPECIFIC PHOSPHOLIPASE C (PI-PC99) MEDIATES BACTERIAL
ASSOCIATION AND ENTRY
Howard Goldfine and Sandra J. Wadsworth.

Dept. of Microbiology, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104-6076, USA

The role of PI-PLC in mediating escape from the primary vacuole of a macrophage is still
unclear. In a previous study we demonstrated that association with and entry of wild type
Listeria monocytogenes (Lm) into murine macrophage-derived J774 cells was delayed. Prior
to entry of the wild type, host calcium signals were detected. Upon infection with mutants in
either PI-PLC or listeriolysin O (LLO) the calcium signals were greatly diminished or absent
and association of the bacteria with host cells and the rate of entry were increased.
We have extended our findings to include activation of three isoforms of PKC in J774 cells.
We show that PKC , I and II are activated prior to entry of wild type Lm. PKC 
translocates to the periphery of host cells beginning 30 sec after infection and PKC I and II
translocate to early endosomes. Mutants in LLO and PI-PLC did not activate PKC  or II.
Rottlerin, an inhibitor of PKC  activation, also inhibited the calcium signals generated by the
wild type strain. Both rottlerin and a calcium channel blocker inhibited translocation of PKC
II. These findings lead us to propose a host signaling pathway activated by bacterial LLO
and PI-PLC in which formation of diacylglycerol leads to PKC activation, which generates a
calcium signal, leading to activation of PKC II. In contrast, activation of PKC I occurred in
host cells after infection with the PI-PLC mutant strain, but not on infection with an LLO
mutant. Pharmacological inhibition of the PKC  or PKC II activation increased association
with host J774 cells and entry of wild type Lm. These findings indicate that early host signals
generated by PI-PLC with LLO are critical in controlling the entry of this pathogen, which
greatly affects eventual escape from the phagosome. (Supported by research grants from
the National Institutes of Health).




                                              77
ANALYSIS OF THE ROLE OF ENA/VASP PROTEIN DOMAINS IN
THE ACTIN-BASED MOTILITY OF LISTERIA MONOCYTOGENES
Marcus Geese, Joseph J. Loureiro*, Frank B. Gertler*,Jürgen Wehland, Antonio S. Sechi.

Department of Cell Biology, Gesellschaft für Biotechnologische Forschung, D-38124
Braunschweig,Phone +49/53176181241 or 246, Fax+49/53176181444 Germany.
*Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts,
USA.

Much has been learned about the dynamics of the actin cytoskeleton through studies of the
intracellular pathogen Listeria monocytogenes. After entering the cell, Listeria escape the
phagosome, enter the cell cytoplasm and subvert the host cell actin cytoskeleton to propel
themselves within the infected cell and spread to adjacent cells. Listeria accomplish this feat
through expression of a single virulence factor, the ActA protein (Domann et al., 1992; Kocks
et al., 1992). ActA harbours two major domains: an amino-terminal region which is required
for the nucleation of actin, and a central proline-rich domain which significantly enhances the
actin-based motility of Listeria (Pistor et al., 1995). Similar domains can also be found in the
iActA protein of Listeria ivanovii, another pathogenic Listeria species (Gerstel et al., 1996).
The proline-rich domain binds with a high affinity to proteins of the Ena/VASP family
(Niebuhr et al., 1997). Members of this protein family include VASP, Mena and Evl.
Ena/VASP proteins are characterised by a similar domain organisation. Their N-terminal
region, the EVH1 domain, is essential for mediating the binding to proteins harbouring the
motif E/DFPPPPXD/E, the hallmark of the ActA proline-rich region (Niebuhr et al., 1997; Carl
et al., 1999). The central domain of these proteins contains a proline-rich region which binds
to profilin and, in addition,to SH3 and WW domains (Reinhard et al., 1995; Gertler et al.,
1996; Ermekova et al., 1997). The C-terminus of Ena/VASP proteins binds to F-actin in vitro
and is thought to mediate the multimerisation of these proteins (Bachmann et al., 1999).
In cultured cells, Ena/VASP proteins localise in subcellular regions where remodelling of the
actin cytoskeleton takes place, such as the front of spreading lamellipodia in motile cells
(Rottner et al., 1999) and the distal tips of growth cone filopodia (Gertler et al., 1996; Lanier
et al., 1999). Moreover, the expression of the neuronal-specific isoform of Mena induces the
formation of actin-rich cell surface projections in fibroblasts and is required for axon guidance
(Gertler et al., 1996; Lanier et al., 1999). Ena/VASP proteins are also essential for the
remodelling of the actin cytoskeleton that accompanies T-cell activation (Krause et al.,
2000). Overall, taken together with the observation that Ena/VASP proteins are also
necessary for efficient Listeria motility in cells and cell-free extracts (Niebuhr et al., 1997;
Laurent et al., 1999), these findings clearly indicate that Ena/VASP proteins function as
regulators of the actin cytoskeleton. The mechanism by which this is accomplished may
involve recruitment of profilin-G-actin complexes to sites of actin cytoskeleton
rearrangement. This notion has been experimentally supported by the observation that
GFP-tagged profilin is recruited both to the bacterial surface at the onset of Listeria motility
and to the tips of spreading lamellipodia (Geese et al., 2000). In contrast to this line of
evidence, which suggest that Ena/VASP proteins act as positive regulators of the actin
dynamics, Bear et al. (2000) have recently shown that proteins of this family can retard the
motility of fibroblasts. Thus, Ena/VASP proteins may play multiple roles, or their activity may
be differently regulated depending upon the particular biological systems in question.


                                               78
Although the role of the EVH1 domain of Ena/VASP proteins in Listeria motility has been
extensively studied, little is known about the functions of the other structural domains in this
actin-based process. In addition, it is not known whether or not the phosphorylation state of
Ena/VASP proteins also plays a role in bacterial movement. To address these points, we
took advantage of the generation of a double Mena/VASP knock-out cell line that does not
express detectable levels of Evl. We expressed several VASP and Mena mutants in this cell
line and analysed their effect on the motility of Listeria. Our preliminary results clearly
indicate that deletion of the proline-rich region or the putative G-actin binding site causes a
reduction in the speed of Listeria. In contrast, the deletion of the F-actin binding site
enhances bacterial motility.

The phosphorylation status of Ena/VASP proteins also appears to play a role in Listeria
motility since the expression of mutated Ena/VASP proteins that cannot be phosphorylated
results in a reduction of Listeria motility. Overall, these findings should help to better our
understanding of the function of Ena/VASP proteins in actin-based processes.

References
Bachmann, C., Fischer, L., Walter, U., and M. Reinhard. 1999. The EVH2 domain of the
vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin
bundle formation. J. Biol. Chem. 274:23549-23557.

Bear, J.E., Loureiro, J.J., Libova, I., Fassler, R., Wehland, J. and Gertler, F.B. 2000.
Negative regulation of fibroblast motility by Ena/VASP proteins. Cell. 101:717-28.
Carl, U.D., Pollmann, M., Orr, E., Gertler, F.B., Chakraborty, T., and Wehland J. 1999.
Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with
proline-rich ligands. Curr. Biol. 9:715-718.

Chakraborty, T., Ebel, F., Domann, E., Niebuhr, K., Gerstel, B., Pistor, S., Temm-Grove, C.,
Jockusch, B. M., Reinhard, M., Walter, U. and Wehland, J. 1995. A focal adhesion factor
directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-
based cytoskeleton of mammalian cells. EMBO J. 14:1314-1321.

Domann, E., Wehland, J., Rohde, M., Pistor, S., Hartl, M., Goebel, W., Leimeister-Wächter,
M., Wünscher, M. and Chakraborty, T. 1992. A novel bacterial virulence gene in Listeria
monocytogenes required for host cell microfilament interaction with homology to the proline-
rich region of vinculin. EMBO J. 11:1981-1990.

Ermekova, K.S., Zambrano, N., Linn, H., Minopoli, G., Gertler, F., Russo, T. and Sudol, M.
1997. The WW domain of neural protein FE65 interacts with proline-rich motifs in Mena, the
mammalian homolog of Drosophila enabled. J Biol Chem. 272:32869-77.

Geese, M., Schluter, K., Rothkegel, M., Jockusch, B.M., Wehland, J. and Sechi, A.S. 2000.
Accumulation of profilin II at the surface of Listeria is concomitant with the onset of motility
and correlates with bacterial speed. J Cell Sci. 113:1415-26.

Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J. and Soriano, P. 1996. Mena, a relative
of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell
87:227-239.



                                               79
Gerstel, B., Grobe, L., Pistor, S., Chakraborty, T. and Wehland, J. 1996. The ActA
polypeptides of Listeria ivanovii and Listeria monocytogenes harbor related binding sites for
host microfilament proteins. Infect Immun. 64:1929-36.

Krause, M., Sechi, A.S., Konradt, M., Monner, D., Gertler, F.B. and Wehland, J. 2000. Fyn-
binding protein (Fyb)/SLP-76-associated protein (SLAP), Ena/vasodilator-stimulated
phosphoprotein (VASP) proteins and the Arp2/3 complex link T cell receptor (TCR) signaling
to the actin cytoskeleton. J Cell Biol. 149:181-94.
Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H., and P. Cossart. 1992. L.
monocytogenes-induced actin assembly requires the actA gene product, a surface protein.
Cell. 68:521-531.

Lanier, L.M., Gates, M.A., Witke, W., Menzies, S., Wehman, A.M., Macklis, J.D.,
Kwiatkowski, D., Soriano, P., and F.B. Gertler. 1999. Mena is required for neurulation and
commissure formation. Neuron. 22:313-325.

Laurent, V., Loisel, T.P., Harbeck, B., Wehman, A., Gröbe, L., Jockusch, B.M., Wehland, J.,
Gertler, F.B., and M.-F. Carlier. 1999. Role of proteins of the Ena/VASP family in actin-based
motility of Listeria monocytogenes. J.Cell Biol. 144:1245-1258.

Niebuhr, K., Ebel, F., Frank, R., Reinhard, R., Domann, E., Carl, U. D., Walter, U., Gertler, F.
B., Wehland, J. and Chakraborty, T. 1997. A novel proline-rich motif present in ActA of
Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a
protein module present in the Ena/VASP family. EMBO J. 16:5433-5444.

Pistor, S., Chakraborty, T., Walter, U. and Wehland, J. 1995. The bacterial actin nucleator
protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament
proteins. Curr. Biol. 5:517-525.

Reinhard, M., Giehl, K., Abel, K., Haffner, C., Jarchau, T., Hoppe, V., Jockusch, B.M., and U.
Walter 1995. The proline-rich focal adhesion and microfilament protein VASP is a ligand for
profilins. EMBO J. 14:1583-1589.

Rottner, K., Behrendt, B., Small, J.V., and J. Wehland. 1999. VASP dynamics during
lamellipodia protrusion. Nature Cell Biol. 1:321-322.




                                              80
SMCL FROM LISTERIA IVANOVII: UNDERSTANDING THE ROLE
OF A BACTERIAL SPHINGOMYELINASE IN BACTERIA-HOST
CELL INTERACTION.
J.A. Vázquez-Boland, B. González-Zorn

Grupo de Patogénesis Molecular Bacteriana, Departamento de Patología Animal I, Facultad
de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.

Introduction: We recently cloned and sequenced the smcL gene encoding the L. ivanovii
sphingomyelinase. This enzyme belongs to a group of phospholipases C strictly specific for
sphingomyelin and currently comprising three other members: the staphylococcal -toxin
and the sphingomyelinases of Bacillus cereus and Leptospira interrogans serovar hardjo (1).
Although the toxic properties of the -toxin have been extensively studied in the past, no
clear evidence of the role in pathogenesis of any of the members of this group of membrane-
damaging enzymes was available.

Results: By gene disruption in L. ivanovii and heterologous expression in a L.
monocytogenes host strain lacking all known membrane-damaging virulence determinants
(hly, plcA and plcB), we showed that the smcL gene product (SmcL) contributes to escape
from the phagocytic vacuole, thereby promoting bacterial intracellular proliferation (1). Using
L. ivanovii isogenic mutants and complementation of a L. monocytogenes double plcA-plcB
mutant, we also demonstrated that production of SmcL by intracellular bacteria triggers a
marked elevation of ceramide levels in host cells. This required the previous release of
bacteria into the cytosol and was associated with increased cell death via apoptosis (2).
Thus, SmcL plays a dual role in virulence: (i) it has a mechanical function, contributing to
phagosome membrane disruption; and (ii) it modulates host cell responses via ceramide
signalling. In contrast to L. monocytogenes, which has a wide host range, L. ivanovii shows a
clear pathogenic tropism for ruminants. smcL is L. ivanovii-specific within the genus Listeria
and is present in LIPI-2, a 22-kb pathogenicity island also specific to L. ivanovii (3).

Conclusion: Our preliminary data suggest that smcL and the genomic island carrying it play
a role in the specific pathogenic properties of L. ivanovii.

References:
- González-Zorn, B., G. Domínguez-Bernal, M. Suárez, M.T. Ripio, Y. Vega, S. Novella,
   and J.-A. Vázquez-Boland. 1999. Mol. Microbiol. 33:510-523
- González-Zorn, B., T. Levade, and J.A. Vázquez-Boland. 2001. (in preparation).
- G. Domínguez-Bernal, S. Altrock, B. González-Zorn, J. Kreft, and J.A. Vázquez-Boland.
   2001 (in preparation).




                                              81
INTERACTION OF LISTERIA MONOCYTOGENES WITH
MICROVASCULAR ENDOTHELIAL CELLS
Michael Kuhn, Lars Greiffenberg, Tobias Hertzig, Werner Goebel

Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften der Universität
Würzburg, Am Hubland, 97074 Würzburg, Germany

Problem: Invasion of endothelial tissues may be crucial in a Listeria monocytogenes
infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into
endothelial cells has been previously demonstrated using human umbilical vein endothelial
cells as a model system. However, during the crossing of the blood-brain barrier L.
monocytogenes most likely encounters brain microvascular endothelial cells which are
different from macrovascular- or umbilical vein endothelial cells.
Methods: In the present study human brain microvascular endothelial cells (HBMEC) were
used to study the interaction of L. monocytogenes with endothelial cells which closely
resemble native microvascular endothelial cells of the brain.

Results: We show that L. monocytogenes efficiently interacts with the surface of HBMEC in
an InlB-independent manner. Invasion of HBMEC by L. monocytogenes is, however, strictly
InlB-dependent. Furthermore, we show that the invasion efficiency is strongly reduced in the
presence of human serum. Once within the HBMEC, L. monocytogenes replicates efficiently,
moves intracellularly by inducing actin tail formation, produces bacteria-containing
protrusions and spreads from cell to cell. Using a green fluorescent protein-expressing L.
monocytogenes strain, we present direct evidence that HBMEC are highly resistant to
damage by intracellularly growing L. monocytogenes since infection of HBMEC with L.
monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells.
Heterologous plaque-assays with L. monocytogenes-infected P388D1 macrophages as
vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts,
hepatocytes, and epithelial cells.

Ref:
Greiffenberg et al., 1998. Infect. Immun. 66:5260-5267.
Greiffenberg et al., 2000. Infect. Immun. 68:3275-3279.




                                             82
CHARACTERISATION OF GROESL IN LISTERIA
MONOCYTOGENES: EVIDENCE FOR IN VIVO EXPRESSION
Cormac G.M. Gahan, James O'Mahony, Colin Hill

Department of Microbiology and National Food Biotechnology Centre University College
Cork, Ireland.

Problem: The ability of intracellular pathogens to adapt to the hostile environment of the
host is an important factor governing virulence. In some organisms stress proteins
synthesised during infection represent important immunodominant antigens. In this study, we
characterised the Operon encoding the heat shock proteins GroES and GroEL in Listeria
monocytogenes and examined whether transcription of the Operon increases during
infection.

Method: We sequenced the groESL Operon in L. monocytogenes. We used reverse
transcriptase Polymerase chain reaction (RT-PCR), slot-blot RNA analysis and green
fluorescent protein technology to analyse expression of groESL under various environmental
stress conditions including heat shock, ethanol stress and acid shock and during infection
ofJ774 mouse macrophage cells. In addition, we utilised haemolysin expression from the
groESL promoter as a novel means of analysing promoter activity during murine infection
and growth in J774 cells.

Results: Strains harbouring transcriptional fusions of the Aequorea victoria green
fluorescent protein (GFPuv) to the promoter region of groESL demonstrate a significant
increase in fluorescence following heat shock that can be detected by both fluorimetry and
epifluorescence microscopy. Using both RT-PCR and GFP technology we have also
detected expression of groESL during internalisation by J774 cells. Increased intracellular
expression of dnaK was also determined using RT-PCR. A strain was constructed in which
hemolysin expression is under the influence of the groESL promoter. In this strain hemolysin
expression during infection suggested transcription from the groESL promoter during murine
infection and J774 infection, albeit at lower levels than the known virulence factor pIcA.

Conclusion: We have utilised a number of approaches to characterise groESL expression
in L. monocytogenes in response to various environmental stresses. It is evident from this
study that the pathogen responds to in vivo-associated stresses and to host infection, with
increased transcription of this operon.




                                            83
ROLE OF THE AUTOLYSIN AMI IN THE ADHESION OF LISTERIA
MONOCYTOGENES TO EUKARYOTIC CELLS
Eliane Milohanic, Renaud Jonquieres, Pascale Cossart, Patrick Berche and Jean-Louis
Gaillard

Lab. Microbiologie, Hòpital Raymond Poincaré, 104 blvd. Raymond Poincaré, F-92380
Garches

Problem: Adherence of pathogenic microorganisms to the cell surface is a key event during
infection. We previously reported the characterization of Listeria monocytogenes transposon
mutants defective in adhesion to eukaryotic cells. The mutant which was the most severely
defective in adhesion had lost the ability to produce Ami, a 102 kDa autolytic amidase with a
N-terminal catalytic domain and a C-terminal cell wall-anchoring domain made up of
repeated modules containing the dipeptide GW (« GW modules »).
Results: We generated ami null mutations by plasmid insertion into L. monocytogenes
strains lacking the invasion proteins InlA, InlB. or both. These mutants were 5 to 10 times
less adherent than their parental strains in various cell types. The adhesion capacity of the
mutants was restored by complementation with a plasmid expressing the Ami cell wall-
anchoring domain. The cell binding activity of the Ami cell wall-anchoring domain was further
demonstrated using the purified polypeptide. Growth of the ami null mutants constructed in
EGD and EGDΔinlAB background was attenuated in the liver of mice inoculated
intravenously, indicating a role for Ami in L. monocytogenes virulence. Adhesive properties
have been reported in two other autolysins, Staphylococcus epidermidis AtlE and
Staphylococcus saprophyticus Aas. Interestingly, we found that these domains were also
composed of repeated GW modules, thus suggesting an essential role for these motifs.
These molecules may contribute to the colonization of host tissues by Gram-positive
bacteria.




                                             84
INDUCTION OF COLD-SHOCK: PROTEINS IN LISTERIA
MONOCYTOGENES UPON EXPOSURE TO DIFFERENT STRESS
CONDITIONS
H. H. Kamphuis. J. A. Wouters, T. Abee

Wageningen Centre for Food Sciences (WCFS), Laboratory of Food Microbiology,
Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands

Problem: As consumers ask for minimally processed food products, cold storage is widely
used and new preservation techniques like ultra high pressure treatments are emerging. A
possible hazard in minimally processed foods is Listeria monocytogenes. This pathogen is
known for its growth and survival during several processing conditions. A response to low
temperature shared by almost all bacteria is the synthesis of so-called cold-shock proteins
(CSPs). CSPs can confer cryoprotection, and their single stranded nucleic acid binding
abilities can include transcriptional activation, transcription anti-termination and inhibition of
the formation of secondary RNA structures. CSPs might also play a role in the adaptation to
other stress conditions than low temperature, like exposure to ultra high pressure or growth
at stationary phase. In this study CSPs of L. monocytogenes will be identified and their
production during different stress conditions will be monitored.

Method: The protein composition of L. monocytogenes L028 cells grown at 37°C and that of
cells cold-shocked to 10°C were analyzed using two-dimensional gel electrophoresis.

Results: In Western-blotting experiments with anti-CspB from Bacillus subtilis, four 7 kDa
spots were observed, suggesting the presence of a CSP family in L. monocytogenes. Two of
the four putative CSPs were cold induced, approximately 10 and 3 fold. Strikingly, applying
ultra high pressure to L. monocytogenes cells resulted in induction of one of the other two
CSPs. At stationary phase conditions at 37°C none of the CSPs were induced. Furthermore,
27 (non 7-kDa) proteins were found to be induced (more than 2-fold) upon exposure to low
temperature, and were named cold-induced proteins (CIPs).

Conclusion: Further research will focus on the identification of CSPs and CIPs and the
unraveling of their role in stress response of L. monocytogenes.




                                               85
EFFECT OF DIFFERENT SALTING AND SMOKING
PROCEDURES ON L. INNOCUA 2030C AND TVC DURING
CHILLED STORAGE OF VACUUM-PACKED COLD-SMOKED
SALMON-TROUT
Vaz-Velho, M., Silva, M., Ribeiro, J., Pessoa, J., Gay, M., Aalberts, C. ,Águas, M.       and
Gibbs, P.

INTRODUCTION: Smoked fish is the product that is prepared by subjecting the fish to the
direct action of smoke from smouldering wood or sawdust. A dry or wet salting step is always
used in the process before exposure to the wood or sawdust smoke. In the cold smoking
process temperatures below 30 °C are used. Drying is minimal at the start of the process
and it will increase at the end. (Dillon et al., 1994). It has been reported that the type of
salting (dry or wet-salting) in the cold-smoked salmon process leads to the development of
different microflora (Truelstrup-Hansen et al., 1996). A considerable variation in the cold-
smoked salmon microflora among different smokehouses and production batches from the
same smokehouses was also reported. With respect to the pathogen Listeria
monocytogenes, there is no point of the process that can fully assure its absence. Neither
the smoking temperature nor the salt content are enough to kill the organism. Furthermore
refrigerated storage and vacuum packaging still allows the growth of the pathogen (Huss et
al., 1995).

According to the World Health Organisation (WHO), the critical point is not to prevent the
occurrence of L. monocytogenes in foods, but to control its growth and survival in order to
minimise its levels in foods (Quist, 1996).

Experimental procedure: Different salting methods (dry salting or brining) using different
ratios sugar:salt (1:4 or 1:6) during different periods of salting ( 6 or 8 hours) and further
smoking by two processes (Process 1 - 2 hours of drying + 6 hours of smoking; Process 2 -
6 hours of drying + 2 hours of smoking) were assayed for the potential effect they might have
on Listeria innocua 2030c and total flora numbers in vacuum-packed sliced cold-smoked
salmon-trout during five weeks of storage at 5ºC. L. innocua 2030c, a tetracycline-resistant
strain was used as a marker for replacing L. monocytogenes due to processing laboratory
constraints of using the pathogen and because all the L. monocytogenes strains isolated
from Portuguese cold-smoked fish products were until now tetracycline sensitive.

Fresh salmon-trout (4-6 kg) slaughtered on the same day was obtained from a farm in
Pisões (North of Portugal) packed with ice and transported in a refrigerated truck (3 hours of
travelling). After overnight storage at 5ºC the fish were eviscerated, beheaded, filleted and
washed in tap water.

Dry-salted and brined fillets were placed in a chilled chamber at 5ºC and 16ºC, respectively,
for dry-salted and brined fillets were placed in a chilled chamber at 5ºC and 16ºC,
respectively For brining, 211.2 g of salt per litre of brine was used.
Draining was done overnight at 5 ºC.
Smoking was done by two different processes:
Process 1- drying for 2 hours and smoking for 6 hours. 32 fillets (16 inoculated with L.
innocua 2030c and 16 not inoculated (control/TVC) were smoked by this method.
                                                 86
Process 2- drying for 6 hours and smoking for 2 hours. 32 fillets (16 inoculated with L.
innocua 2030c and 16 not inoculated (control/TVC) were smoked by this method.
Time/temperature profile of the the processes were recorded on a portable microprocessor
(Hanna instruments, HI 92804c). Smoke temperature was kept below 30ºC. Humidity,
recorded by an hygrometer (Rotronic AM3) varied from 70% at the first three hours
decreasing to 52% at the end of the smoking process.
The smoked samples were cooled overnight at 5 ºC. The following day, lug and pin bones
and belly flaps were removed and the fillets were then sliced by hand and vacuum packed in
a Multivac-Gastrovac, 1mbar/10s.
The permeability of the packs to O2, CO2 and N2 were respectively 4mol/m2.d.bar., 13
mol/m2.d.bar and 4 mol/m2.d.bar. Packs were stored for 5 weeks at 5ºC for weekly analysis.
Inoculation with L. innocua 2030c: An overnight (30ºC, 18 hours) 40 ml culture of L. innocua
2030c (ca.10 8-9 CFU/ml) in TSB-YE (0.6% yeast extract) (Lab M) was centrifuged for ten
minutes and the cells resuspended in 40 ml of sterilised water containing 1% NaCl w/v
(Merck) and this solution was further placed in 2 litres of water with 1% of NaCl (Merck). The
raw fish fillets were immersed in this suspension for 30 seconds.
Counting L. innocua 2030c and TVC: L. innocua 2030c counting was done by spread plate
onto PALCAM (Merck) with 8g/ml tetracycline (Sigma). Counting of L. innocua 2030c on the
raw material was performed in Escola Superior de Biotecnologia (ESB) Counts of L. innocua
on the raw fillets, varied from 2.4-3.8 log CFU/g.
TVC counting was done by overlay in Long & Hammer (1941) plating medium (modified by
Van Spreekens, 1974).
Samples were frozen to –20ºC and sent to other laboratories for L. innocua 2030c and TVC
counting, during the same 5 week period of storage (inoculated: ASEPT and RIVO-DLO;
non-inoculated: ESB and Leatherhead Food RA.). All samples were analysed in triplicate.
Chemical, sensorial and statistical analysis: Determination of salt content and moisture of the
finished products were evaluated after three weeks of storage by Centro de Qualidade
Alimentar (CEQA), ESB.
After three weeks of storage at 5 ºC, sensory evaluation was carried out with 10 judges
asked to classify the samples for overall acceptability regarding the flavour, aroma and
texture.
The effect of the different treatments on sensory properties of the products, on L. innocua
2030c behaviour and on TVC of non-inoculated samples were evaluated by analysis of
variance (ANOVA). It was accepted there was a significant difference between processes if
p< 0.05.

Results- inoculated samples: With respect to L. innocua 2030c growth, Process 1 was
significantly different from Process 2, numbers being lower in the latter. The type of salting
was also considered to be significant within Process 1, dry-salted samples showing higher
numbers of L. innocua, but this effect was not significant within Process 2.
The period of salting had a significant effect on samples from Process 1, numbers of L.
innocua 2030c being lower when dry-salting was used but not on samples from Process 2.
For L. innocua 2030c numbers, no significant differences between samples with 1/6 and 1/4
of sugar added were found within each smoking process

Results: non-inoculated samples: With respect to TVCs, Process 1 was significantly
different from Process 2, TVC numbers being lower in the former. This effect was more
noticeable when dry-salting was used.
In Process 2 the period of salting significantly influenced TVC numbers, samples with 8
hours of salting showing lower numbers than samples salted for just 6 hours.
                                              87
In both processes the type of salting had a significant effect on TVC, numbers being higher
in brined samples.
For TVC counts, no significant differences between samples with 1/6 and 1/4 of sugar added
were found within each smoking process.
For sensory properties combining dry salting for 8 hours with a ratio 1:6 sugar:salt and
following Process 2 seems to be the best choice.

Conclusions: A short exposure to smoke (Process 2) is more effective than 6 hours of
exposure (Process 1) in reducing Listeria spp. numbers. Also, 8 hours of salting is preferable
to 6 hours when a short period of smoke is used. If a longer exposure to smoke is chosen
than brining is preferable to dry salting.
On the other hand the best process for reducing TVC numbers was a longer exposure to
smoke (Process 1) combined with dry-salting for 8 hours. To guarantee TVC levels below
106 CFU/g, shelf-life must be limited to three weeks for brined samples independent of the
smoking process, and to 4 weeks for dry-salted samples from Process 1.
For sensory properties combining dry salting for 8 hours with a ratio 1:6 salt:sugar and
following Process 2 seems to be the best choice.

Acknowledgements: To the EC FAIR project CT95-1207 Spoilage and Safety of Cold-
smoked fish

References: Dillon, R., Patel, T.R., Martin, A.M. 1994. Microbiological control for fish
smoking operations. In: Fisheries Processing, Biotechnological Applications. Ed A.M. Martin.
London: Chapman and Hall, 51-81.

Huss, H.H., Ben Embarek, P.K., Jeppesen, V.F. 1995. Control of biological hazards in cold
smoked salmon production. Food. Control 6(6), 335-340.

Qvist, S. 1996. The Danish government position on the control of Listeria monocytogenes in
foods. International Food Safety Conference. Listeria: the state of the science, Rome 29-30
June 1995. Food Control 7 (4/5): 249.

Truelstrup-Hansen, L.T., Gill, T., Røntved, S.R., Huss, H.H. 1996. Importance of autolysis
and microbiological activity on quality of cold-smoked salmon. Food Research International
29 (2): 181.

Van Spreekens, K. J. A. 1974. The suitability of a modification of Long and Hammer’s
medium for the enumeration of more fastidious bacteria from fresh fishery products. Arch.
Lebensmittelhyg, 25, 213-219.




                                             88
IN VIVO STUDIES WITH MUTANTS OF LISTERIA
MONOCYTOGENES

Schlech, 'W., R.A. Garduno, and T, Gill,

Department of Medicine, Faculty of Medicine and Department of Food Science and
Technology, Faculty of Engineering, Dalhousie University, ACC 5040, QEJT Health Science
Center, 1278 Tower Road, Halifax, Nova Scotia, Canada, B3H-2Y9
Problem; No reliable molecular markers of high virulence have been identified in Lm, so it is
not currently possible to predict whether an isolate present in food products could cause
epidemic Listeriosis. We believe that L.m. solely induces key virulence factors in vivo, when
infecting the host. Because most Lm pathogenicity studies have been done in vitro, in vivo
induced factors that could be used as markers of high virulence may have been missed,

Method; /,w was grown in diffusion chambers surgically implanted in the peritoneal cavity of
rats, in the presence or absence of peritoneal exudate cells, We also isolated mutants
resistant to the polycationic peptide protamine, known to have an altered cell surface. A
protamine-resistant (PtmR) mutant derived from the Canadian Maritimes outbreak strain was
selected for most of our studies. A genomic library of Lm was created to be screened with a
rabbit antiserum raised against the Lm grown in vivo.

Results; By SDS-PAGE, the PtmR mutant displayed an altered pattern of spontaneously
released surface proteins, and a large band of material not stained by conventional protein
stains, was seen in cell wall preparations. By electron microscopy a diffuse layer of material
(likely a glycocalix) was seen on the cell wall of the mutant. The PtmR mutant had a lower in
vivo survival rate than the parent strain, particularly in the presence of thioglycollate-elicited
peritoneal macrophages, and it formed plaques on L929 cells that had a different
morphology than those of the parent strain Some E. coli library clones gave a very strong
signal after immuno-staining with the anti-in vivo
Lm rabbit serum, and studies are in progress on the potential identification of in vivo induced
factors

Conclusion: Lm is not traditionally considered a capsulated bacterium. If the PtmR mutant
indeed forms a glycocalix this will give us the opportunity to identify the biosynthetic genes
involved, The intraperitoneal chamber model should prove useful in studying bacteria host-
cell interactions, as weil as the impact of alterations on the Lm cell surface (e.g via acquiring
PtmR), within the in vivo environment, The characterization of PtrnR mutants might serve as
a link to identify surface-exposed virulence factors and determine their potential function in
vivo, Some of the antigens potentially identifiable via genomic library screening with the anti-
in vivo Lm may constitute in vivo-induced factors that could be used as markers of high
virulence,




                                               89
disease of man and animals




            90
PATHOLOGY OF L. MONOCYTOGENES INFECTION
Martina Deckert1, Maja Abram2, Dirk Schlüter3

1
 Department of Neuropathology, University of Cologne, Köln, Germany
2
 Department of Microbiology, University of Rijeka, Rijeka, Croatia
3
 Department of Medical Microbiolgy and Hygiene, University of Heidelberg/Mannheim,
Mannheim, Germany

L. monocytogenes may cause severe infections, particularly in immunocompromised
persons including pregnant women, fetuses and neonates as well as in patients with
impaired cellular immune responses. Infection during pregnancy may result in severe fetal
disease and even abortion. Neonatal listeriosis may manifest as sepsis with involvement of
liver, spleen, and CNS. In contrast, listeriosis of adult patients is ususally confined to the
CNS. Cerebral listeriosis differs from other bacterial infections of the CNS due to the ability
of L. monocytogenes to cause meningitis, encephalitis, brain abscess or combined forms.
In order to systematically study the immune responses that play a role in the various forms of
human listeriosis we have established animal models of pregnancy-associated and cerebral
infection, respectively. Whereas mice normally eliminate L. monocytogenes effectively
following either i.v. or i.p. infection, pregnant mice fail to clear the bacteria and instead
develop a severe, necrotizing hepatitis. Infection of placenta and fetuses resulted in a high
abortion rate due to lack of effector cells in placenta and fetus as well as low levels of anti-
listerial cytokines.
Cerebral listeriosis of mice closely parallels human CNS infection including the high affinity
of bacteria for structures of the ventricular system and the brain stem. Clinically relevant,
mice can be protected from the inevitably fatal course of CNS listeriosis by a prior systemic
immunization, which results in an accelerated and more extensive recruitment of protective
CD4+ and CD8+ T cells to the L. monocytogenes-infected brain as well as in an increased
production of protective cytokines. In addition to pro-inflammatory cytokines, the
immunosuppressive mediator IL-10 plays a pathogenetically relevant role by the prevention
of an hyperinflammatory immune reaction in the CNS.
Thus, the various forms of experimental murine listeriosis share important features with
human listeriosis and provide a suitable tool to dissect the immune response and to identify
pathogenetically relevant factors in order to improve therapeutic strategies and the patients’
prognosis.




                                              91
VETERINARY PERSPECTIVE.
W. Donachie1 and J.C. Low2

1
Microbiology Division, Moredun Research Institute, Edinburgh,                  Scotland,    UK,
2
SACVS(Edinburgh), Bush Estate, Penicuik, Midlothian, Scotland, UK.

Listeriosis in animals is widespread and has been recorded in more than forty species of wild
and domesticated animals and in countries over six continents. It is of major importance in
the domesticated ruminants; cattle, sheep and goats where a number of syndromes are
recognised; encephalitis, abortion, septicaemia, mastitis, purulent conjunctivitis and keratitis
and enteritis. The pathogenesis of listeric encephalitis in ruminants is only partially
understood. There is a seasonal association, with disease more common in the winter and
early spring, and an association with the feeding of silage. Pathogenesis is believed to
involve the invasion of the cranial nerves and centripetal travel to the brain stem, where the
main lesions occur. Listeric abortion caused by L. monocytogenes occurs in ruminants and
many other species of domesticated animal. Listeria ivanovii is also recorded as a cause of
abortion in sheep and cattle but occurs less frequently than L. monocytogenes and is
extremely rare as a cause of other conditions. Septicaemia is relatively uncommon and
generally, though not invariably, occurs in the neonate as an extension of intrauterine
infection. Occasional massive outbreaks of septicaemia involving pregnant ewes have been
described. Iritis and keratoconjunctivitis caused by L. monocytogenes have been recorded
occasionally in both cattle and sheep. In monogastric animals listeriosis is uncommon though
septicaemia and meningoencephalitis have been reported. Although there is an extensive
library of information on the immune response of infected animals to L. monocytogenes, this
relates almost exclusively to laboratory rodents and the picture is a lot less clear in
ruminants. Experiments show that oral infection leads to seroconversion and cell mediated
responses with development of protective immunity. Diagnostic indicators of infection are
problematic as many animals are naturally exposed to the organism and have
seroconverted. The sporadic nature of ruminant listeriosis raises doubts about the overall
effectiveness of control programmes but it is generally agreed that active immunisation is
best generated by vaccination with a live attenuated strain. There have been major
advances in our understanding of L. monocytogenes, particularly its taxonomy and the
molecular definition of its mechanisms of virulence. However, there remain major gaps in
our understanding of the epidemiology and pathogenesis of listeric infections in farm
animals.




                                              92
CURRENT SITUATION OF ANIMAL LISTERIOSIS IN SLOVENIA
Zdovc Irena, Mehle Janez

Institute of Microbiology and Parasitology, Veterinary Faculty, Gerbiceva 60, Ljubljana,
Slovenia

Problem:Listeriosis in animals is a sporadic bacterial infection most commonly manifested
by encephalitis or meningoencephalitis. It is also important in the field of food hygiene due to
potential human infections. Since BSE was accepted as a causing agent of serious
zoonosis, all ruminants with nervous disorders must regularly be thoroughly examined on all
infections causing similar clinical signs, including rabies and listeriosis.

Method:During the last 12 month period 637 specimens from different species of domestic
animals and 6 wild animals suspected on listeriosis were examined. The majority of samples
was obtained from ruminants, especially from cattle (tissue 35, milk 267), sheep (tissue 34,
milk 83) and goats (tissue 15, milk 133). Specimens of choice were the brain tissue from
animals with CNS involvement, aborted placenta or fetus, milk, and occasionally rectal or
vaginal swabs of clinically ill animals. Specimens from chinchillas, pigs, cats and rabbits
were less frequent. Furthermore, 269 samples of different foodstuffs, mostly milk products,
were examined. All samples were analysed with cultural examination. Isolates were
confirmed by commercial biochemical kits (Api Listeria, BioMerieux) and when necessary
also with genetic method (ACCUPROBE, L. monocytogenes kit, BioMerieux).

Results: L. monocytogenes has been isolated from 20 samples. The majority of positive
cases represent samples from cattle. Among 11 isolates were 5 from brain tissue and the
other 6 from raw milk. All cases of infection were sporadic, with exceptional case when 3
cows from the same stable were found to be infected contemporarily. The rest of positive
samples also derived from domestic ruminants, 3 from goats and 6 from sheep and all of
them were isolated from brain tissue. All other samples were negative to L. monocytogenes.
Among these from one sample of brain tissue of goat L. innocua was isolated.

Conclusion:Listeriosis in animals in Slovenia is not an epidemiological problem but is
important due to serious nature of the disease and possible consequences in cases of
human infections. Furthermore, accurate and sensitive diagnostic procedures is
indispensable in animals with CNS clinical signs to differentiate listeriosis from rabies or
BSE. The prevalence of listeriosis in ruminants, particularly in cattle, in Slovenia is increasing
slightly, probably due to more frequent examinations in cattle herds.




                                               93
NEURAL ROUTE OF CEREBRAL LISTERIA MONOCYTOGENES
MURINE INFECTION
Yuxuan Jin1, Lone Dons2. Krister Kristensson1, and Martin Rottenberg3

Department of Neuroscience1 and Microbiology and Tumor Biology Center,3 Karolinska
Institutet, Nobels vagen 12A, 171 77 Stockholm, Sweden, and Department of Veterinary
Microbiology, The Royal Veterinary and Agricultural University, Stigb0jlen 4, 1870
Frederiksberg C, Denmark2

Problem: The pathways by which Listeria monocytogenes reaches the brainstem to cause
brain stem encephalitis (rhombencephalitis) have not teen clarified. The pathological
features of cerebral L.monocytogenes infection suggest that besides a hematogenous
spread, bacteria might also spread via a neural route. In order to investigate the neural
spread of L.monocytogenes we have in the present study explored the use of genetically
immunodeficient mice as a model for neuroinvasion of L. monocytogenes.

Method: Recombination activating gene 1 (RAG-1)-deficient mice were infected with
L.monocytogenes strains. Kinetics of bacterial loads in snout, trigeminal ganglia (TG) and
brain were determined by counting CFU from organs of mice. Tissue sections of TG, brain
stem and brain of infected mice were analysed by immunostaining.

Results: After snout infection of mice with L.monocytogenes we observed a sequential
appearance of bacteria in the snout, TG, and brain. This observation supports that L.
monocytogenes can spread via the trigeminal nerve. By immunostaining L.monocytogenes
could be observed in the right TG (the injected side), including nerve cell bodies and in the
brain stem, but no bacteria could be found in sections from the rest of the brain. We
examined if the plcB gene of L.monocytogenes (important for spread from cell to cell)
participated in L.monocytogenes neuroinvasion. /?AG-/-deficient mice infected with a pIcB
deletion mutant had a dramatically increased survival time compared with those infected with
the WT strain, suggesting L. monocytogenes propagates mainly from cell to cell, rather via
systemic dissemination. Extended survival in mice infected with the plcB mutant was related
to lower levels of bacteria in the TG and brain but not in the snout compared to the levels in
those infected with the WT strain, indicating that TG axons are not the first cellular targets
infected by L.monocytogenes.

Conclusion: The present study suggest that after inoculation in the snout, L.monocytogenes
invades the central nervous system through cell-to-cell spread in the trigeminal system in
genetically immunodeficient mice.




                                             94
EXPERIMENTAL CONGENITAL LISTERIOSIS: A MURINE MODEL
Maja Abram1, Darinka Vuckovic1, Miljenko Doric1, Branka Wraber2, Herbert Hof3, Dirk
Schlüter3, Martina Deckert4

'Department of Microbiology, Medical Faculty, University of Rijeka, Brace Branchetta 20, HR-
51000 Rijeka, Croatia, 2Institute of Microbiology and Immunology, University of Ljubljana,
Ljubljana, Slovenia, 3Department of Microbiology and Hygiene, University of
Heidelberg/Mannheim, Mannheim, Germany, 4University of Bonn, Department of
Neuropathology, Bonn, Germany
Problem: Listeria monocytogenes may cause severe infections in pregnancy with
detrimental effects on the fetus. Since normal pregnancy is associated with a Th2 cytokine
pattern in order to prevent rejection of the semiallogeneic fetus, an ongoing infection which
elicits a Th1 response may be detrimental for pregnancy. Our work intends to study prenatal
listeriosis of both fetus and the pregnant female.

Method: Balb/c mice were infected intravenously on various days of pregnancy, and
sacrificed 1, 2, 3 and 6 days thereafter. Bacterial clearance was monitored by plating of
tissue homogenates. Immunohistochemistry was applied to topographically analyse the
leukocytic infiltrates and their topographical relation to the bacteria in the utero-placental
tissue. A panel of Th1 and Th2 cytokines was quantified by enzyme-immunoassay in
maternal blood and also analysed in placenta and maternal liver by RT-PCR. Uninfected
pregnant mice and Listeria monocytogenes infected non-pregnant Balb/c mice served as
controls.

Results: Gestating Balb/c mice showed an increased susceptibility to primary L.
monocytogenes infection with a significantly increased rate of abortion. Clearance of bacteria
and duration of listeriosis were prolonged. Histopathology revealed more severe
inflammation in pregnant animals as compared to virgin controls. In the placentas of infected
animals Listeriae were detected in clusters. Both serum and liver of pregnant animals
exhibited markedly lower mRNA and protein level of the proinflammatory cytokines TNF-α
and IFN-γ as compared to L. monocytogenes-infected non-pregnant mice.

Conclusion: The reduced levels of pro-inflammatory, anti-listericidal cytokines may
contribute to the aggravated disease and the poor outcome of congenital listeriosis. On the
other hand, even the reduced production of Th1 cytokines may be incompatible with a
successful pregnancy and enhance abortion.




                                             95
SYSTEMIC AND INTRACEREBRAL INFECTIONS OF MICE WITH
LISTERIA MONOCYTOGENES SUCCESSFULY TREATED WITH
LINEZOLID
M. Callapina, M. Kretschmar, A. Dietz, C. Mosbach, H. Hof, T. Nichterlein

Institute of Medical Microbiology and Hygiene, Mannheim, Faculty of Clinical Medicine,
University of Heidelberg, 68167 Mannheim, Germany

Problem: Linezolid is an oxazolidinone derivative which is active mostly against
grampositive bacteria. In this work its activity against the facultatively intracellular bacterium
Listeria monocytogenes was examined.

Methods: In vitro, in tissue culture and in animal models of systemic and intracerebral
infection the activity of linezolid was determined and compared with ampicillin which is the
antibiotic of choice for treatment of listeriosis.

Results: All strains of L. monocytogenes are susceptible to the substance, with minimal
inhibitory concentrations (MICs) determined by Etest ranging from 0.38 to 1.5 mg/l which is
beyond the preliminary breakpoint of this substance. Linezolid is bacteriostatic against L.
monocytogenes since up to 64 times the MIC does not kill the bacteria in 24 hours. Linezolid
is also bacteriostatic on L.monocytogenes in infected tissue culture cells. In animal models of
systemic and intracerebral infection, linezolid is able to inhibit bacterial growth but is clearly
less effective than ampicillin.

Conclusion: Linezolid might be useful for the treatment of infections with L.monocytogenes
also in humans when ampicillin may not be used.




                                               96
defense, immunology and
       antibiotics




           97
CYTOKINE RELEASE OF L. MONOCYTOGENES-INFECTED
DENTRITIC CELLS
Kolb-Mäurer A., Kemmerer U., Gentschev I., Kämpgen E., Goebel W.CO

Lehrstuhl für Mikrobiologie, Am Hubland/Biozentrum, D 97074 Würzburg and Dermatologische
Universitätsklinik, 97080 Würzburg

DC are professional antigen-presenting cells (APC) which play a crucial role in initiating and
amplifying both the innate and adaptive immune response. They are among the first cells to encounter
pathogens. In the present study, we analysed surface marker expression, mixed leucocytic reaction
(MLR) and cytokine release of L. monocytogenes (L.m.)-infected human DC and compared these data
with listerial Lipoteichoic acid (LTA) stimulated human DC. Phenotypical changes in human DC
induced during L. m. infection results from the recognition of listerial LTA. Incubation of DC with the
L. m. effectively stimulated the release of IL-6, IL-12 p70 and IL-18. Listerial LTA differ in the
ability to trigger cytokine release of DC. In contrast to the slight increase of IL-6 and IL-12 after the
addition of listerial LTA to DC, the production of IL-18 was strongly enhanced. It has been reported
that IL-18 may play an important role in various diseases including cancer and infectious disease.
Combined with IL-12, the both cytokines provide antitumoral effects in murine models. From our data
on the cytokine response, flow cytometry and MLR of human DC to Listeria infection and listerial
LTA treatment, we conclude that L. m. as possible live carrier for DC-based tumor vaccination
strategies and LTA as adjuvans, may lead to a successful immune response against tumor antigens.




                                                   98
EARLY HOST-PATHOGEN INTERACTIONS AND THE INDUCTION
OF T CELLS
M.E.A. Mielke,

Robert Koch-Institut; Nordufer 20, 13353 Berlin

Listeria monocytogenes is an enteroinvasive bacterial pathogen of man and animals with a
pronounced capacity to induce protective and inflammatory T cells.
The infection-induced pathophysiological process which may result in both fatal
sepsis/meningitis or the inapparent induction of sterile immunity can be divided into
- enteroinvasion
- spread via lymphatics and blood
- invasion of target organs (brain, liver and spleen)
- multiplication in permissive host cells
- inducton of (and evasion from) early host defense
- host cell–phagocyte interactions
- priming of specific (CD4+ and CD8+ ) T cells
- expression of T cell effector functions
- survival and death of primed T cells
and finally
- sterile immunity (memory).

Listeriae have been shown capable of infecting the host by translocating from the intestinal
lumen through Peyer´s Patches (PP) but also through PP-independent routes. The latter is
most obvious on occasions in which listeriae were absent from the PP of mice inoculated
intragastrically, but were present in the mesenteric lymph nodes of these same animals. In
addition, i) inoculation of listeriae into the rectums of mice, which lack PP, resulted in the
infection of caudal lymph nodes and ii) germfree SCID mice, which do not develope PP,
suffer from infection with bacteria present in mesenteric lymph nodes, livers and spleens.
The most probable route of PP-independent infection is via direct infection of and spread
from enterocytes, which in fact has been demonstrated in vitro and in vivo.
Infection of mesenteric lymph nodes finally leads to blood stream invasion. In cases in which
the bacteria are not rapidly eliminated from the blood stream by macrophages of the spleen
and liver, listeriae may penetrate into the central nervous system, mainly via the blood
vessels of the plexus chorioideus at the bottom of the 4th ventricle near brain stem and
cerebellum.
In the liver, parenchymal cells may be infected by spread of the bacteria from Kupffer cells or
directly by infection of hepatocytes after the bacteria have passed the fenestrated
endothelium of liver sinuses. The clear anatomy of the liver facilitated most direct
observations of host-parasite interactions at all stages of the infection.
Systemic listeriosis of mice has been used extensively as a model infection for studying
mammalian host defense against intracellular bacterial pathogens. Most effort has been
expended on trying to understand the requirement for specific T cell-mediated immunity for
combatting infection with this pathogen. By contrast, pre-immune defenses have received
much less attention, although these early innate reponses are critically important for both the
survival of severe infection and the induction of the T cell response. If these early defenses
fail to act, the murine host is rendered exquisitely susceptible to L. monocytogenes, and

                                              99
rapidly succumbs to overwhelming infection before T cell-mediated immunity is generated or
expressed. The most critical of these early defenses is mediated by bone marrow derived
CD11b+ cells that rapidly accumulate at foci of Listeria infection in the liver and spleen.
Extensive studies in the murine model led to the conclusion that the most decisive step from
nonspecific microabscess formation to granulomatous inflammation, usually acompanying
successful eradication of the bacteria, is the activation of CD4 + T cells, which together with
bone marrow derived cells, nonspecifically invade infected tissues. Once specific CD4+ T
cells have been activated, the subsequent accumulation of monocytes can no longer been
inhibited by CD11b-specific mAb. The protective capacity of CD8+ T cells, on the other hand,
can most impressively demonstrated in immunized but CD4 + T cell depleted mice which are
protected even against high challenge doses. Most interestingly, the CD8 + T cell mediated
protection can be expressed in the absence of granulomatous inflammation.
As demonstrated by use of isogenic mutants of Listeria monocytogenes, the induction of T
cells depends on virulence factors which determine the capacity for cell-to-cell spread and
intracellular survival. The degree of the T cell response induced correlates with the intensity
and duration of the infection-induced cytokine response.




                                             100
HIERARCHY AND DYNAMICS OF PEPTIDE-SPECIFIC CD4 T
CELL POPULATIONS DURING L. MONOCYTOGENES-
INFECTION
Gernot Geginat

Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim
der Universität Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany

Problem: Ample evidence exists that second to CD8 T cells also CD4 T cells play a role
during systemic infection with L. monocytogenes. In contrast to Listeria monocytogenes-
specific CD8 T cells for which several CD8 T cell epitopes are known and which have been
studied in great detail, the analysis of the CD4 T cell response is hampered by the limited
available information on CD4 T cell epitopes of L. monocytogenes.

Method: We used a novel approach for the direct ex vivo identification and characterization
of CD4 T cell epitopes based on the screening of peptide spot libraries with freshly isolated
splenocytes in a sensitive ELISPOT assay. This technique was applied for the analysis of
splenocytes from L. monocytogenes-infected BALB/c and C57BL/6 mice.

Results: The screening of peptide spot libraries covering the whole LLO and p60 of L.
monocytogenes revealed five new H-2d and five new H-2b-restricted CD4 T cell epitopes.
The enumeration of cells reactive with these CD4 T cell epitopes revealed the existence of
dominant and subdominant CD4 T cell populations during L. monocytogenes infection.
Furthermore, the kinetic analysis of L. monocytogenes-specific T cell populations in infected
mice revealed synchronous expansion and contraction of peptide-specific CD4 and CD8 T
cell populations during the course of infection.

Conclusion: These data show that in vivo the L. monocytogenes-specific T-cell response is
not principally biased towards CD8 T cells.




                                            101
CROSS-TALK BETWEEN INNATE AND ADAPTIVE IMMUNE
SYSTEMS: CD8+T CELL PRIMING AGAINST NON-SECRETED
LISTERIA MONOCYTOGENES ANTIGENS.
A.R. Tvinnereim, S. Hamiliton, J.T. Harty,

University of Iowa, Iowa City IA, U.S.A.

Studies by several groups reveal that CD8+ T cells respond to both secreted and non-
secreted model antigens after infection of mice with recombinant Listeria monocytogenes. In
contrast, only secreted Listeria proteins serve as targets for protective CD8+ T cells,
revealing a dichotomy between CD8+ T cell priming and protective immunity based on
antigen location. Naïve CD8+ T cells are selected to divide and differentiate based
recognition of specific peptide-MHC class I complexes and receipt of co-stimulation from
mature dendritic cells (DC). DC can present antigens after direct infection or through several
exogenous routes including cross-presentation, where antigens are obtained from cells that
succumb to infection. Since presentation of non-secreted antigens requires destruction of the
bacterium, we predicted that cytokine stimulated microbicidal activity would be essential for
detection of this response. In contrast to this notion, IFN- gene knockout mice, that lack the
cytokine deemed most potent in enhancing antilisterial activity, demonstrate similar CD8+ T
cell priming against non-secreted Listeria antigens as wild-type mice. These results
suggested that phagocytic cells such as neutrophils that do not require IFN- for their
microbicidal activity might carry out destruction of Listeria to reveal non-secreted antigens.
Neutrophils, which are critical for early resistance to Listeria but undergo activation induced
apoptosis shortly after bacterial ingestion, would then serve as substrates for cross-
presentation of non-secreted Listeria antigens by DC. In support of this idea, Listeria infected
neutrophils serve as substrates for DC mediated cross-presentation to activate CD8+ T cells
that react with both secreted and non-secreted antigens. Strikingly, specific in vivo depletion
of neutrophils, by low dose treatment with the RB6.8C5 antibody, decreases CD8+ T cell
responses to the non-secreted antigen while having no affect on the response to secreted
Listeria antigens. These results may explain the dichotomy between CD8+ T cell priming and
protective immunity based on antigen location in Listeria but also serve to expand the
interface between elements of innate and adaptive immunity in response to infection.




                                              102
ABUNDANCE OF NATURALLY PROCESSED ANTIGENIC
PEPTIDES IN LISTERIA MONOCYTOGENES INFECTED MICE
Mojca Skoberne1, Herbert Hof2 and Gernot Geginat2

1
  Institut of microbiology and immunology, Medical faculty, University of Ljubljana, Korytkova
2, 1000 Ljubljana, Slovenia. 2 Institut für Medizinische Mikrobiologie und Hygiene, Fakultät
für Klinische Medizin Mannheim der Universität Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167
Mannheim, Deutschland

Problem: Listeria monocytogenes is a facultative intracellular bacterium which resides in the
cytoplasm of the infected host cells. This localization of bacteria results in the MHC class I-
restricted presentation of Listeria-derived antigenic peptides on the surface of infected cells.
So far, data on the abundance of antigenic peptides is mostly based on the results obtained
with in vitro infected cell lines.

Method: We compared the abundance of naturally processed antigenic peptides recognized
by four L. monocytogenes specific CD8 T cell lines in vivo in infected spleens and in vitro in
infected macrophage cell lines. The in vivo antigen presentation was assessed either by
direct biochemical purification of naturally processed antigenic peptides from infected
spleens or by a new ex vivo antigen presentation assay.

Results: Our data show that the relative abundance of naturally processed antigenic
peptides is different in vivo and in vitro. Thus we conclude that the infected macrophage cell
lines do not directly reflect the antigen presentation in infected animals.

Conclusion: In the light of this new finding a number of previous studies which compared in
vitro antigen presentation and in vivo CD8 T cell expansion need to be reevaluated.




                                              103
IN VIVO EFFECTS OF INTERNALIN RELATED PROTEIN A AND
LISTERIOLYSIN ON THE MURINE IMMUNE SYSTEM.
Siegfried Weiss, Susanne zur Lage, Trinad Chakraborty, Ayub Darji

Molecular Immunology, German Research Centre for Biotechnology, Braunschweig,
Germany and Institute for Medical Microbiology, University Giessen, Giessen, Germany


The soluble virulence factor IrpA of Listeria monoctogenes was tested for its role as
protective antigen by first establishing and characterizing a CD8 T cell clone. Although the
epitope that was recognized by these T cells was of extremely low affinity towards MHC
class I, mice which were adotively transferred with cells of this clone were protected against
a challenge with L. monocytogenes. During these experiments we observed that the ratio of
CFU found in spleen and liver were usually around 1 when wild type bacteria were used
while this ratio was around 10 when bacteria containing an in frame deletion of irpA were
tested. After depletion of macrophages spleen:liver ratios were around 1 for both strains.
This suggests that irpA might help the bacteria to avoid macrophages in spleen.
Previously we had shown that listeriolysin inhibits CD4 T cell responses by inducing
antagonistic MHC class II/peptide complexes on infected APC. This was in conflict with the
role of CD4 T cells in granuloma formation after L. monocytogenes infection. We therefore
analysed in more detail the antigen specific CD4 T cell activities induced by listeriolysin
treated APC in vitro and in a model system in vivo. As shown before, the production of IL-2
was completely inhibited. However, production of INF and IL-3 was not influenced at all in
these experiments. Thus, the T cell antagonism that is induced by listeriolysin treated APC is
only partial. Since IL-3 is probably involved in the influx of cells that take part in the formation
of granulomas, these findings reconcile the inhibitory activity shown by us with the well
established function of CD4 T cells in Listeria infection.




                                               104
EXPLOITING LISTERIA MONOCYTOGENES FOR THE DESIGN
OF NOVEL STRATEGIES
Stefan H.E. Kaufmann, Leander Grode, Jürgen Hess, Michael Rolph, Anna Marit Sponaas

Max-Planck-Institute for Infection Biology, Berlin, Germany

Listeria monocytogenes is an intracellular pathogen controlled by T lymphocytes.
Experimental listeriosis of mice is an elegant model that allows the rapid elucidation of the
responses underlying microbial infection controlled by T cells. CD8 T cells are of central
importance although other T cells, in particular CD4 T cells, also contribute to antibacterial
protection. We have used experimental listeriosis of mice to analyze the feasibility of novel
vaccination strategies against intracellular bacteria. Data will be presented demonstrating the
feasibility of gene-gun immunization with naked DNA encoding the dominant antigens
listeriolysin and p60. Moreover, we have successfully used Salmonella typhimurium and
Mycobacterium bovis BCG as recombinant carriers for these antigens. We have also
harnessed molecules of the immune system for the design of novel vaccines. First we
showed that the heat-shock protein gp96 carrying listerial peptides is a promising vaccine
candidate. Evidence has been gathered demonstrating that gp96 binds N-formylated
peptides, and we assume that this complex is responsible for vaccine induced protection
against L. monocytogenes. Finally, we showed that CD40 stimulation allows successful
vaccination against L. monocytogenes using killed listeriae.




                                             105
PATHOGENESIS AND NEUROIMMUNOLOGY OF CEREBRAL
LISTERIOSIS
D. Schlüter1, S. Reiter1, M. Montesinos-Rongen2, S. Lütjen1,2, L.-Y. Kwok1, M. Deckert2

1
 Institut für Med. Mikrobiologie und Hygiene, Universitätsklinikum Mannheim, Universität
Heidelberg, Mannheim, Germany; 2 Abteilung für Neuropathologie, Universität zu Köln, Köln,
Germany

Listeria monocytogenes (LM) may cause meningitis, encephalitis, and brain abscess. To
study the pathogenesis of central nervous system (CNS) listeriosis we have established a
murine model based on the direct application of LM into the basal ganglia. Infection of the
brain by LM leads to the recruitment of leukocytes to the brain. Granulocytes are the
predominant cell type in the inflammatory infiltrates, but they are not able to control CNS
listeriosis and mice die from a progressive meningoencephalitis. The inevitably fatal course
of CNS listeriosis can be prevented by an active systemic immunization prior to CNS
infection. The favourable course of immunized mice is accompanied by a significant
reduction of the intracerebral bacterial load, and both CD4+ and CD8+ T lymphocytes play a
pivotal role in the control of intracerebral LM. However, compared to systemic listeriosis
immunization is less effective in the brain, and only 60% of mice are protected from death by
immunization. The high vulnerability of mice to CNS listeriosis is partially caused by an
impaired immunological control of LM. In fact, studies in IL-10-/- mice revealed that IL-10 was
critical for the down-regulation of an overshooting immune response, but had no significant
influence on the intracerebral bacterial load. In the absence of IL-10 the protective effect of
immunization was abrogated and mice died of a necrotizing brain stem encephalitis with
severe brain edema and multiple intracerebral hemorrhages. Thus, immunosuppressive
activity of IL-10 is strictly required to prevent an overshooting immune response.




                                             106
LISTERIA MONOCYTOGENES: ABSENCE OF SYNERGISM
BETWEEN AMPICILLIN AND GENTAMICIN USING E-TEST AND
CHECKERBOARD MICRODILUTION TECHNIQUES
I. Davis, N. Campbell, M.Currie, W. F.Schlech

Div. of Infectious Diseases, Dept. of Medicine, Dalhousie University Faculty of Medicine,
Halifax, Canada

Introduction: Listeria monocytogenes has previously been reported to demonstrate in vitro
and in vivo synergism with the combination of ampicillin and gentamicin. A new technique
has recently been devised using the Etest (Epsilometer) to quickly evaluate susceptibility of
bacteria to combinations of antibiotics. Previously, microdilution (checkerboard) and time-kill
techniques have been used for this purpose. These techniques, while the “gold standard” are
resource and labor intensive. The E-test technique is both fast and easy to perform.

Method: We evaluated the reliability of the E-test in determining susceptibility of Listeria
monocytogenes to ampicillin and gentamicin as single agents and in combination. Listeria
isolates were obtained from samples stored from the 1981 listeriosis outbreak in Atlantic
Canada as well as from sporadic cases of invasive listeriosis. Etest strips were used for the
agar diffusion testing. Susceptibilities to gentamicin and ampicillin were determined using the
microdilution, and Etest methods for all isolates. Using the highest MIC for each antibiotic,
combination antibiotic testing was performed using the Etest technique and confirmed
using the microdilution technique. The FIC index was calculated from a formula using the
FIC (fractional inhibitory concentration) of each antibiotic. Synergy was defined as an FIC
index less than or equal to 0.5. Indifference was between 0.5 and 4 and an FIC index greater
than 4 was determined as antagonistic.

Results: There was good reproducibility and strong correlation between the microdilution
and Etest techniques for both single antibiotic and synergy testing with a maximum
variation of a 2 fold dilution for any isolate.. Ampicillin and gentamicin were found to be
indifferent for all isolates tested.

Conclusion: The Etest performs well in both single and combination antibiotic testing for
Listeria monocytogenes and appears to be simple, fast and reliable. For our isolates,
ampicillin and gentamicin did not demonstrate synergism as has been found in previous in
vitro experiments. Further testing using other clinical strains, different antibiotic
combinations, and comparisons with time-kill technique or in vivo experiments will need to be
carried out to determine whether this simple method will be clinically useful in making
treatment decisions for patients.




                                             107
ANTIMICROBIAL THERAPY OF LISTERIOSIS
Hof H., Nichterlein T., Lampidis R.

Institute Med. Microbiol. and Hyg., Fac. Clin. Med. Mannheim, University of Heidelberg,
68167 Mannheim

In nature Listeriae are exposed to various substances with antimicrobial activities. So for
example on plants there are several compounds which inhibit the growth of gramposistive
bacteria including pathogenic as well as non-pathogenic Listeriae. (Table 1)

Table 1: Antimicrobial activities of natural compunds against Listeriae

Allyl isothiocyanate from diverse plants (Lin et al., J.Food Protect. 63, 2000, 727)
Herb extracts (Prototeca one and two) (Cutter, J.Food Protect. 63, 2000, 601)
Eucalyptus and other oils (Harkenthal et al., Pharamazie 54, 1999, 460)
Sulfur compounds from cabbage (Kyung and Fleming, J. Food Protect. 60, 1997, 67)
Furocoumarins from parsley (Manderfield et al., J. Food Protect. 60, 1997, 72)
Essential oils from plants (Aureli et al., J. Food Protect. 55, 1992, 344)

For example, in carrots there are compounds which kill Listeriae immediately. These agents
will possibly control the growth of bacteria in vegtables. The thionins and other plant
defensins present on many plants are cystein-rich oligopeptides with a broad range of
antimicrobial activities with marked activities against Listeria spp.. Notably, those substances
are possible candidates as food additives to inhibit an overgrowth of Listeriae.
Also in situations where Listeriae have to compete with other bacteria in mixed populations
there are several bacterial products, i.e. bacteriocins, lantibiotics and bacteriophages which
are able to stop excessive spreading of Listeriae.

After uptake of Listeriae by a mammalian host an array of „endogenous antibiotics“on the
surface of the mucosal barriers such as histatins and lingual antimicrobial protein (LAP) in
the mouth or cryptdin and cecropins in the gut will eliminate some bacteria. After entry and
distribution in the host`s body the invaders will be exposed to humoral factors with
antimicrobial activity like lysozyme, transferrin, complement etc. After having been
phagocytosed they will be confronted with defensins and other antimicrobial oligopeptidic
antibiotics in the granules or histones, ubiquicidin, calprotectin and other antimicrobial
peptides in the cytosol of a host cell.

Hence, at the first glance it seems that an antibiotic treatment is in most cases not indicated.
If so, however, when these first line defense mechanisms are overrun, there are a lot of
different antibiotics and chemotherapeutic agents available.

In principle most of the common antibiotics are active against Listeriae. It has to be kept in
mind, however, that in most instances the action is only bacteriostatic. Among Listeriae
especially among L. monocytogenes there is a strong homogeneity. This natural resistance
pattern is slightly different in the various Listeria spp..



                                              108
Nalidixic acid, fosfomycin and cephalosporins of the second and third generation are those
antibiotics which are reported to be incactive, although this does not holds true in any case.
Among several hundreds of isolates of Listeria spp.of the SLCC (Seeliger`s Special Culture
Collection) six strains at least were found to be susceptible to cephalosporins and even to
aztreonam and mecillinam in an agar dilution method (Table 2 )

Table 2: Susceptibility of some exceptional strains of L. monocytogenes to cephalosporins
and few other ß-lactams (representative examples of intrinsicly resistant and susceptible
isolates)


                           (MIC values: mg/l)

            SLCC 5835 (EGD) SLCC 7140 SLCC 4949 SLCC 6793
Ampicillin      0.5             2          0.5       0.125
Cephalothin     4               4          0.25      0.5
Cefoxitin      32              64          2         1
Cefuroxime    128             128          0.5       1
Cefotaxime     64             128          0.25      0.25
Cefepime       64             128          8         4
Cefpirome      32              64          1         2
Mecillinam     64             128          8         2
Aztreonam >2048             >2048          4        32


This phenomenon is hard to explain, because the penicillin binding proteins (PBP) 3 and 5 of
these outstanding strains are not altered; these essential PBP could not bind radioactively
labeled cefotaxime as in normal Listeriae.

Listeriae in general are resistant to nalidixic acid, so that this substance can be added to
selective media. Obviously, few hydrophilic aminoacids in the hot spot of the gyrase A,
where quinolones tend to bind, in Staphylococcus aureus as well as in Escherichia coli are
exchanged in Listeriae by hydrophobic ones,. Thus, it can be anticipated that the substrate
does not fit any more in Listeriae resulting in resistance. Some newer quinolones, such as
moxifloxacin, are, however, rather active against Listeriae. (Table 3)

There are also some other agents under development with promissing effects (Table 3).

Table 3: New, promissing drugs
quinolones:         moxifloxacin (better than ciprofloxacin)
ketolides:          telithromycin (in vitro activity equal to that of macrolides but better
                    pharmacological properties)
glycylcyclines:     GAR-936 (not better than tetracyclines)
oxazolidinones:     linezolid (good in vitro activity; moderate therapeutic power)

There are more and more reports about single isolates from food which differ from the
general susceptibility pattern. There are strains with single as well as with multi-resistance .
Indeed resistance properties can be transferred by plasmids (for example vancomycin,
tetracycline and macrolide) and so it is conceivable that there is a major concern that
resistance may spread.
                                              109
On the other hand in human isolates no antibiotic resistance has been noted until now.
Consequently, therapeutic failure is not due to antibiotic ressistance of the pathogen but to
clinical reasons. In general, the recommendation is still valid that amoxillin is the drug of
primary choice for the treatment of listeriosis. Combination with aminoglycosides increases
the bactericidal activity, since there is a definite synergism. Indeed more than 30% mortality
in spite of a rational drug regimen. One reason for this limited therapeutic power is the
intracellular residence of L.monocytogenes within host cells, where they are inaccessible for
most of the in vitro active agents. And even if an agent is pricipally able to be accumulated
hundredfold in most host cells, such as macrolides, in particular host cells they may be
lacking, because some host cells possess pumps which can export antibiotics, so that
intracellular bacteria like L. monocytogenes are protected.




                                             110
MOLECULAR CHARACTERIZATION OF THE INTRINSIC
RESISTANCE OF LISTERIA AGAINST NALIDIXIC ACID
LAMPIDIS ROBERT; HOF HERBERT

Institut für Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Mannheim,
Theodor-Kutzer-Ufer, 68167 Mannheim, Germany, Phone +49 621 3832708, Fax +49 621
3833816, E-mail robert.lampidis@imh.ma.uni-heidelberg

Problem: Although Listeria monocytogenes is susceptible to many common antibiotics, it is
intrinsically resistant against the quinolone nalidixic acid and shows a decreased
susceptibility against therapeutically important fluoroquinolones like ciprofloxacin. The aim of
this study was to investigate, whether type II topoisomerases were responsible for the
quinolone-resistant phenotype of L. monocytogenes.

Methods: Quinolones act on the type II topoisomerases DNA gyrase and DNA
topoisomerase IV, enzymes that control DNA topology. The genes encoding the subunits A
and B of DNA gyrase and the subunits C and E of topoisomerase IV of L. monocytogenes,
gyrA, gyrB, parC and parE, respectively, were cloned and sequenced. In order to further
analyze the actual role of the gyrase genes, a plasmid encoded gyrA allele was mutagenized
(Thr84-Ser and Phe88-Glu) and transformed into L. monocytogenes. These heterodiploid
strains were tested for their susceptibility patterns against nalidixic acid and
fluoroquinolones.

Results: Compared to the sequences of quinolone-susceptible bacteria, the quinolone
resistance-determining region (QRDR) of DNA gyrase subunit A was altered: the deduced
amino acid sequence revealed the exchanges Ser84-Thr and Asp/Glu88-Phe, two amino
acid variations at hot spots, commonly associated with resistance against quinolones. No
relevant divergences from QRDR consensus sequences were observed in GyrB or both
topoisomerase IV subunits. Thus, it could be argued that the amino acid substitutions in
GyrA would explain the intrinsic resistance of L. monocytogenes against nalidixic acid.
However, heterodiploid strains for gyrA, carrying the wildtype gene on the chromosome and
a putatively sensitive allele on a plasmid, showed no significant differences in their
susceptibility patterns against nalidixic acid or fluoroquinolones, although sensitive alleles
are supposed to be dominant over resistant alleles.

Conlusion: The results indicate that: a) the revealed alterations of the amino acid sequence
in the QRDR are not exclusively responsible for the nalidixic acid-resistant phenotype of L.
monocytogenes, b) sensitive, plasmid-borne alleles of gyrA are not dominant over
chromosomal alleles in L. monocytogenes, and c) other mutations or factors like efflux
pumps dominate over the presumed DNA gyrase mutations.




                                              111
detection, food and food
      technology




           112
CONVENTIONAL ISOLATION METHODS FOR LISTERIA
MONOCYTOGENES.
Catherine W. Donnelly, Ph.D.,

University of Vermont, Burlington, VT 05401

        A variety of conventional methods are available to detect L. monocytogenes in food
and clinical samples. However, detection of L. monocytogenes is often limited by the
performance of the enrichment media used to support bacterial growth to detectable levels.
Listeria may exist at extremely low levels in foods, and it is therefore critical that sample
enrichment protocols amplify these low initial populations to detectable limits. Further,
Listeria may exist in an injured state in food products and processing environments as a
result of processing treatments such as heating, freezing, exposure to acids or exposure to
sanitizing compounds. The presence of selective agents in enrichment media normally used
for recovery of Listeria may inhibit repair and ultimately detection of sublethally injured
Listeria, which under ideal conditions, may go on to repair, grow and regain pathogenicity.
Simple modifications to existing regulatory protocols, such as those that utilize more than
one enrichment broth, raise sensitivity of detection to 90%. Summary data will be provided to
show the efficacy of repair/enrichment strategies, which increase sensitivity of detection to
97.5-98.8% compared to standard regulatory protocols that have only 65-70% sensitivity.
Ribotype analysis of isolates obtained from meat samples reveals a complex microbial
ecology, and striking differences in both number and distribution of distinct genetic types of
Listeria is revealed depending upon whether samples are enriched in selective versus
repair/enrichment media. Continuing work on enrichment of dairy environmental samples in
UVM and LRB has shown that combining these two primary enrichment media into a single
tube of Fraser broth for dual secondary enrichment yields a significantly higher (p<.05)
percentage of Listeria-positive samples than when either LRB or UVM are used alone.
Refinement of conventional Listeria recovery methods should consider the importance of the
enrichment step in microbial recovery, the nutritional needs of specific genetic types, along
with the physiological condition of Listeria isolates in foods.


1. Introduction

        Major disease outbreaks and numerous sporadic cases of listeriosis occurring
worldwide have implicated Listeria monocytogenes as a major foodborne pathogen. The
most recent of these outbreaks in the U.S. involved the consumption of hot dogs, with
approximately 101 illnesses reported to the Centers for Disease Control by 14 states,
resulting in a total of 16 deaths. Manufacturers of hot dogs and luncheon meats collectively
recalled greater than 500,000 pounds of product due to possible Listeria contamination
(Mead, 1999). Mead (personal communication) reported that in four hot dog samples
cultured quantitatively from this outbreak, L. monocytogenes serotype 4b was present in
quantities less than 0.3 colony forming units per gram. This highlights the need for increased
focus on improving sensitivity of detection of Listeria in foods. It is crucial that the sample
enrichment protocols increase target cell levels to detectable limits and take into
consideration that the target cells may be present at low levels, or may exist in an injured or
sub-lethally damaged state in a food product. Processing stresses such as the addition of

                                              113
sodium nitrite (NaNO2), NaCl, lactic acid, heating, freezing, and contact with sanitizers
reduce levels of Listeria and may cause sublethal injury. Within the food processing industry,
there is a need for isolation methods with increased ability to detect both low level and
injured Listeria populations. Many procedures rely on the use of highly selective enrichment
procedures, which enable preferential growth of Listeria over non-Listeria contaminants
present in foods.

A. Selective procedures

        Most selective enrichment and isolation procedures developed to date for detection of
Listeria take advantage of the resistance of this organism to selective compounds which
suppress growth of background contaminants. Selective agents commonly used in selective
enrichment/plating media include acriflavine, naladixic acid, lithium chloride, moxalactam and
phenylethanol. Detection of L. monocytogenes in food products or food processing
environments is accomplished by use of a variety of standard or rapid microbiological
procedures. Among the most widely used are protocols devised by the United States
Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) for the detection of
Listeria in meats (McClain and Lee, 1989; Cook, 1999) and the Food and Drug
Administration (FDA) for the detection of Listeria in dairy products (Lovett and Hitchins,
1989; Hitchins, 1995).

        The FDA procedure initially involves enrichment of 25 g or 25 ml of sample into 225
ml of Enrichment Broth, M52 (Hitchins, 1995) which consists of a trypticase soy broth/yeast
extract base supplemented with: monopotassium phosphate (anhydrous), 1.35 g/l; disodium
phosphate (anhydrous) 9.6 g/l; acriflavin HCl 10 mg/l; nalidixic acid sodium salt 40 mg/l;
cycloheximide 50 mg/l; and pyruvic acid (sodium salt 10% w/v aqueous soultion) 11.1 ml.
Samples are enriched without selective agents for 4 hrs at 30 0C. Following addition of
selective agents (acriflavin, nalidixic acid and cycloheximide), samples are incubated for an
additional 44h for a total incubation period of 48h at 30 oC. This procedure is a modification of
the original procedure which called for sample enrichment for 1 and 7 days at 30 0C (Lovett
and Hitchins, 1989). The original broth was modified by increasing its buffering capacity,
thereby positioning this media to be used successfully in conjunction with DNA probe and
other methods that are more sensitive than conventional cultural procedures. After 24 and 48
h, EB cultures are streaked onto OXA (Curtis et al. 1989) and Listeria plating medium (LPM)
agar (Lee and McClain, 1986) or LPM plus esculin/Fe 3+agars, both of which have replaced
the originally recommended modified McBride agar (MMA) as the selective isolation media.
PALCAM agar ( Netten et al., 1989) may be used in place of LPM agar. This substitution
brings the method into closer alliance with methodology used outside the U.S. and
decreases reliance on the Henry technique. OXA and PALCAM plates are incubated (with
optional use of a CO2-air atmosphere) at 350C for 24-48 h, with LPM plates incubated at
30oC for 24-48 h. LPM plates can be viewed using Henry illumination or alternatively, esculin
and ferric iron salt may be added to LPM to eliminate the need for Henry illumination. With
OXA and PALCAM, Listeria colonies develop a black halo. It is recommended that 5 or more
typical colonies be picked from OXA and PALCAM or LPM and transferred to TSAYE for
confirmation of purity and typical isolated colonies. The selection of five colonies insures that
multiple species of Listeria, if present, will be identified. TSAYE plates are incubated at 30 oC
for 24-48 h (35oC incubation may be utilized if colonies are not being used for wet mount
motility confirmation). Purified isolated are subjected to a series of standard biochemical
tests, with a total of 10-11 days being required to isolate and confirm the presence of Listeria


                                              114
in food samples via the FDA procedure. This procedure was specifically developed to
optimize Listeria detection in milk and dairy products.

        The USDA-FSIS selective enrichment protocol for isolation of Listeria from raw meat
and poultry was developed by McClain and Lee(1989). The USDA-FSIS procedure for
isolation of Listeria, while originally designed to detect the presence of Listeria in meats, has
been subsequently used with success for isolation of Listeria from dairy products and
environmental samples. The USDA scheme differs from the FDA procedure primarily in the
selective enrichment and plating media used, along with the size of the sample tested. The
revised USDA procedure (Cook, 1999) differs from the original method in that (a) LEB II has
been replaced by Fraser Broth (Fraser and Sperber, 1988) as the secondary enrichment
medium; (b) LPM agar has been replaced by Modified Oxford Agar (MOX); and the
regulatory sample size has been increased to 25g. Fraser broth and Modified Oxford Agar
will both blacken during incubation due to the ability of Listeria spp. and other contaminants
to hydrolyze esculin, with colonies of Listeria exhibiting black halos on Modified Oxford Agar
following 24-48 hrs of incubation. MOX is also more selective than LPM or Oxford agar, with
staphylococci and streptococci both generally unable to grow on MOX.

        Inadequacies in the original (Carnevale and Johnston 1989) FSIS procedure related
to the use of Fraser broth as the secondary enrichment broth have been reported. False
negative results due to reliance on Fraser broth darkening and a 24 hour secondary
enrichment have been reported by a number of laboratories. Kornacki et al. (1993)
compared recovery of L. monocytogenes from Fraser broth incubated for 26 versus 48h. L.
monocytogenes was isolated from 60 of 1088 meat product and environmental swabs from
meat and dairy plants. A false negative rate of up to 6.7% was recorded, attributed to the
failure of L. monocytogenes to be detected in Fraser broth at 26h, but not at 48h, and by the
failure of Fraser broth to blacken. Further, when primary enrichments were streaked directly
to selective enrichment media, failure to recover L. monocytogenes in eight samples was
recorded. As a result of this study, a 48 h incubation of Fraser broth was recommended.
Thus, all Fraser broth enrichment cultures should be streaked regardless of color following
24-26 h incubation. Once cultures have been streaked to MOX, Fraser broth cultures should
be reincubated at 35oC for an additional 24 h enrichment period. MOX plates streaked from
24-26 hour Fraser broth enrichment cultures should be examined for presence of colonies
displaying typical Listeria morphology. If present, isolation should proceed. If absent, a
second MOX plate should be streaked from the 48 h enrichment culture.

        A detection method widely used in Europe is the Netherlands Government Food
Inspection Service (NGFIS) method, developed by Netten et al. (1989). These authors have
reported increased sensitivity with this method over that of the USDA procedure when used
to examine foods containing L. monocytogenes at levels of less than 10 CFU/g. Food
samples are enriched in L-PALCAMY enrichment broth for 48h at 30 0C. After 24 and 48 h,
0.1 ml of L-PALCAMY enrichment broth is plated onto PALCAM agar. Plates are incubated
at 300C for 48 h under microaerophilic conditions (5% oxygen, 7.5% carbon dioxide, 7.5%
hydrogen and 80% nitrogen).
Numerous studies have been conducted to compare efficacy of these and other widely used
detection/isolation protocols. A comparison of enrichment methods was made by Hayes et
al. (1992) when examining foods obtained from the refrigerators of patients with active
clinical cases of listeriosis. Two thousand two hundred twenty-nine (2229) foods were
examined in the study and 11% were positive for L. monocytogenes. A comparative
evaluation of three microbiological procedures was conducted on 899 of the examined foods.
                                              115
The USDA-FSIS (Carnevale and Johnston, 1989), FDA (Lovett and Hitchins, 1989) and
Netherlands Government Food Inspection Service (NGFIS) (Netten et al. 1989) methods
were not statistically different in their ability to isolate Listeria from the 899 samples. The
FDA procedure detected L. monocytogenes in 65% of foods shown to be positive, while the
USDA-FSIS and NGFIS procedures detected L. monocytogenes in 74% of foods shown to
be positive. Thus, none of these widely used conventional methods proved to be highly
sensitive when used independently for analysis of Listeria contamination in foods. It was
noted, however, that use of a combination of any two methods improved detectability from
65-74% (for individual protocols) to 87-91% for combined protocols. Other protocols used
for isolation of Listeria from foods include methods developed by the International Dairy
Federation (IDF), ISO/DIS, Australian Standard Methods and Nordic Committee on Food
Analysis. The future challenge will be to harmonize these methods so that performance of
methods is comparable.

       Enumeration procedures developed by the USDA-FSIS (Cook, 1999) include a choice
of two methods: a Most Probable Number (MPN) technique and a direct plating method. The
MPN procedure is described as the most sensitive, detecting 100 cfu/g or less of L.
monocytogenes. This procedure utilizes UVM broth in a 9 tube series. The direct plating
method utilizes UVM broth as the diluent for homogenization of the sample, followed by
direct plating onto Modified Oxford Medium (MOX). This method is described as being
considerably less sensitive than the MPN method, possibly underestimating the actual
number of cells present, particularly with respect to injured cells. Therefore, the direct plating
method would be utilized for samples anticipated to contain high levels of L. monocytogenes
(Cook, 1999). Despite advances in conventional Listeria detection and enumeration
methodology, all procedures developed to date lack sensitivity and reliability and often
positive samples escape detection. Therefore, these methods underestimate the true
incidence of Listeria in foods and therefore cannot be reliably used to assess risk. Until these
methods are further refined, it will be difficult to implement a risk-based compliance system
upon which we can have full confidence.


B. Detection of sublethally-injured Listeria:

        Most conventional and rapid methods for detection of Listeria in food product and
environmental samples use highly selective enrichment media to facilitate growth over
competitive background flora. These highly selective enrichment procedures do not account
for recovery of sublethally injured Listeria which could exist within a variety of heated, frozen
and acidified foods; or heated, frozen and sanitized areas within food processing
environments. It is well recognized that Listeria can be injured as a result of exposure to a
variety of processing treatments which include sublethal heating and freezing, drying,
irradiation, or exposure to chemicals (sanitizers, preservatives, acids) (Donnelly, 2000).
Under ideal conditions in food systems, injury is reversible and injured Listeria can repair
sublethal damage. Repair of heat-injured L. monocytogenes has been shown to take place in
whole and 2%
milk stored at 40C. (Meyer and Donnelly, 1992).

Several investigators have attempted to improve sensitivity of current detection systems by
recognizing that Listeria may exist in an injured state in food products and food processing
environments. All current detection procedures, with the exception of cold enrichment ,
involve selective enrichment and/or selective plating. Cold enrichment is not feasible for
                                               116
routine sample analysis as several months may be necessary to record positive results. By
failing to consider recovery of injured Listeria, current methodologies underestimate the true
incidence of Listeria. Several previous studies have reported on the ability of commonly used
plating media to recover injured Listeria. Among the compounds examined, phenylethanol,
acriflavin, polymixin-acriflavin and sodium chloride were found to inhibit recovery of thermally
stressed and non-stressed Listeria (Donnelly, 2000). It should be noted that these
compounds comprise media which are routinely used for detection of Listeria. When
examined for ability to quantitatively recover thermally stressed Listeria on solid media,
Listeria enrichment broth (LEB) agar, modified McBride's agar (MMA), lithium chloride
phenylethanol moxalactam (LPM) agar and FDA enrichment broth agar showed significantly
impaired abilities to recover injured cells.

Busch and Donnelly (1992) developed an enrichment medium capable of resuscitating heat-
injured Listeria. This medium, Listeria Repair Broth (LRB), permits complete repair of injured
Listeria within 5 hrs at 370C. LRB is composed of the following constituents (per liter of
distilled water): trypticase soy broth, 30.0 g; glucose, 5.0 g; yeast extract, 6.0 g; magnesium
sulfate, 4.94 g; ferrous sulfate, 0.3 g; pyruvic acid (sodium salt), 10.0 g; MOPS-free acid, 8.5
g; and MOPS-sodium salt, 13.7 g. In studies which compared the efficacy of LRB in
promoting repair/enrichment of heat-injured Listeria with that of existing selective enrichment
media, repair was not observed in FDA enrichment broth (Lovett and Hitchins, 1989),
phosphate-buffered Listeria Enrichment Broth (PEB; Gene-Trak Systems, Framingham, MA),
or UVM Enrichment Broth (McClain and Lee, 1989). Final Listeria populations in selective
enrichment media following a 24 h incubation were 1.7 x 10 8 to 9.1 x 108 CFU/ml, compared
with populations in LRB which consistently averaged 2.5 x 10 11 to 8.2 x 1011 CFU/ml. Ryser
et al. (1996) evaluated the ability of UVM and LRB to recover different strain-specific
ribotypes of L. monocytogenes from meat and poultry products. Forty-five paired 25g retail
samples of ground beef, pork sausage, ground turkey and chicken underwent primary
enrichment in UVM and LRB (30oC/24h) followed by secondary enrichment in Fraser broth
(35oC/24h) and plating on modified Oxford agar. A three hour non-selective enrichment
period at 30C was used with LRB (with tested food) to enable repair of injured Listeria prior
to addition of selective agents. Of 180 meat and poultry products tested, LRB identified
73.8% (133/180) and UVM 69.4% (124/180). Although there was not a statistically significant
difference in these results, combining UVM and LRB results increased overall Listeria
recovery rates to 83.3%. These results demonstrate that use of LRB for repair/enrichment of
samples in conjunction with the USDA/FSIS method has the potential to improve recovery of
Listeria from meat and poultry products. In this study, Listeria isolates were genetically
discriminated using the automated RiboprinterTM Microbial Characterization System. A total
of 36 different Listeria strains comprising 16 L. monocytogenes (including four known clinical
ribotypes), 12 L. innocua and 8 L. welshimeri ribotypes were identified from selected positive
samples (15 samples of each product type, 2 UVM and 2 LRB isolates per sample). Twenty-
six of 36 (13 L. monocytogenes) ribotypes were detected using both UVM and LRB; whereas
3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were
observed using only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey
and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes) 20 (9 L.
monocytogenes) and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively,
with some Listeria ribotypes confined to a particular product. More importantly, striking
differences in both the number and distribution of Listeria ribotypes, including previously
recognized clinical and non-clinical ribotypes of L. monocytogenes, were observed when 10
UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third
set of six samples per product type was examined from which two Listeria isolates were
                                              117
obtained using only one of the two primary enrichment media, UVM and LRB failed to
identify L. monocytogenes (both clinical and non-clinical ribotypes) in 2 and 4 samples,
respectively. These findings illustrate the complex microbial ecology of Listeria in foods and
the limitations of existing detection procedures to fully represent the total distribution of
Listeria isolates in foods. Furthermore, two of the L. monocytogenes ribotypes missed using
UVM were known clinical isolates of serotype 1/2a, both of which were responsible for
sporadic and epidemic cases of human listeriosis in England and Scotland (McLauchlin et al.
1986). These findings, combined with reports of L. innocua being able to outgrow L.
monocytogenes in UVM and Fraser Broth (Curiale and Lewus, 1994; Petran and Swanson
1993) suggest that different genetic types of L. monocytogenes may vary somewhat in their
nutritional requirements or in abilities to compete with other genetic types of Listeria.
Refinement of existing systems should consider the nutritional needs associated with
specific genetic types (unique riboprints) of Listeria strains distributed in foods.

        Roth and Donnelly (1995) examined growth of healthy and heat-injured L.
monocytogenes in five different enrichment broths, two of which were non-selective and
three which were selective. Selective media included Fraser broth (Fraser and Sperber 1988
), Listeria Repair Broth Selective (LRBS), and University of Vermont Modified Listeria
Enrichment Broth (UVM) (Difco Laboratories, Detroit, MI). Non-selective media included
Listeria    Repair  Broth    (LRB)     and    Trypticase   Soy     Broth   (TSB)   (Difco).




                                             118
The Gompertz equation is used for analysis of non-linear regression models. It was used in
this study to analyze data collected from both healthy and heat-injured Listeria populations.
For healthy cell populations, the Gompertz equation generated growth parameters, and
these parameters were then used to examine the following properties: exponential growth
rates, lag phase duration, generation time and maximum population density. For the heat-
injured cell populations, the Gompertz equation was used to analyze the repair time of each
broth as well as the time point where the counts on the selective media (TPAN) and the
nonselective media were equal. Fisher’s Protected LSD (least square difference) at the 95%
confidence level was used to statistically analyze the information obtained from the
Gompertz equation on both healthy and heat-injured cells. Lag phase duration was
measured in hours. LRB, LRBS, TSB and UVM all performed roughly within the same time
range, with a LPD of approximately 3-6 hours with no statistical difference among these four
broths. The LPD of Fraser broth was over 14.5 hours, which when compared statistically at
p<0.05 was slower than the other media tested. Fraser broth is formulated from UVM, with
the addition of 3.0 gm/liter of lithium chloride and additional acriflavin (0.125%) which is
added to inhibit the growth of enterococci, gram positive and gram negative bacteria. It
would appear that although the level of selective agents used in Fraser broth are not
inhibitory towards L. monocytogenes, the levels are sufficient enough to cause an extended
lag period. Increased lag period with a medium containing selective agents has been
documented by other investigators (McLauchlin and Taylor, 1986). Beumer and Brinkman
(1989) reported that Listeria growth could be delayed due to when acriflavine levels
approach 15 mg/l in media used for Listeria enrichment. The lag period in bacterial growth is
a phase of adjustment and this would indicate that the selective agents in Fraser broth are
affecting the growth of healthy Listeria. LRB was found to have the fastest exponential
growth rate (39 min), the fastest generation time (28 min), the highest maximum population
density (over 1010 cfu/ml). With heat injured cells, Fraser broth had the statistically longest
repair time of over 19 hours, versus UVM which had a repair time of slightly over five hours.

        Roth and Donnelly (1996) assessed survival of acid-injured Listeria in acidic food
products such as apple cider, yogurt, fresh coleslaw and salsa . Temperature was found to
greatly dictate survival of Listeria in salsa (Figure 3). When stored at 30oC, populations
declined rapidly within 3 days. At 4oC, populations persisted for up to 17 days of storage. In
further studies, the efficacy of two different enrichment media for recovery of acid-injured
Listeria from acidic foods was assessed using Listeria Repair Broth and University of
Vermont (UVM) medium. At time points where differences were detected, LRB detected the
organism in 22 of 54 samples, compared with UVM which detected only 3 of 54
contaminated samples.

        Detection of injured Listeria has applications to testing of seafood products. Listeria is
found in 8.7% of all domestic/imported ready-to-eat fish and seafood in U.S. The first
documented case of listeriosis positively linked to consumption of fish was reported in Italy in
1989. The organism has been recovered from cooked crabmeat and shrimp/smoked salmon,
and a 1991/92 outbreak in Australia/NZ was linked to smoked mussels. For cold smoked
fish, routine use of a number of inhibitors including 3-6% NaCl in combination with vacuum
packaging, NaNO2 (125-200 ppm), and sodium lactate (2%) may result in presence of
injured organisms (Peterson et al. 1993, Pelroy et al. 1994). Similarly, the marinade for
greenshell mussels consists of acetic acid (1.5% wt/vol); acetic and lactic acids (.75% each)
and acetic acid (1.5%) plus glucono-delta lactone (Bremer and Osborne 1995) Poysky et al.
(1997) examined effects of liquid smoke/heat on hot smoked salmon. Product was stored at
5C for 4 days, then pre-enriched in Trypticase soy broth for 6 hrs at 30C before adding
selective agents to UVM. Of 245 samples tested, 57 negative for L. monocytogenes on day 1
were positive 6-9 days after incubation.
       Baylis et al. (2000) compared use of the Oxoid Ltd. S.P.R.I.N.T. Salmonella system
against the ISO 6579:1993, Qualicon BAX PCR, bioMerieux VIDAS and Tecra Unique
methods. The S.P.R.I.N.T. system was developed for the rapid detection of low levels of
injured Salmonella in foods. This system utilizes an enrichment broth that contains a
specifically developed peptone that allows consistent and rapid recovery of injured
Salmonella, coupled with a Recovery Supplement which contains an Oxyrase® Enzyme
System that assists recovery through reduction in oxidative stress of the medium. After 5hr
of incubation, selective agents are added to the medium. When tested with ice cream and
skimmed milk powder containing low levels of heat-injured S. typhimurium, the S.P.R.I.N.T.
method was superior (61% confirmed positive samples) to the ISO (37% positive); BAX
(36% positive); VIDAS (30% positive) and Tecra (25% positive) methods. Similar
improvements have been advanced by Donnelly and colleagues (Pritchard and Donnelly,
1999) for recovery of injured Listeria in dairy products, where continuing work on enrichment
of dairy environmental samples in UVM and LRB has shown that combining these two
primary enrichment media into a single tube of Fraser broth for secondary enrichment yields
a significantly higher (p<.05) percentage of Listeria-positive samples than when either LRB
or UVM are used alone.

SUMMARY:

       Despite reductions in disease incidence due to Listeria monocytogenes, this organism
remains the leading cause of death due to a foodborne pathogen (207). Recent multistate
outbreaks of illness and death highlight the need for renewed collaboration among industry,
university and governmental agencies to control this dangerous but interesting foodborne
pathogen. Improvements in testing methods are also needed to insure adequate sensitivity
of detection of regulatory procedures used to identify and ultimately control Listeria. (include
juxtaposition statement)

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Baylis, C.L., S. MacPhee, A.J. Turner, K.M. Lilley and R.P. Betts. 2000. Evaluation of Oxoid
S.P.R.I.N.T. SALMONELLA in ice cream and milk powder. AOAC Annual Meeting Final
Program Sept. 10-14, 2000. F-1201, p. 84.

Beumer, R.R. and E. Brinkman. 1989. Detection of Listeria spp. with monoclonal antibody-
based enzyme-linked immunosorbent assay (ELISA). Food Microbiol. 6:171-177.

Bremer, P.J. and C.M. Osborne. 1995. Efficacy of marinades against Listeria
monocytogenes cells in suspension or associated with green shell mussels (Perna
canalicus). Appl. Environ. Microbiol. 61:1514-1519.

Busch, S.V. and C.W. Donnelly. 1992. Development of a repair-enrichment broth for
resuscitation of heat-injured Listeria monocytogenes and Listeria innocua. Appl. Environ.
Microbiol. 58:14-20.


Carnevale, R.A. and R.W. Johnston. 1989. Method for the isolation and identification of
Listeria monocytogenes from meat and poultry products. United States Department of
Agriculture Food Safety and Inspection Service, Laboratory Communication No. 57, Revised
May 24. USDA-FSIS, Washington, D.C.
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Cook, L.V. 1999. Isolation and identification of Listeria monocytogenes from red meat,
poultry, egg and environmental samples (revision 2); Chapter 8 in USDA/FSIS Microbiology
Laboratory Guidebook 3rd Edition.

Curiale, M.S. and C. Lewus. 1994. Detection of Listeria monocytogenes in samples
containing Listeria innocua. J. Food Prot. 57:1048-1051.

Curtis, G.D.W., R.G. Mitchell, A.F. King and E.J. Griffen. 1989. A selective differential
medium for the isolation of Listeria monocytogenes. Lett. Appl. Microbiol. 8:95-98.

Donnelly, C.W. 2001. Listeria monocytogenes, Chapter 10, pp. 213-246 in Y.H. Hui, M.D.
Pierson and J.R. Gorham, eds. Foodborne Disease Handbook. Marcel Dekker, Inc. N.Y.

Fraser, J.A. and W.H. Sperber. 1988. Rapid detection of Listeria spp. in food and
environmental samples by esculin hydrolysis. J. Food Prot. 51:762-765.

Hayes, P.S., L.M. Graves, B. Swaminathan, G.W. Ajello, G.B. Malcolm, R.E. Weaver, R.
Ransom, K. Deaver, B.D. Plikaytis, A. Schuchat, J.D. Wenger, R.W. Pinner, C.V. Broome
and the Listeria study group. 1992. Comparison of three selective enrichment methods for
the isolation of Listeria monocytogenes from naturally contaminated foods. J. Food Prot.
55:952-959.

Hitchins, A.D. 1995. Listeria monocytogenes, Chapter 10, pp. 10.01-10.13 In Food and Drug
Administration Bacteriological Analytical Manual, 8 th Ed., AOAC International, Gaithersburg,
MD.

Kornacki, J.L., D.J. Evanson, W. Reid, K. Rowe and R.S. Flowers. 1993. Evaluation of the
USDA protocol for detection of Listeria monocytogenes. J. Food Prot. 56:441-443.

Lee, W.H. and D.W. McClain. 1986. Improved Listeria monocytogenes selective agar. Appl.
Environ. Microbiol. 52:1215-1217.

Lovett, J. and A.D. Hitchins. 1989. Listeria isolation. Bacteriological Analytical Manual, 6th ed.
Supplement, Sept. 1987 (Second Printing 1989):29.01. AOAC, Arlington, VA.

McClain, D. and W.H. Lee. 1989. FSIS method for the isolation and identification of Listeria
monocytogenes from processed meat and poultry products. Lab Comm. No. 57, Revised
May 24, 1989. U.S. Dept. Agric., FSIS Microbiology Division, Beltsville, MD.

McLauchlin, J.A., A. Audurier and A.G. Taylor. 1986. Aspects of the epidemiology of human
Listeria monocytogenes infections in Britain 1967-1984; the use of serotyping and phage
typing. J. Med. Microbiol. 22:367-377.

Mead, 1999. Multistate outbreak of listeriosis traced to processed meats, August 1998-
March 1999. Written communication, May 27, pp. 1-11.

Meyer, D.H. and C.W. Donnelly. 1992. Effect of incubation temperature on repair of heat-
injured Listeria in milk. J. Food Prot. 55:579-582.


                                               121
Netten, P. Van, I. Perales, A. Van de Moosdijk, G.D.W. Curtis and D.A.A. Mossel. 1989.
Liquid and solid selective differential media for the detection and enumeration of L.
monocytogenes and other Listeria spp. Int. J. Food Microbiol. 8:299-316.

Pelroy, G, M. Peterson, R. Paranjpye, J. Almond and M. Eklund. 1994. Inhibition of Listeria
monocytogenes in cold-process (smoked) salmon by sodium nitrite and packaging method.
J. Food Prot. 57:114-119.

Peterson, M.E., G.A. Pelroy, R.N. Paranjpye, J.S. Almond, and M.W. Eklund. 1993.
Parameters for control of Listeria monocytogenes in smoked fishery products: sodium
chloride and packaging method. J. Food Prot. 56:938-943.

Petran, R.L. and K.M.J. Swanson. 1993. Simultaneous growth of Listeria monocytogenes
and Listeria innocua. J. Food Prot. 56:616-618.


Poysky, F.T., R.N. Paranjpye, M.E. Peterson, G.A. Pelroy, A.E. Guttman and M.W. Eklund.
1997. Inactivation of Listeria monocytogenes on hot-smoked salmon by the interaction heat
and smoke or liquid smoke. J. Food. Prot. 60:649-654.

Pritchard, T.J. and C.W. Donnelly. 1999. Combined secondary enrichment of primary
enrichment broths increases Listeria detection. J. Food Prot. 62:532-535.

Pritchard, T.J., K.J. Flanders and C.W. Donelly. 1995. Comparison of the incidence of
Listeria on equipment versus environmental sites within dairy processing plants. Int. J. Food
Microbiol. 26:375-384.

Roth, T.T. and C.W. Donnelly. 1995. Injury of Listeria monocytogenes by acetic and lactic
acids: mechanisms of repair and sites of sublethal damage. IFT Ann. Mtg. Book of Abstracts
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Roth, T.T. and C. W. Donnelly. 1996. Survival of acid-injured Listeria monocytogenes and
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Ryser, E.T., S.M. Arimi, M.M.-C. Bunduki and C.W. Donnelly. 1996. Recovery of different
Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products
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                                            122
GROWTH OF LISTERIA MONOCYTOGENES PREDICTED BY
MATHEMATICAL MODELS AND OBSERVED IN FOOD
PRODUCTS
Pemilla Arinder, Eva Nerbrink, Elisabeth Borch.

Swedish Meats R&D, Västra langgatan 20, SE-244 24 Kavlinge, Sweden

Problem: Listeria monocytogenes may be present in ready-to-eat products, both as a result
of inadequate heat treatment and as a result of post heat-treatment contamination. For
contaminated products that are intended to be eaten without a further heat treatment, it is
important to evaluate the risk of growth of L. monocytogenes during storage. The risk of
growth is depending on intrinsic parameters such as pH and salt level and extrinsic
parameters like temperature. Mathematical models are useful for the evaluation. However,
the models are often based on growth in culture medium and it is important to validate the
model for specific types of food. Several mathematical models have been published both
predicting the lag-phase and the maximum growth rate.

Methods: In this study L. monocytogenes was inoculated in meat products, in pasta, in rice
and in vegetables. The products were stored at chill-temperature (4-8°C) and bacterial count
was analysed during the storage. The growth was predicted using several mathematical
models and the results has been compared to the observed.

Conclusion: Some mathematical models predicted faster growth than was observed,
but in some cases the observed growth was faster than the predicted.




                                            123
ISO 11290-1, HORIZONTAL METHOD FOR THE DETECTION OF
LISTERIA MONOCYTOGENES: CHANGE OF ISOLATION MEDIA
RR Beumer

Laboratory of Food Microbiology, Wageningen University, PO
Box 8129, 6700 E V Wageningen,
The Netherlands

In ISO 11290 a method is described for the isolation of Listeria monoytogenes from food
samples. After a two-stage enrichment procedure, using half Fräser and Fräser broth,
isolation takes place on two media: PALCAM and Oxford agar. These media suffer from the
disadvantage that L. monocytogenes cannot be differentiated from non-pathogenic listerias.
Even the selection of five colonies from such media does not guarantee the detection of this
pathogen, even though a few L. monocytogenes colonies are present on the plate. Recently,
media has been described on which L. monocytogenes can be distinguished from other
Listeria spp.

Naturally contaminated samples, likely to contain listeria's, were applied in the ISO-
procedure for the detection of L. monocytogenes. PALCAM and Oxford media were
compared with isolation media that could distinguish L. monocytogenes from other listeriae:
LMBA-agar and the following commercial media: ALOA (AES, France and Biolife, Italy);
RapidL'mono (Bio-Rad, Veenendaal, The Netherlands), Chromogenic Listeria medium
(Oxoid, Basingstoke, England) and BCM® Listeria monocytogenes medium (Biosynth, Staad,
Switzerland).

The results of this trial led to resolution 143, taken at the ISO meeting in Vienna (1999): 'SC9
members agree to change the isolation media PALCAM and Oxford used in the standard
(ISO 11290-1, horizontal method for the detection and enumeration of Listeria
monocytogenes. Part 1: detection), to PALCAM and at least one other medium left to the
choice of the laboratory.




                                              124
COMPARISON OF THREE ENRICHMENT PRODEDURES FOR
THE DETECTION OF STRESSED LISTERIA MONOCYTOGENES
IN SOFT CHEESE
N. Rijpens, L. Herman

DVK-CLO, Brusselsesteenweg 370, B-9090 Melle, Belgium, Tel: 0032797272.30.18, E-mail
N.Rijpens@clo.fgov.be

Problem: L. monocytogenes can be sublethally stressed by environmental challenges and
many of the selective agents used in enrichment media interfere with the repair process in
such cells. We investigated whether the incorporation of a non-selective enrichment step
increases the isolation rate of L. monocytogenes from soft cheeses.

Methods: Three PCR-based 48-hour methods for the detection of L. monocytogenes in soft
cheeses were compared. The cheeses were artificially contaminated with 50 stressed L.
monocytogenes cells. PCR was performed on 1 ml of enrichment medium after 44 hours of
enrichment using centrifugation or IMS as sample preparation. Additionally 10 µl of the 44-
hour enrichment media were streaked on selective agar media and presumptive Listeria
colonies were confirmed using PCR.

Results: The success of the methods was dependent on the cheese examined. For the
blue-veined cheese-type (made from raw or pasteurized milk) the methods that incorporated
a non-selective enrichment step gave much better results than the completely selective
method. For most of the mold-ripened cheeses it was very difficult to recuperate the stressed
L. monocytogenes. The results for this cheese type were dependent on the cheese brand
and time of sampling but the average best results were obtained using the completely
selective enrichment procedure. For the cheeses of the red smear-type two out of three
tested cheeses were initially contaminated with Listeria spp. and therefore it was less clear
which enrichment procedure the optimal one was. IMS gave better results as sample
preparation for PCR than did centrifugation. More importantly however better results were
obtained performing PCR on solid selective media than on enrichment broths. This higher
sensitivity may compensate for the extra 1 or 2 days of the analysis when, respectively
ALOA or Oxford, are used as selective media.

Conclusion: It is clear that the conventionally recommended completely selective
enrichment procedures are not always the best choice for the detection of stressed L.
monocytogenes in soft cheeses. An extensive comparison of different methods on a broad
range of cheeses of different types seems however mandatory in order to draw well-founded
conclusions about the most optimal enrichment procedure for each cheese type.




                                            125
IMPROVING RECOVERY OF LISTERIA MONOCYTOGENES
INJURED BY ACIDIFICATION OR SALTING
Gnanou Besse N., Lafarge V.

Agence Française de Sécurité sanitaire des Aliments, Laboratoire d'Etudes et de
Recherches sur l'Hygiène et la Qualité des Aliments, 39-41 rue du 11 Novembre, 94704
Maisons Alfort , France. Phonr: 33 1 49 77 11 10, Fax: 33 1 49 77 11 02, E-Mail:
n.besse@afssa.fr

Problem: Recovery of stressed Listeria is of great importance in food hygiene and in the
development of analytical methods, since damaged cells may recover and regain their
pathogenicity. Conditions improving repair of heat-damaged Listeria have been widely
investigated. Since some cell injuries, such as cell wall or cell membrane lesions, have an
almost universal occurrence, our purpose was to determine whether factors facilitating heat-
stressed Listeria repair have any influence on salt or pH-injured cells resuscitation.

Methods: The influence of various medium components and incubation conditions on the
recovery of salt- or acid-injured Listeria monocytogenes were studied on a solid repair
medium according to an Hadamard matrix. The most influential parameters were further
studied with complete factorial design experiments.

Results: Influential factors on salt-stressed cells repair were: temperature and atmosphere
incubation, supplementation of the media with D-glucose, catalase or lithium chloride.
Influential parameters on acid-injured cells repair were temperature incubation and addition
of LiCl. Medium supplementation with L-cysteine or magnesium sulphate had no effect on
resuscitation of both salt- or acid-injured Listeria.

Conclusion: Results showed that conditions promoting resuscitation are stress specific.
Therefore, food processes and specific stresses involved should be considered before
applying repair procedures to food analysis.




                                            126
METHODS FOR THE DETECTION OF LISTERIA
MONOCYTOGENES IN HUMAN FAECES
Katharina Grif, Manfred P. Dierich, Franz Allerberger

Institut für Hygiene und Sozialmedizin and Bundesstaatl. bakt.-serol. Untersuchungsanstalt,
Schöpfstr. 41, A-6020 Innsbruck, Austria

Problem:The aim of this study was to evaluate various detection kits concerning their ability
to detect Listeria monocytogenes in the faeces of healthy Austrians.

Method: Stool specimens, approximately 1 to 2 g each, from 505 healthy humans were
emulsified in 10 ml Listeria-Broth (Biotest, Heidelberg, Germany), and enriched at 4°C for 6
months. One ml of enriched sample was subcultured in 10 ml Fraser Broth (bioMerieux,
Marcy-l'Etoile, France) for 24h at 37°C; the same procedure was performed with 1 ml of this
first subculture sample. The resulting enrichment broths were tested in parallel using the
following five detection procedures: conventional plating onto Palcam agar (Biotest,
Heidelberg, Germany) and Rapid'L.MONO agar (Sanofi Diagnostic Pasteur, Marnes La
Coquette, France), immunomagnetic separation (IMS) followed by conventional plating,
enzyme-linked fluorescent immunoassay (ELFA), ELISA (Pathalert™ Listeria
monocytogenes, Merck, Darmstadt, Germany), and PCR (Probelia™ Listeria
monocylogenes, Sanofi Diagnostic Pasteur, Marnes La Coquette, France). Enzyme-linked
fluorescent immunoassay (ELFA) was performed on 50 ul of Fräser broth with tile automated
VTDAS system (bioMerieux Vitek, Hazelwood, USA) using the VIDAS L. monocytogenes
assay.

Results: By conventional plating L. monocytogenes was isolated from one specimen (0.2%),
and a further three were positive using IMS (0.8%). ELFA and EIA showed only one
concordant positive result, this specimen being negative by conventional culture, IMS, and
PCR. Eighteen of the 505 samples were positive by PCR (3.6%), and this included three of
the four culture confirmed specimens.

Conclusions: Our data indicate that PCR is a promising test when screening for faecal
carriage; use of IMS improves the detection rate above that of a conventional plating
method. ELFA and ELISA - assays intended for the qualitative detection of L monocytogenes
in foods and environmental samples are not suitable for faecal material.




                                            127
VALIDATING DETECTION METHODS FOR FOODBORNE
PATHOGENIC BACTERIA: LISTERIA MONOCYTOGENES
D. Hitchins.

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 200 C
Street SW, Washington DC, USA 20204-0001

Problem: Comparing their performances with conventional cultural methods is often used to
validate novel Listeria monocytogenes detection methods. Comparative validation does not
explicitly show the absolute performance efficacy of a method.

Method: Detection of 1 cfu per analytical portion of a food, typically 25 g, is the maximal
method performance efficacy. Since, usually, two spiking concentrations around the 50 %
detection limit are studied, the data can be used to calculate an absolute 50 % endpoint of
detection value based on the Poisson distribution. However, pooled published data for
different Listeria methods show a large scatter about the Poisson based endpoint curve.
While the scatter could be due to method, food, and strain differences, among other factors,
it is most likely largely the result of the variability involved in the 3-replicate MPN inoculum
enumeration.

Results: Thus, when the inoculum level is calculated from the proportion of positives
obtained by the control method, the variability is dramatically reduced, since replication is
then increased to >60. This shows that the inoculum enumeration in validations needs more
replication. Increasing the replication level of the MPN enumeration from 3 to 60 or so
replicates can accomplish this. Alternatively, the control method results can be used as in
this study. Either option will coincidentally improve the 50 % detection endpoint estimates.




                                              128
UTILIZATION OF PCR-SYSTEMS FOR THE DETECTION OF
LISTERIA MONOCYTOGENES IN ROUTINE DIAGNOSTIC OF
ACID-CURD PRODUCTS
Simon, Pamela;Barbara Brunner, M. Bülte,

Institute of Veterinary Food Science, Justus-Liebig-University Giessen, Frankfurter Str. 92,
35392 Giessen, Germany

Problem: Listeria (L.) monocytogenes is an important foodpathogen, that occurs in acid-curd
products (sour milk products) as well as in other food. The diagnostic of L. monocytogenes in
food using culture medium requires time and laborwork. Therefore it is necessary to
establish rapid methods of diagnostic for the routine.

Method: Our experiments were carried out with different naturally contaminated samples of
acid-curd cheese (sour milk cheese), acid curd, cultures for acid-curd cheese production,
and others (n=448/859). Two commercially available PCR-systems, BAX (Qualicon,
Birmingham, UK) and PROBEUA (BIO-RAD, Freiburg), were used. The experiments were
performed following §35 LMBG (Lebensmittel- und Bedarfsgegenständegesetz). LISTERIA
MONOCYTOGENES BLOODAGAR (LMBA) was used supplementary or as a substitute for
Oxford-Agar respectively.

Results: Using PROBELIA-PCR-hybridization-system we got an overall equivalence of 85,9
% with the reference method. 4,2 % of the samples showed a false negative and 9,8 % a
false positive result. The experiments with 859 samples of acid-curd products using BAX-
PCR-system resulted in 92,4 % equivalence with the reference method, in which 5,0 % of
the samples were false negative and 2,6 % were false positive reagent.

Conclusion: These results show that both PCR-systems are suitable as a rapid routine
method for detection of L. monocytogenes in acid-curd products.




                                            129
COLONY-BLOT ASSAY WITH ANTI-P60 ANTIBODIES AS A
METHOD FOR QUICK IDENTIFICATION OF LISTERIA IN FOOD
Barbara Rozalska1, Marzena Wieckowska-Szakiel1, Andreas Bubert2, Marek Rozalski3,
Urszula Krajewska3, Wieslawa Rudnicka1

1
 Department of Infectious Biology, Institute of Microbiology and Immunology, University of
Lodz, 90-237 Lodz, Banacha 12, Poland, 2Microbiological Analytics, Scientific Laboratory
Products, Merck KGaA, 64271 Darmstadt, Germany, 3Department of Biochemistry, Institute
of Environmental Research and Bioanalysis, Medical Academy of Lodz, Muszynskiego 2,
Poland.

Problem: In order to overcome the limitations of immunological assays for the detection of
Listeriae, we took advantage of the recently developed antibodies against the extracellular
protein of 60 kDa, which had been shown to be useful for unambiguous Listeria-genus and
L. monocytogenes specific identification.

Methods: The present study evaluated the ability to isolate Listeria from foods, using
shortened procedure of sample enrichment on HFB medium followed by immunomagnetic
separation (Dynabeads-Listena) or filtration methods, and serological identification of
isolated bacteria by colony-blot and Western blot methods with anti-p60 antibodies.

Results: By these methods, identification of Listeria was achieved in much shorter time (40-
48 h) than with standard cultivation and biochemical identification procedures. The possibility
to select Listeria colonies growing on non-selective media by blotting with anti-p60Ab seems
to be particularly valuable in examination of food samples containing not too many Listeria
(1-10 CFU/25 g).

Conclusion: These rapid methods used are easy to perform and, what is the most
important, their specificity is very high and fulfills the expectations. However, the blot method
with anti-PepD mAb specific to unique region of L. monocytogenes p60, is necessary to
distinguish L. monocytogenes from other Listeria species.




                                              130
DETECTION OF LISTERIA ANTIGENS
Bubert A.1, K. Kramer2, B. Rozalska3, W. Goebel4, and B.S. Yoon5

1
 Microbiological Analytics, Merck KGaA, Darmstadt, Germany; 2Dept. of Botany, Techn.
University Munich, Freising, Germany, 3Dept. of Infectious Biology, Univ. Lodz, Lodz,
Poland, 4Dept. of Microbiology, Biocenter Univ. Würzburg, Würzburg, Germany, 5Dept. of
Biology, Kyonggi Univ., Suwon, Korea


Up to now immunoassays are worldwide the most popular rapid test methods used for the
detection of Listeriae from foods in routine labs. The advantage of immunoassays over
modern molecular techniques rely on their ruggedness and easy handling. Although
molecular methods are more powerful in terms of sensitivity and specificity, sample
preparation prior to the application of these techniques is still an unsolved problem.
Various attempts have been done to generate polyclonal and monoclonal antibodies for the
genus-and species-specific identification of Listeriae from food and/or environmental
samples. However, among these only a very few antibodies are potentially useful in
diagnostics. While for the detection of the genus Listeria anti-flagella Ab’s are widely
accepted in commercial immunoassays despite of some limitations in sensitivity and
enrichment conditions, specific detection of L. monocytogenes via antibodies is difficult.
Several developments of L. monocytogenes-specific Ab’s (anti-Hly, anti-InlA, anti-p60, etc.)
have been described, but all these Ab’s show limitations in terms of sensitivity, reactivity or
reproducibility. While inlA is poorly expressed under in vitro culture conditions which leads to
non-satisfying detection limits in immunoassays, expression of hly is highly variable and
differs in L. monocytogenes isolates which in turn leads to inconsistent results. Detection of
p60 by the described polyclonal Ab’s is restricted to the presence of this protein in culture
supernatants. Until now p60 remains the most appropriate target for immunological
identification of this pathogen because p60 represents a murein hydrolase protein that is
secreted by all Listeria species in larger amounts and that has been shown to be essential
for cell viability. However, our previously developed polyclonal L. monocytogenes-p60
specific antibodies are very difficult to reproduce and thus not suitable in commercial
immunoassays. We therefore generated L. monocytogenes-p60 specific monoclonal Abs,
compared and applied these in different types of assays. The results show that p60 is a
suitable protein target in EIA–based methods such as microplate or colony blot assays for
the specific identification of this pathogen. We, however, still failed to receive satisfying
results in a immunochromatographic assay system. Work to adapt these antibodies in such
immunological assays is currently in progress.




                                              131
PRESENCE OF LISTERIA MONOCYTOGENES IN SAMPLES OF
COOKED SAUSAGES
Becker, B.; Trierweiler, B.; Fechler, J. and Holzapfel, W.H.

Institute of Hygiene and Toxicology, Federal Research Centre for Nutrition, Haid-und-Neu-
Str- 9, D-76131 Karlsruhe, Germany


Introduction

Despite new hygiene regulations, HACCP and strict control measures, the incidence of food
borne infections and intoxications has been increasing in Germany. About 7 % of these are
due to meat products. Cooked sausages such as Lyoner, Gelbwurst, Bierwurst, Weisswurst,
Schinkenwurst and Mortadella are of special importance in this respect as they represent the
greatest proportion of the per-head consumption of meat in the German speaking area.

Meat products frequently contain pathogenic bacteria including Listeria monocytogenes,
toxinogenic Staphylococcus and Salmonella. As these pathogens are destroyed when
sausages are heated up to 72 C, any later detection must be assumed to be due to
recontamination of the products. The presence of L. monocytogenes is strictly regulated.
Certain ready-to-serve meat products containing counts above 100 cfu of L.
monocytogenes/g of food are considered to be ‘not marketable‘ (BGVV, 2000). This applies
to any time before the ‘best before use‘ date. Because of the high mortality rate of listeriosis,
a follow-up check and tracing of the source of contamination are indicated when numbers
>100 cfu of L. monocytogenes/g of cooked sausage are detected.

Material and methods

Within the frame-work of a research project sponsored by the Federal Ministry of Health
‘Studies into the hygienic status of different chilled retail display cabinets containing meat,
meat products and other food’, 287 samples of cooked sausages were checked for L.
monocytogenes. Samples were drawn in a regional survey in Karlsruhe (20 shops), in a
country-wide field study (102 shops), and in 3 butcher shops.

For detection of Listeria, samples were enriched in L-Palcam Listeria Selective Enrichment
Broth (Merck, Germany) for 48 hours at 37 C. After 24 and 48 hours, Palcam Listeria
Selective Agar plates (Merck, Germany) were inoculated by a surface streak from the
enrichments and incubated for 24 hours at 37 C. Any suspect colony appearing grey-green
in a black-brownish zone was inoculated onto standard-I agar (Merck, Germany), incubated
for 24 hours at 37 C and then biochemically identified using the API Listeria test
(BioMérieux, Germany).


Results and discussion

Pathogens were detected in 30 products (10.5 %) out of 287 samples tested (Table 1).
Schmidt (1989) has found contamination by L. monocytogenes even in 17 % of vacuum
                                              132
packaged cooked sausages; Listeria counts were between 100 and 200 cfu/g. In the
present studies, a correlation between Listeria contamination and total counts did not exist.
Average total counts were between 1.8 x 106 and 2.2 x 107 cfu/g and they have been found
to be lower (by one order of magnitude) in winter than in summer. At least 66 % of the
samples – regional and country-wide – showed total counts below the critical limit of 10 6
cfu/g. It should be noted, however, that L. monocytogenes has relatively frequently been
found in all three butcher shops.

In view of the high Listeria contamination (29.5 % of samples) in 3 butcher shops, the
complete production chain from slaughtering to cooked sausage was randomly checked for
L. monocytogenes (Table 2). In all three shops, Listeria have been found in pork and beef, in
the rind and on the skin of the carcasses. Listeria were isolated also from the sausage meat
mixture. In one shop, Listeria were present in the meat, sausage mixture and cooked
sausage. As a properly conducted cooking process (central temperature of 72 C and higher
and a holding time of 1 minute) destroys Listeria, the detected pathogens were obviously
transferred to the sausage by cross contamination during cutting at the butchers. At butcher
B, non-pathogenic L. innocua was isolated from the working table in the sausage kitchen.
Working tables in the butcher shops were in a better hygienic state (104 -105 cfu/100 cm2)
than those in supermarkets ( 106 cfu/100 cm2).

As the sausage samples checked belonged to the category of pasteurised food, lower
counts could be expected when a good manufacturing practices including good hygienic
conditions are applied. A complete inactivation of all microorganisms cannot be achieved
even if the cooking process was conducted properly; pathogens are eliminated, however.
Pathogens in ready-to-eat cooked sausages, therefore, are a problem of secondary
contamination probably during packaging or cutting of the sausages.

Summary

Within the frame-work of a research project sponsored by the Federal Ministry of Health,
samples of cooked sausages were checked for presence of L. monocytogenes. Samples
were drawn in a regional survey in Karlsruhe, a country-wide field study and in 3 butcher
shops.

L. monocytogenes was isolated from 30 (10.5 %) out of 287 samples totally. Listeria positive
samples could not be correlated to the total aerobic counts.

Literature

BgVV (2000): Empfehlungen zum Nachweis und zur Bewertung von Listeria monocytogenes
     in Lebensmitteln, Pressedienst, http://www.bgvv.de/presse/2000

SCHMIDT, U. und KAYA, M. (1989): Bedeutung des Vorkommens von Listerien bei Fleisch
    und Fleischerzeugnissen. Tagungsbericht der 30. Arbeitstagung der Deutschen
    Veterinärmedizinischen Gesellschaft, Garmisch-Partenkirchen, September 1989




                                            133
Table 1: Presence of Listeria spp. and L. monocytogenes in samples of cooked sausages


    Type of study        No. of samples              No. of positive samples


                                            Listeria spp.      Listeria monocytogenes


Regional   survey   in        22             6 (27.3 %)              6 (27.3 %)
summer


Regional   survey   in        21             3 (14.3 %)               1 (4.8 %)
winter


Country wide survey           102            15 (14.7 %)              5 (4.9 %)
in summer


Country wide survey           98                8 (8.2 %)             5 (5.1 %)
in winter


Studies in 3 butcher          44             14 (31.8 %)             13 (29.5 %)
shops


Total                         287            46 (16.0 %)             30 (10.5 %)




                                          134
Table 2: Studies in three butcher shops (rural district of Karlsruhe) – Presence of Listeria
monocytogenes in the production chain of cooked
         sausage


Butche Pork Rind           Pig    Beef   Skin of   Blood of    Sausage      Cooked
   r                      blood           cattle    cattle       meat       sausage
                                                                mixture

  A       +      +          -      +       +          -           +            +


  B       +      +          -      +       +          -           +             -


  C       +      +          -      +      n.d.*      n.d.         +             -


* n.d. = not determined




                                             135
ADSORPTION, AND BIOFILM FORMATION AMONG ISOLATES
OF LISTERIA MONOCYTOGENES ON FOOD GRADE STAINLESS
STEEL
J.M Farber, M.L. Kalmokoff, and J.W. Austin.

Bureau of Microbial Hazards, Microbiology Research Division, Food Directorate, Health
Products and Food Branch, Health Canada, Banting Research Centre, Tunney’s Pasture,
P.L.# 2204A2, Ottawa, Canada. K1A 0L2.

Problem: While it has been clearly demonstrated that L. monocytogenes is able to bind to
food processing surfaces, there has been much less information concerning the ability of
this species to actually form biofilms. We evaluated thirty six isolates of Listeria
monocytogenes in terms of the ability to adsorb, and to form biofilms on food grade
stainless steel in a model system.

Method: Short term binding (2h) of cells to the model surface was evaluated by epi-
fluorescence microscopy. Biofilm formation in a 72 hour growth assay was assessed by
scanning electron microscopy.

Results: Differences were found among the strains in terms of the ability of cells to adsorb
to stainless steel over a short period. However, there were no correlations between the
degree of adsorption and either the serotype, or source of the strain. Most strains bound
the test surface at levels of 104-105 cell/mm2. The ability of each strain to form a biofilm
over a 72 hour period was also assessed. With the exception of a single isolate, none of
the strains formed biofilms, but adhered to stainless steel as isolated cells. Unlike the short
term assay, significant differences in the degree of adherence were found among the
strains, with adherence levels ranging from 102 to >105 cells/mm2. In general, strains
demonstrating enhanced levels of adherence also produced extracellular fibrils, whereas
those which adhered poorly did not. A single isolate (L. monocytogenes CLIP23485)
formed a biofilm, which consisted of adhered single cells, microcolonies, and regions
containing larger aggregates of cells.

Conclusions: We have surveyed both short-term adsorption and longer-term biofilm
formation among a diverse selection of L. monocytogenes strains. Our findings indicate that
there are significant differences among these strains in terms of adherence, and the ability to
form a biofilm.




                                              136
THE DIFFERENTIAL ADHERENCE OF LISTERIA
MONOCYTOGENES STRAINS IN MONOCULTURE AND
MULTISPECIES BIOFILMS
Norwood. D.E.1, Harvey, J.2, and Gilmour, A.1,2

Department of Food Science (Food Microbiology), The Queen's University of Belfast1 and
Department of Agriculture and Rural Development for Northern Ireland 2, Newforge Lane,
Belfast BT9 5PX, N.Ireland, UK.

Problem: In almost all cases of L. monocytogenes contaminated products, the
microorganism can be isolated from the environment of the food plants from which they
originated. There is mounting evidence that contamination of food by L. monocytogenes can
occur by transfer from biofilms in food processing environments. Certain strains of L.
monocytogenes have been observed to have a predilection towards persistence in the food-
processing environment.

Method: An adherence survey of 111 strains of L. monocytogenes was initially conducted
using traditional plating techniques. Two strains of L. monocytogenes from this survey, Scott
A and FM876, were then further investigated by direct microscopic analysis. The differential
adherence capabilities of these two strains to stainless steel in 48-hour monoculture and
multispecies biofilms at 4°C, 18°C and 30°C were determined using immunofluorescent
microscopy and image analysis.

Results: In the initial adherence survey using traditional plating techniques persistent strains
were found to adhere in significantly greater numbers than sporadic strains. When the two
selected strains of L. monocytogenes were further investigated by direct microscopic
analysis optimum adherence occurred at 18°C in monoculture biofilms, with the persistent
strain FM876 exhibiting significantly greater adherence than Scott A throughout.

Conclusion: These studies would suggest that adherence capability could be a factor
responsible for the persistence of certain strains of L monocytogenes in food processing
environments.




                                              137
LISTERIA MONOCYTOGENES ATTACHES TO A WIDE VARIETY
OF MATERIALS COMMONLY USED IN THE FOOD INDUSTRY.
Mark Beresford1,2 Peter Andrew1, Gilbert Shama2.

(1) Department of Microbiology and Immunology, University of Leicester, LEI 9HN, UK.
(2) Department of Chemical Engineering, Loughborough University, Loughborough LEU
3TU, UK.

Problem:. The aim of the investigation was to investigate attachment of L. monocytogenes
to seventeen materials (metals, plastics, rubbers and glass) commonly used in food-
processing environments and then to identify genes implicated in adhesion.
Method: Square coupons of each material were immersed in a culture of L. monocytogenes
for up to two hours. The coupons were then washed in PBS and the number of adherent
cells determined after removal by sonication.

Results: L. monocytogenes attached rapidly to all surfaces and by two hours the numbers
attached had increased significantly (P<0.05) for all materials.
However, two populations of adherent bacteria were apparent; weakly and more strongly
adherent. Significantly (p<0.05) less cells adhered strongly to polypropylene compared to all
other materials. Screening of a transposon mutant library led to the discovery of twelve
different mutants that attached to glass coverslips in significantly lower numbers than the
wild type.

Conclusion: L. monocytogenes can attach a wide variety of materials commonly used in
food industry. L. monocytogenes attaches to polypropylene coupons less strongly than any
of the other materials used in the investigation. Transposon mutagenesis has generated
twelve mutants that are defective in adherence to glass.




                                            138
OCCURENCE OF LISTERIA MONOCYTOGENES IN SOUR MILK
CURD AND SOUR MILK CHEESE
B. Brunner , M. Bülte

Institute of Veterinary Food Science, Justus-Liebig-University Giessen, Frankfurter Strasse
92, 35392 Giessen, Germany
Problem: Listeria (L.) monocytogenes is an important food-borne pathogen causing severe
disease with high lethality. Different food especially milk products are implicated in
outbreaks. The aim of this study was to investigate the appearance of L. monocytogenes in
sour milk curd and sour milk cheese.

Method: Therefore, naturally contaminated samples (n=556 for sour milk curd and n=1353
for sour milk cheese) were taken from different dairy plants. Investigation was performed
according to the reference procedure ISO 11290-1 (International Organization for
Standardization). Sour milk curd was investigated after ripening to a pH >5,8. Additionally,
130 samples of sour milk cheese were investigated after production and one or two days
best before date. Further, 30 samples of sour milk curd were spiked with different
concentrations of L. monocytogenes and investigated directly and after ripening, according
to ISO 11290-1 and -2.

Results: In 556 samples of sour milk curd we had no positive result for Listeria spp. One
sample of earlier investigated sour milk curd contained L. monocytogenes and L. Innocua. In
contrast to sour milk curd we detected L. monocytogenes in sour milk cheese directly after
production in about 26% of the products, especially from one factory. Quantitative
investigation yielded colony-counts mostly under 1,0 x 102 cfu/g (colony forming unit).
Storing the cheese until best before date at a temperature from 4-8°C yielded higher
quantities of L. monocytogenes only exceptionally up to 1,0 x l06 cfu/g. In the spiked samples
of sour milk curd it was difficult to detect L. monocytogenes directly at a level below 10
cfu/25g, while after ripening L. monocytogenes could be detected more easily.

Conclusion: The conclusions are, that in sour milk curd a positive result for L.
monocytogenes might be scarce, perhaps due to the low pH before ripening. Sour milk
cheese might be contaminated and L. monocytogenes can grow up during storage time.




                                             139
REDUCED LISTER1A MONOCYTOGENES OCCURRENCE IN
FINNISH VACUUM-PACKED FISH PRODUCTS
Hatakka Maija1, Johansson Tuula2, Rantala Leila2, Pakkala Pekka1, Honkanen-Buzalski
Tuula2


1
 National Food Administration, P.O. Box 5, FIN-00531 Helsinki, Finland, 2National Veterinary
and Food Research Institute, Department of Food Microbiology, P.O. Box 368, FIN-00231
Helsinki, Finland

Problem: Cold-smoked and cold-salted vacuum-packed fish products are widely consumed
in Finland. These kinds of ready-to-eat fish products with a long shelf life carry a particular
risk of listeriosis, since their processes do not destroy Listeria monocytogenes.
An outbreak linked to a cold-smoked or cold-salted fish product was reported in Finland and
Sweden in the 1990s. Previous studies detected L. monocytogenes in 15-25% of smoked
and cold-salted fish products in Finland during 1996-98, with even thousands of CFU/g
occasionally found. Finland's annual food control plan for 2000 especially focused on fishery
establishments.

Method: In spring 2000, the National Food Administration and National Veterinary and Food
Research Institute (EELA) studied the occurrence of L. monocytogenes in fish products in
Finland at the retail level. Six official food control laboratories analysed vacuum-packed cold-
smoked (n=232) and cold-salted (n=82) fish products collected from retail outlets in different
parts of Finland for L. monocytogenes. The products came from 29 fishery establishments. L
monocytogenes isolates were sent to EELA for serotyping and pulsed-field gel
electrophoresis (PFGE) typing.

Results: L. monocytogenes was detected in 10 cold-smoked (4%) and five cold-salted (6%)
vacuum-packed fish products from seven fishery establishments. L. monocytogenes levels in
all the positive samples were below 100 CFU/g. Twelve isolates belonged to serogroup 1
and three to serogroup 4. Seven different PFGE profiles were found.

Conclusions: Hygiene measures in fishery establishments, this retail level research project
and the checking of storage temperatures has probably reduced essentially the L.
monocytogenes contamination rate of vacuum-packed fish products.




                                              140
MAILLARD REACTION DURING COOKING OF MEAT PRODUCTS
CAUSES SUPPRESSION OF VIRULENCE GENE EXPRESSION IN
LISTERIA MONOCYTOGENES
Mahmoud Sheikh-Zeinoddin, Tania M. Perehinec, Sandra E. Hill and Catherine E.D. Rees

University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough,
Leicestershire, UK. LE12 5RD

Problem: Many environmental signals affect the expression of virulence genes of the food
borne pathogen Listeria monocytogenes. In addition, media composition has been shown to
suppress levels of haemolytic activity.

Method: Using a PTplcA::luxAB reporter gene fusion it was observed that the heat processing
of media also reduces the level of virulence gene expression in L. monocytogenes without
affecting its growth. Physico-chemical factors that are considered to enhance the Maillard
reaction were also found to increase the levels of suppression. This observation has now
been extended to effects of heat treatment on meat extracts. Different meat samples were
subjected to different levels of heat treatment which may be similar to those experienced
during cooking.

Results: In all cases thorough cooking of meat lead to a suppression of reporter gene
activity while growth was not affected. However, low or intermediate levels of heat treatment
did not produce this effect and, in the case of pork meat, lead to enhancement of gene
expression.

Conclusion:This finding may help to explain the high number of outbreaks of food-borne
listeriosis which have been associated with pork products.




                                            141
 DIVERSITY OF LISTERIA MONOCYTOGENES STRAINS
ISOLATED IN MEAT PRODUCTS
T. Autio, J. Lunden, J. Björkroth, H. Korkeala.

Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, P.O.Box
57, 00014 Helsinki University, Finland.

Problem: This study was set up to establish the diversity of L. monocytogenes strains in
meat products, and to show whether associations between producers do exist.

Method: A total of 100 strains isolated in meat products were included in this study. The
strains had been isolated in products of 11 Finnish producers during the years 1996-1999.
Genetic diversity of these strains was analyzed by pulsed-field gel electrophoresis (PFGE)
typing. Restriction enzymes AscI and ApaI were used. The numerical analysis was
performed using GelComparl software.

Results and conclusion: Restriction enzymes AscI and ApaI displayed 18 and 17,
respectively, unique macro restriction patterns, resulting 19 PFGE- types (X-XIX) in total.
The number of different PFGE-types detected in products of a single producer varied from
one to four. Some PFGE-types were detected in the products of same producer over two
years, which indicates prolonged contamination of the processing plant. Seven of the L.
monocytogenes PFGE-types were found in foods of several producers having no apparent
association with each other. In numerical analysis the clustering of the strains did not
correlate with the origin or the year of isolation. This data shows variety of L. monocytogenes
PFGE-types are found in Finnish meat products and identical L. monocytogenes genotypes
can be found in foods of several producers.




                                              142
LISTERIA MONOCYTOGENES CONTAMINATION ASSOCIATED
WITH THE TRANSFER OF A DICER
Lunden. J, Autio, T., Korkeala, H.

Department of Food and Environmental Hygiene, P.O.Box 57 (Hämeentie 57);
Faculty of Veterinary Medicine, 00014 Helsinki University, Finland

Problem: Listeria monocytogenes has been shown to persist in food processing plants for
several years, sustained by complex processing machines. Here we report the spread of a
persistent strain to another plant associated with the transfer of a dicing machine.

Materials and methods: Environment, equipment and products were sampled for L.
monocytogenes for several years in plant A and B. The isolates were genotyped by using
pulsed field gel electrophoresis (PFGE) typing. The dicing machine was transferred from
plant A to plant B, and it was used for dicing cooked meat products.

Results. A certain L. monocytogenes PFGE type (type I) was found in plant A from the
dicing line and from diced products on several occasions. It was never found in the raw
areas. The same PFGE type was soon found in plant B after the transfer of the dicing
machine. It had not been recovered in plant B before the transfer of the machine. This PFGE
type I was the dominating strain in the dicing line for one year. Improved disinfection which
included repeated acid and heat treatments eradicated the L. monocytogenes contamination.
Conclusions: L. monocytogenes PFGE type I was the dominating strain in the dicing line in
both plants, and the dicing machine was the most common site of recovery. The fact that the
PFGE type I was not found in the raw areas indicates that the contamination was sustained
by the dicing machine. It is possible that the dicing machine transferred the contamination
from plant A to plant B. It is important not only to thoroughly disinfect the processing
machines daily but also when relocating the processing machines in order to eradicate
persistent L. monocytogenes strains and inhibit the spread of contamination to other
facilities.




                                            143
INDUSTRIAL APPLICATION OF AN ANTI LISTERIAL STRAIN OF
LACTOBACILLUS SAKEI AS A PROTECTIVE CULTURE AND ITS
EFFECT ON THE SENSORY ACCEPTABILITY OF COOKED,
SLICED, VACUUM - PACKAGED MEATS.
Sylvia Bredholta, Truls Nesbakkenb and Askild Holcka

a
Norwegian Food Research Institute, Osloveien 1, N-1430 AS, Norway. bNorwegian Meat
Research Centre, P.O. Box 396 Okern, 0513 Oslo, Norway
A protective lactic acid bacterium (LAB) was applied during the commercial production of
cooked meat products, The LAB, a strain of Lactobacillus sakei, was previously isolated from
cooked ham and inhibited growth of Listeria monocytogenes and Escherichia coli 0157: H7
in this product- L. sakei applied to the cooked products at a concentration of 10 5- 106 cfu/g
immediately before slicing and vacuum - packaging using a hand-operated spraying bottle.
The LAB strain inhibited growth of 103 cfu/g of a cocktail of three rifampicin resistant mutant
L monocytogenes strains both at 8°C and 40C. Consumer acceptance tests of cooked ham
and of servelat sausage, a Norwegian non-fermented cooked meat sausage, showed that
control and inoculated products were equally acceptable. The products were still acceptable
after storage for 28 days at 4°C and, after opening the packages, for a further 5 days at 4°C.
The findings presented here confirm that the L sakei strain is suitable for use as a protective
culture and may technically easily be implemented in the commercial production of cooked
meat products.




                                             144
COMPARATIVE STUDY FOR THE ENUMERATION OF LISTERIA
MONOCYTOGENES FROM FRESH FERMENTED SAUSAGES
Albert, T.1, Hechelmann, H.1, Reissbrodt, R.2 und Gareis M.1

1
Federal Centre For Meat Research
Institute for Microbiology and Toxicology
E.-C.Baumann-Str.20
95326 Kulmbach
GERMANY
2
Robert Koch-Institut, Wernigerode, GERMANY

Summary

The current method DIN EN ISO 11290:1998 for the enumeration of Listeria monocytogenes
from food like fresh fermented sausages is very time-consuming. With the PALCAM-medium
a differentiation between Listeria spp. is not possible.
In this study a total of 562 fresh fermented sausages from two producers were examined
according to DIN EN ISO 11290 (enumeration method). In parallel using the PALCAM-
medium according to VAN NETTEN the new chromogenic selective LMP-Medium (Listeria
Monocytogenes Plating Medium) was tested. It is a part of the BCM  LMDS (Listeria
Monocytogenes Detection System ) from BIOSYNTH. Only the virulent species among
Listeria spp. (L.monocytogenes and L. ivanovii) form turquoise convex colonies (1.0 to 2.5
mm in diameter). This is due to the phosphatidylinositol specific phospholipase C (PIPLC)-
activity on the chromogenic substrate, 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate.
With the PALCAM-medium isolation of Listeria monocytogenes from 24 (4,3%) samples
was succesfull. In contrast to this a total of 33 (5,9%) samples examined with the LMP-
Medium were positive. Six samples were detected positive with both methods. In none of the
positive samples the Listeria monocytogenes-counts were above 60 CFU/g.
For the identification of suspicious colonies a PCR with primers (part of the iap-gene)
specific for Listeria monocytogenes were used. The CAMP-test and the xylose-/rhamnose-
utilisation was proofed additionally. All of the 102 colonies isolated from the LMP-Medium
showed the amplification-products. 48 colonies (from a total of 51) from the PALCAM-
Medium were positive. In some cases a background-flora (Bacillus spp. and yeasts) with a
phospholipase C-activity appeared. However, differentation of these germs from Listeria
monocytogenes could easily be carried out by culture morphology.
By the isolation of suspicious colonies from the LMP-medium a safer pre-selection for further
tests (e.g. PCR) was possible. This is an important advantage in contrast to the PALCAM-
medium. With this method results could be received after 30 hours.
Based on our studies, the LMP-Medium is suitable for the enumeration of Listeria
monocytogenes from fresh fermented sausages.




                                            145
typing, epidemiology




         146
THE LISTERIA MONOCYTOGENES DATABASE IN PULSENET,
THE NATIONAL MOLECULAR SUBTYPING NETWORK FOR
FOODBORNE DISEASE SURVEILLANCE IN THE UNITED
STATES.
S. B. Hunter, N. Tucker, A. R. B, Ong, L. M. Graves, and B. Swaminathan,

Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta , Georgia 30333,
U.S.A.

Background/Problem: The Centers for Disease Control and Prevention (CDC) established
a pulsed-field gel electrophoresis (PFGE) pattern database for Listeria monocytogenes as
part of the PulseNet project in November 1998. The goal of this system is to aid in detection
and investigations of listeriosis outbreaks and characterized the diversity of L.
monocytogenes in the United States. The L. monocytogenes database of PFGE patterns in
PulseNet has grown rapidly.

Methods: PFGE patterns are submitted electronically, via the Internet, by participating
United States public health laboratories, the United States Department of Agriculture
(USDA), the U. S. Food and Drug Administration (FDA), and Canadian public health
laboratories. Clinical isolates of L. monocytogenes directly submitted to CDC=s Foodborne
and Diarrheal Diseases Laboratory section for serotyping and characterization are also
PFGE typed. PulseNet=s L. monocytogenes PFGE pattern database includes both AscI and
ApaI restriction patterns. AscI is the primary restriction endonuclease for L. monocytogenes
subtyping; ApaI is used as needed to provide additional discrimination between isolates.

Results: During 2000, 24 PulseNet participating laboratories submitted 212 gel images
(TIFF files) containing 1669 PFGE patterns to the PulseNet database. Currently, 413 unique
ApaI patterns and 337 unique AscI patterns exist in the PulseNet database. In the year
2000, the Simpson=s Index of diversities for ApaI and AscI patterns were 98.1% and
97.7%, respectively.

Conclusions: The PulseNet network has facilitated the detection and investigation of
several outbreaks, including a 1998 multi-state outbreak associated with hotdogs, a 1999
paté-associated outbreak, and a multistate outbreak in 2000 that was traced to contaminated
deli turkey meat produced at a single processing facility. The PulseNet database for L.
monocytogenes has become a valuable tool for recognition of listeriosis clusters and
investigation of outbreaks.




                                            147
REPORT ON THE PHASE III OF THE WHO SEROTYPING STUDY
OF LISTERIA MONOCYTOGENES
Lehmann1), Simone, A. Schönberg2)

Federal Institute for Health Protection of Consumers and Veterinary Medicine, 1)Jahnstraße
8, D-06846 Dessau, and 2)Diedersdorfer Weg 1, D-12277 Berlin, Germany.

Problem: After the WHO studies I and II in 1996 and 1998 it was suggested to select
reference strains for serotyping of Listeria monocytogenes (L.m.) which were examined in
comparative studies.

Method: For this purpose 6 strains of L.m. (Serovar 1/2a, 3a, 3c, 4b, 4c, 4d) were used
representing all the important O- and H-factors. Seven laboratories from various countries
were selected for serotyping these strains using two methods: the own procedure and a
suggested simple procedure.

Results: Five out of 7 laboratories sent their results which could be evaluated. Using their
own method, four out of 5 laboratories identified the expected Serovar of the 6 strains and
one identified the Serovar for 5 strains. Four out of 5 laboratories were able to identify the
expected Serovar of the 6 strains using the simple method, whereas one identified the
Serovar for 4 strains.

Conclusions: It is possible to use the simple method for the identification of O- and H-
antigenic factors of L. monocytogenes within 3 days, which is less time consuming. The 6
strains used in this study are suitable as reference strains for serotyping of L.
monocytogenes.




                                             148
CHANGES IN SEROGROUP DISTRIBUTION OF HUMAN
LISTERIA MONOCYTOGENES STRAINS IN SWEDEN
Tham W.1, E. Bannerman2, J. Bille2, H. Ericsson1, S. Helmersson1, B. Henriques3, Ch.
Jacquet4, S. Loncarevic5, J. Rocourt4, I. Tjernberg6, H. Unnerstad1, M-L Danielsson-Tham1.

1
 Department of Food Hygiene, P.O. Box 7007, Faculty of Veterinary Medicine, SE-750 07
Uppsala, Sweden. 2Centre National des Listeria, Institut de Microbiologie, Rue du Bugnon
44, CH - 1011 Lausanne, Switzerland. 3Swedish Institute for Infectious Disease Control, SE-
171 82 Solna, Sweden. 4-Institut Pasteur, 28, rue du Dr Roux, 75724 Paris Cedex 15,
France. 5Section for feed and food microbiology, National Veterinary Institute, P.B. 8256
Dep, 0033 Oslo, Ullevalsv. 68, Oslo, Norway, 6Department of Medical Microbiology, Malmö
University Hospital, SE-205 02 Malmö, Sweden
Altogether, 336 strains of Listeria monocytogenes isolated from human cases of listeriosis in
Sweden during 1986-1999 have been serotyped. The serotyping showed that 45% belonged
to Serovar 4b, 44% to Serovar 1/2a and 10% to serovars 1/2b or 1/2c. In Sweden we have
seen a trend towards a decreasing number of serogroup 4 cases while the serogroup 1/2
cases are increasing. During the period 1976-1985 the human strains of serogroup 4
constituted 69%. During the 2 last years of the 20th century, however, the percentage of
serogroup 4 strains was less than 30%. Increased consumption of vacuum-packed gravad or
cold-smoked rainbow trout (Oncorhynchus mykiss) and salmon (Salmo salar) contaminated
with L. monocytogenes serogroup 1/2 might be an explanation of this phenomenon.




                                            149
STATUS REPORT ON WHO-SPONSORED INTERNATIONAL
COLLABORATIVE STUDY OF SUBTYPING METHODS FOR
LISTERIA MONOCYTOGENES: PULSED-FIELD GEL
ELECTROPHORESIS, PHASE III.
L. M. Gravesa, S. B. Huntera, N. Tuckera, M. Brettb, J. Harveyc, C. Jacquetd, A. Kerouanton-
Le Galle, E. Lehnkeringf, B. Ojeniyig, M. Wagnerh, A. Brisaboise, A. Gilmourc, J. Rocourtd and
B. Swaminathana.

a
 Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, Georgia, USA.,
b
 Institute of Environmental Science, Porirua, New Zealand, cFood Microbial Ecology,
Newforge Lane, Belfast, Ireland, dCentre National de Référence des Listeria Centre, Paris,
France, e Unité d'épidémiologie bactérienne AFSSA LERQHA, Maisons-Alfort, France, fL.A.
County Public Health Laboratory, Los Angeles, CA. USA , gThe Royal Veterinary and
Agricultural University, Frederiksberg C, Denmark, and hInstitute for Milk Hygiene, Milk
Technology and Food Science, Wien, Austria.

Problem: In 1996, the WHO International Multicenter Listeria monocytogenes Subtyping
Study was published in the International Journal of Food Microbiology as a special issue on
molecular typing of Listeria. Pulsed-field gel electrophoresis (PFGE) was among the
methods studied and was recommended for further standardization. A phase II study was
designed using a subset of 43 strains selected from the 80 coded strains studied in phase I.
Thirteen laboratories tested the 43 strains using their own PFGE protocol or one supplied by
the study coordinator. Results from phase II of the study had indicated that better
reproducibility might be achieved if all laboratories used the same protocol.
Method: Eight laboratories participated in Phase III of the pulsed-field gel electrophoresis
(PFGE) standardization project of the WHO-Sponsored International Collaborative Study of
Subtyping Methods for L. monocytogenes. Participants from the eight laboratories agreed to
perform PFGE on a set of 12 strains using a standardized PFGE protocol and the
endonucleases AscI and ApaI to restrict the DNA. All eight laboratories submitted gel image
files (TIFF format) of PFGE patterns from the 12 isolates run on gels along with a specified
molecular size standard in the outer lanes and in a middle lane. Gel images were analyzed
using Molecular Analyst Fingerprinting Plus (Bio-Rad Laboratories, Hercules, CA).

Results: Three laboratories showed 100% similarity of PFGE patterns for all 12 strains when
PFGE AscI patterns were compared in the 30-700-kb size range. When the patterns were
compared in the 75-700-kb range, all 8 laboratories matched patterns for all 12 isolates at
95% or greater similarity; 5 of 8 laboratories matched all 12 strains at 100%. For ApaI, 4
laboratories matched patterns for all 12 strains at 100% similarity when DNA fragments in
the 30-700-kb range were compared. Six of eight laboratories matched all 12 strains at 95%
or greater similarity.

Conclusion: While the results are very encouraging, further improvements could be
anticipated with better familiarity with the protocol and carefully controlling the
electrophoresis times to compensate for differences in DNA mobilities caused by PFGE
equipment and buffer composition.


                                             150
STRAINS IDENTIFICATION OF LISTERIA SPP. BY DNA
FINGERPRINTING: ERIC-PCR.
Laciar A., Vega A., Lopresti R., Centorbi ONP.

Area Microbiologia. Universidad Nacional de San Luis. Chacabuco y Pedemera. 5700 San
Luis. Argentina.

Problem: Repetitive DNA sequences, which are dispersed m the genomes of bacteria in
extragenic regions, have been used as targets for PCR primers to obtain genomic
fingerprints of various bacteria. Listeria monocytogenes strains possess short repetitive
extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus
(ERIC) sequences. In this work, ERIC-PCR was used to generate DNA fingerprints of L.
monocytogenes and L. innocua strains isolated from humans, animals and food. The
objective of this work was to evaluate the potential of ERIC-PCR for typing L.
monocytogenes and L. innocua isolates of various origins.

Method: Thirteen L. monocytogenes isolates and 5 L. innocua isolates were examined by
ERIC-PCR. L. monocytogenes strains were isolated from blood, CSF, cervix, vegetables
mussel and faecal specimens from Lagostomus maximus maximus. L. innocua strains were
isolated from mussel and vegetables. For ERIC-PCR, the (22 - mer) primers ERIC 1R (5'-
ATGTAAGCTCCTGGGGATTCAC-3') and ERIC 2 (5'-AAGTAAGTGACTGGGGTGA-GCG-
3') were used. PCR was carried out with 50ng of template DNA per reaction for ERIC-PCR.
Amplification reaction was performed in 15l of a solution containing lM of each of the two
opposing primers, 200uM each deoxynucleoside triphosphate, 2,5 mM MgCl2, 50mM KCl-
10mM Tris-HCl (pH 8,3) and 1U ofTaq DNA Polymerase. Amplification was performed with a
DNA thermocycler (Gene Amp PCR System 2400, Perkin-Elmer) with the following
temperature profiles: 1 cycle at 92°C for 2 min, 35 cycles at 92°C for 30 s, at 40°C for 60 s,
at 72°C for 2 min and 1 cycle at 72°C for 5 min. ERIC-PCR products were separated by
electrophoresis on a 1,8% agarose gel in l x TBE buffer at a constant voltage of 8V/cm. After
being stained with ethidium bromide, the gel was photographed under UV transillumination
with Polaroid 667 film. The DNA molecular weight marker 1 kb DNA ladder was used as a
size standard. DNA fingerprints were analized by using a computer program for comparative
analysis of DNA electrophoresis patterns.

Results: The dendograms clearly show that the strains examined are divided into two
distinct groups. Group A consist of human and animal isolates and group B consist of food
isolates. ERIC profiles for food isolates were clearly different from the profiles obtained for
human and animal isolates of L. monocytogenes . No differences were observed between L.
monocytogenes and L. innocua strains isolated from food.
Conclusion: Because only 18 strains were included in this study, these observations should
be validated by the examination of a larger set of strains.




                                             151
CHARACTERIZATION OF FOOD AND HUMAN ISOLATES OF
LISTERIA MONOCYTOGENES BY USE OF PCR-SSCP IN THE
MPL AND PRF GENES
Loncarevic S., Lehner A., Wagner M.

National Veterinary Institute, PB 8156, Oslo, Norway e mail: semir.loncarevic@vetinst.no

Altogether 39 strains isolated from human cases of listeriosis and food products (soft
cheese, rainbow trout, salad etc.) representing different serovars (1/2 a, 1/2b, 1/2c, 3a, 3b,
3c and 4b) were used in this study.

Method used for distinguishing of the strains was PCR-SSCP (Polymerase Chain Reaction -
Single Strand Confirmation Polymorphism). By use of six primer sets for amplification of mpl
gene of L. monocytogenes isolates and SSCP electrophoresis method we have been able to
distinguish all serovars used in this study.
Furthermore, we found out that isolates belong to one serovar could be further differentiate
by specific SSCP pattern. Four primer sets used for amplification of the prf gene of the same
strains gave less satisfactory results.

In conclusion, PCR-SSCP analysis of mpl and partly of prf genes of L. monocytogenes could
be reliable characterization method of listeria isolates on the serovar level as well as for
distinguishing the difference between the same serovar what is essential in epidemiological
studies of listeriosis.




                                             152
COMPARISON OF MOLECULAR METHODS FOR TYPING
RECURRENT AND SPORADIC LISTERIA MONOCYTOGENES
FOOD ISOLATES.
J.Harvey1, D.E. Norwood1, A. Gilmour1,2
1
 Food Science Division (Food Microbiology), Department of Agriculture and Rural
Development and 2Department of Food Science (Food Microbiology), the Queen's University
of Belfast, Belfast BT9 5PX, Northern Ireland.

Problem: Surveys to determine the incidence of Listeria monocytogenes in foods produced
in Northern Ireland and typing of isolates using multilocus enzyme electrophoresis (MEE)
procedures indicated that foods from certain processors contained recurrent electrophoretic
types (ETs). To further research in this area, a more rapid and discriminatory molecular
typing method is required.

Methods: Three methods were used to type 21 recurrent and 24 sporadic L.
monocytogenes food isolates. (i) MEE using eleven commonly occurring cellular enzymes,
(ii) Pulsed field gel electrophoresis (PFGE) using AscI and ApaI restriction fragments of
genomic DNA. (iii) Repetitive element sequence-based PCR (rep-PCR) using REP and
ERIC primers and genomic DNA. DNA fragment band patterns were compared using gel
analysis software.

Results: PFGE was more discriminating than MEE and rep-PCR, which were roughly
equivalent in this respect. MEE and rep-PCR were more useful than PFGE in identifying
recurrent strains. rep-PCR typing resolved some discrepancies among previously obtained
MEE types.

Conclusion: PFGE's greater discriminatory power resulted from detection of minor
differences among recurrent strains identified by MEE and rep-PCR. For typing large
numbers of L. monocytogenes isolates, rep-PCR could be used initially because of its ease
of use and rapidity followed by PFGE to examine representatives of each rep-PCR type.
Knowledge of rep-PCR types should assist in interpreting minor differences in PFGE band
patterns among closely related strains.




                                           153
COMPARISON OF EPIDEMIOLOGICALLY LINKED LISTERIA
MONOCYTOGENES STRAINS CHARACTERIZED BY TWO
DISTINCT PHAGE-SENSITIVITY PHENOTYPES.
N. van der Mee-Marquet, L. Mereghetti, A. Audurier.

Laboratoire de Microbiologie, Faculte de Medecine, Tours, France.

We studied 2 clusters of L. monocytogenes originated from the same source and distinct
according to their phage susceptibility : 2 strains designated Phage-Sensitive (Ph-S) were
characterized by the lysovar : 47-2671-2425-2389, and 2 strains designated Phage resistant
(Ph-R) were characterized by the truncated lysovar 2389. The 4 strains were characterized
with phenotypic and genomic methods. Serotype, biotype, antibiotype and susceptibility
patterns to heavy metals and antiseptics were strictly identical within the 4 strains. DNA
macrorestriction was done with the 3 endonucleases SmaI, ApaI and NotI. SmaI and ApaI
pulsotypes did not differ and a difference of one band was found between Ph-S and Ph-R
strains NotI pulsotypes. RAPD was performed using 103 primers. With 99 primers, RAPD
profiles were strictly identical with the 4 strains. Using 4 primers, we found DNA fragments
which were yielded by Ph-R strains and failed with Ph-S strains. No DNA fragments was
found in Ph-S strains whist lacking in Ph-R strains.
Sequencing of the DNA fragments was done, and homologies were found between the
fragments and nucleotidic sequences associated with viral capsid proteins. These preliminar
results suggest that Ph-R strains may have integrated phage DNA, this resulting in slight-
range host phage susceptibility.




                                            154
A NATIONAL ELECTRIC NETWORK FOR COMPARISON OF
PFGE PROFILES OF LISTERIA MONOCYTOGENES
Rantala Leila1, Lukinmaa Susanna2, Siitonen Anja2, Honkanen-Buzalski Tuula1
1
 National Veterinary and Food Research Institute, Department of Food Microbiology, P.O.
Box 368, FIN-00231 Helsinki, Finland, 2Laboratory of Enteric Pathogens, National Public
Health Institute, Mannerlieimintie 166, FIN-00300 Helsinki, Finland
Problem: PFGE profiling of food-borne pathogens is a powerful tool in epidemiological
studies, in tracing of outbreaks and contamination routes in production plants. However, the
lack of standardisation of this method has complicated comparison of results between
laboratories. In Finland, the National Veterinary and Food Research Institute (EELA) and the
National Public Health Institute (KTL) are the national reference laboratories for food-borne
pathogens. The laboratories have been setting up a bilateral computer based network for
comparison PFGE profiles of L. monocytogenes of human isolates and isolates from food,
food production plants and environment. The network offers a possibility to other laboratories
using the same method to compare their profiles to our library.

Method: The adjustments of the method are made so that tlie results are comparable. Both
laboratories have Variable Angle Pulsed-field Electrophoresis System (Bio-Rad Laboratories,
Richmond,CA). The gel images documented by Alphalmager system (Alpha Innotec
Corporation CA, USA) are analysed by the BioNumerics (Applied Maths, Belgium).

Results: The library: Different PFGE profiles are collected for L. monocytogenes library. One
fragment difference is defined as significant. The profiles arc coded in consecutive order.
Profiles of new isolates are compared to the profiles in the library. When a new profile is
found it obtains the next available free coding number for the subtype. EELA and KTL have a
agreement for coding numbers. Among food origin isolates (n=1100) and human isolates
(n=300) 110 and 87 different PFGE profiles have been identified, respectively.




                                             155
HIGH-RESOLUTION GENOTYPING OF LISTERIA
MONOCYTOGENES BY FLUORESCENT AMPLIFIED-FRAGMENT
LENGTH POLYMORPHISM ANALYSIS
Birte Fonnesbech Vogel,1* and Peter Ahrens,2

Danish Institute for Fisheries Research, Department of Seafood Research, Technical
University of Denmark, DK-2800 Lyngby1 and Danish Veterinary Laboratory, DK-1790
Copenhagen V, Denmark2

Due to the ubiquitous nature of Listeria monocytogenes discriminatory typing systems are
essential for the investigation of its epidemiology. Amplified Fragment Length Polymorphism,
AFLP, is a novel PCR based whole-genome fingerprinting technique, which is considered
highly reproducible and has a high discriminatory power. To evaluate the potential of AFLP
as an epidemiological typing method for Listeria we applied it to strains of human, animal,
food and environmental origin (50 L. monocytogenes and 6 Listeria spp). AFLP analysis of
Listeria with EcoRI and BamHI primers gave strain-specific profiles. Patterns with 99 to
100% homology were considered to represent identical strains. Different strains from the
same species were found to have 80 – 99 % homology while different species within the
genus Listeria had 70 – 80 % homology. Strains of L. monocytogenes were separated at
82% homology into two groups. This could illustrate the existence of two distinct genetic
divisions. The discriminative power of AFLP was compared to RAPD typing using two
primers and the two typing systems were similar in discriminatory power. We conclude that
AFLP analysis is a powerful and reliable method for demonstration whole-genome
polymorphism among Listeria species and could be very useful for epidemiological
investigations of L. monocytogenes.




                                            156
PYROSEQUENCING AS A METHOD FOR GROUPING OF
LISTERIA MONOCYTOGENES STRAINS BASED ON
NUCLEOTIDE POLYMORPHISMS IN THE INLB GENE.
Helle Unnerstad1. Henrik Ericsson1, Anders Alderbom3, Wilhelm Tham1, Marie-Louise
Danielsson-Tham1, Jens G Mattsson2

Department of Food Hygiene, Faculty of Veterinary Medicine, Swedish University of
Agricultural Sciences, PO Box 7009, SE-750 07 Uppsala, Sweden1, Department of
Parasitology (SWEPAR), National Veterinary Institute, SE-751 89 Uppsala, Sweden2, and
Pyrosequencing AB, Vallongatan 1, SE-752 28 Uppsala, Sweden3

Method: Pyrosequencing is a sequencing-by-synthesis method based on real-time
pyrophosphate detection optimised for sequencing of short nucleotide tags. By use of
pyrosequencing, 106 strains of different serovars of Listeria monocytogenes could be
grouped in four categories based on the nucleotide variation in positions 1575 and 1578 of
the inlB gene.

Results: Strains of serovars 1/2a and 1/2c constituted one group and strains of serovars
1/2b and 3b another. The Serovar 4b strains were separated in two groups. Pyrosequencing
of the Serovar 4b strains resulted in a mixture of different nucleotides in the two investigated
positions. A possible explanation for this nucleotide mixture is that the inlB gene, or at least a
part of it, is present in two variants in the genomes of Serovar 4b strains. This was supported
by Southern blot hybridisation with an inlB DNA probe. The probe hybridised to two
fragments of digested genomes of Serovar 4b strains, but only to one fragment in genomes
of strains of the other serovars.




                                               157
CHARACTERISATION OF LISTERIA MONOCYTOGENES
ISOLATED FROM EUROPEAN PRODUCTION LINES OF FRESH
AND COLD-SMOKED FISH
Gabriela Duarte1 and Paul Gibbs1,2
1
  Escola Superior de Biotecnologia, Universidade Catolica Portuguesa, R. Dr. Antonio
Bernardino de Almeida, 4200 Porto, Portugal; 2 Leatherhead Food Research Association,
Surrey, England

Method: The occurrence of Listeria monocytogenes in european production lines of fresh
and cold-smoked fish was surveyed from 1996 to 1999. A total of 747 L. monocytogenes
strains (162 from Portugal; 28 from England; 18 from Denmark; 193 from Iceland; 287 from
France; and 59 from Netherlands.), isolated from both environmental and fish samples along
the processing chain, were characterized by O serotyping and resistance/sensitivity to
tetracycline, cadmium and arsenite profiling.

Results: On basis of these typing methods the isolates were categorised into eleven distinct
types. The major type in Denmark, Iceland, France and Netherlands was type 2 (serogroup l.
TS; CdS; AsS). The major types in UK and in Portugal were, respectively, type 4 (serogroup 1,
TS; CdS; AsS), and type 9 (serogroup 4, TS; CdS; AsS). With simple typing methods it was
possible to show how diverse the population of Listeria monocytogenes is that contaminates
cold-smoked fish products across Europe. The incidence of eleven distinct types L.
monocytogenes along fresh cold-smoked fish processing lines sites was evaluated.

Conclusion: L. monocytogenes types that contaminate fresh fish are, in general, different
from those found in processed fish, suggesting that fresh fish strains probably cannot survive
the cold-smoking process and probably there is a L. monocytogenes plant resident
population responsible for contamination during the process. It should be stressed that the
clarification of potential sources of contamination and potential routes of dissemination of L.
monocytogenes in cold-smoked fish processing plants will implies complementation with
more discriminatory typing techniques including H serotyping, phage typing and genetic
typing techniques such as Arbitrarily Random Primed Polymerase Chain Reaction of DNA
and Pulse-field Gel Electrophoresis of DNA.




                                             158
DISSEMINATION OF L. MONOCYTOGENES IN A FROZEN
CHICKEN NUGGET PROCESSING LINE
Destro. M.T., Andrigheto, C.; Landgraf, M.

Faculty of Pharmaceutical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes 580
BL14; 05508-900, Sao Paulo, SP, Brazil

Problem: The presence and distribution of L. monocytogenes (Lm) in food processing
plants is a cause of concern for industry managers and health authorities. This research was
carried out in order to evaluate the sources of Lm contamination in a frozen chicken nugget
processing line.

Methods: 252 strains of Lm from different origins and isolated from different steps of the
processing line were analysed for their genetic diversity. RAPD modified from a WHO
protocol was used. The RAPD profiles generated by primers UBC155 and M13 were
grouped combined and the similarities analysed. The strains were also serogrouped. The
correlation between serogroup, strain genetic diversity and its distribution along the
processing line was established.

Conclusion: The strains analysed were divided in two clusters, each of them containing 2
groups. It could be established that from the three sub-types of Lm that belonged to the raw
material two could get established on the processing line. These sub-types were detected
later in environmental samples (food contact and non-food contact surfaces) and in food
handlers' samples. On the other hand, other sub-types found initially in environmental
samples were detected in the product in subsequent steps. The introduction of Lm into the
plant by raw material highlights its importance as a contamination source. Measures must
be taken to control the presence of Lm in the raw material as well.




                                             159
PREVALENCE OF LISTERIA MONOCYTOGENES IN SOME
FOODS AND FOOD PROCESSING PLANTS IN ICELAND.
Birna Gudbjörnsdottir, Helene L. Lauzon, Sigrun Gudmundsdottir

Icelandic Fisheries Laboratories, R & D Division P.O. Box 1405,
Skülagata4, 121-Reykjavik, ICELAND http:/www.rfisk.is
E-mail: birna@rfisk.is, helene@rfisk.is or sigrun@rfisk.is

Introduction: Many microbiological hazards are associated with various areas in food
processing plants. In recent years, the growing number of food-borne diseases has focused
the attention on Listeria monocytogenes. L. monocytogenes is a cold-tolerant food-borne
pathogen. The organism is ubiquitous in the environment and causes listeriosis, a disease
that can be serious and is often fatal to susceptible individuals. It has been shown by
epidemiological investigation that listeriosis is a foodborne disease. The food industry,
mainly producers of ready-to-eat products such as cold-smoked salmon and cooked shrimp
and ham, has for many years been concerned about the potential danger of L.
monocytogenes contamination in products because of possible risk to the consumers. The
food industry has been experiencing an increasing pressure from regulatory bodies and
customers regarding L. monocytogenes which can in the worst case result in recalls of
products and reclamations from customers.

Problem: Many of these products undergo various processing procedures, some of which
can inactivate Listeria present on the raw product. However, cross-contamination of the
product both during and after processing can occur due to poor sanitation conditions and/or
manufacturing practices. The cold-tolerant nature of L. monocytogenes allows for the
survival and even growth during refrigerated storage or temperature abuse situation. At
present time there is a great interest in elucidating the relative role of different food products
as a vehicle for human listeriosis.
Methods: During 1996-2000, a total of 23 surveys were carried out in some Icelandic food
processing plants producing ready-to-eat (RTE) products and meat products. A total of 1368
samples, including raw and processed products (cooked shrimp, cold-smoked salmon, cold-
smoked lamb meat and processed lamb meat) as well as samples from the processing
environments, were analysed to evaluate the incidence of Listeria monocytogenes and other
Listeria species. Survey technologies were based on HACCP and hygiene auditing
techniques together with bacteriological sampling of products and processing environments.
A parallel study was performed on other food items where final products (n=96) were
sampled from food stores. Similarly, 103 samples of fresh herring prepared for further
processing.
Results and Conclusion: The overall frequency of L. monocytogenes in the surveys
conducted was 10.5% (165/1567). L. monocytogenes was most often isolated from samples
obtained from plants producing RTE-seafood (12.6% or 130/1045) where raw material
(20.3% or 23/113) and the processing environment (13.2% or 106/805) played a major role
in spreading Listeria. Nevertheless, low incidence of L. monocytogenes was found in the
Icelandic finished products (1.0% or 2/199). Unpasteurized milk was also found to be
contaminated (14.3% or 2/14) but in Iceland it is not allowed to use unpasteurized milk for
further processing. The incidence of L. monocytogenes was high in raw herring samples
(26.2% or 27/103). Hygienic precautions at all processing steps are extremely important to
prevent recontamination of the final product. L. monocytogenes strains isolated from cold-
                                               160
smoked salmon have been biotyped (serotyping and resistance/senstivity to tetracycline,
cadmium and arsenic profiling) and the results did not contribute to trace the contamination.
Moreover L. monocytogenes strains isolated from cooked shrimp were typed using the
automatic Riboprinter™ but the level of discrimination was insufficient. Further strain
identification (typing) using other techniques e.g. Pulsed-Field Gel Electrophoresis is
expected to provide a better discrimination and allow for tracing of Listeria contamination in
the processing environment. Such information will contribute to the development of a better,
improved cleaning programme, hence targeting specific contamination sites and facilitating
corrective actions. Good manufacturing practices for production of RTE-foods are essential
and would help to reduce contamination in the processing environment. It is a requirement
by law that food producers have appropriate internal control based on HACCP. Such control
can not be effective unless it covers the product, process and plant specification. Employees
must be trained to understand the problem, the potential sources and the control of L.
monocytogenes. This needs to be more than normal training in good hygienic practices.
Also, to verify L. monocytogenes control, plants should implement an environmental
monitoring programme for Listeria species. The sampling sites and frequency of sampling
should be determined based on knowledge of specific operation and the control that has
been put in place.

Incidence of Listeria spp. and L. monocytogenes in food and some food processing plants

Sample                  No. samples     Listeria spp        L. monocytogenes
Cold-smoked salmon
raw material            78              24.3                17.9
processing              345             18.0                12.4
final product
environment             102             2.9                 1
Cooked shrimp
raw material            35              25.7                25.7
processing              460             14.3                14.0
final product
environment             25              0                   0
Meat
raw material            60              1.7                 1.7
processing              233             4.7                 2, 1
final product
environment             30              16.7                0
Other food items
Unpasteurized milk      14              14.3                14.3
Vegetables              26              0                   0
Cold cut                                                    0
-pate, paste            6               0                   0
-ham                    6               0                   0
-sausage                9               0                   0
Processed meat          3               0                   0
Raw meat                15              0                   0
"Porramatur"            5               0                   0
"Porramatur" - sour     9               0                   0
Offal                   3               0                   0
Herring                 103             67.0                26.2




                                               161
LISTERIOSIS IN DENMARK 1998 - 2000 CLINICAL AND
EPIDEMIOLOGICAL ASPECTS
P. Gerner-Smidt, B. Bruun, V. Fussing, J. Engberg, A.M. Petersen, J. Schiellerup, Sta-tens
Serum Institut, Artillerivej 5, Copenhagen, Denmark

Problem:Denmark has a high registered incidence of listeriosis of 0,7- 0.8/ 100,000
inhabitants/ year. The disease is statutory notifiable through a laboratory notification system.
On a voluntary basis, the strains and detailed clinical information are collected by the
national reference centre at Statens Serum Institut.

Results: The strains are routinely O-grouped and ribotyped using the automated Riboprinter
system. Strains not discriminated are further compared by PFGE. One-hundred-twenty-one
cases were notified during 1998-2000. Twenty-five deaths were registered. Two were
abortions, 3 newborns, and 20 above 46 years of age. There were no increase in the death
rate with increasing age above 50 years. Sixty-nine patients had predisposing conditions
with the most common being haematologic malignancy, other cancer, immunosuppressive
treatment, and chronic alcoholism. The mortality was the same whether a predisposing
condition was present or not. The clinical presentation of the cases were 13 maternofetal, 81
septicaemia, 22 meningitis, and 5 other. The mortality was for newborns 27%, for
septicaemia 21%, and for meningitis 14%. Sixty-five per cent of the strains were serogroup
1/2. No clusters of cases infected with the same type were observed.

Conclusion: Listeriosis remain a serious disease in Denmark where we seem to have a
hyperendemic situation compared with other industrialised countries.




                                              162
TRANSFORMING CONTAMINATION PREVALENCE DATA INTO
CONCENTRATIONS IN FOODBORNE MICROBIAL RISK
ASSESSMENTS: LISTERIA MONOCYTOGENES DATA AS A
PROTOTYPICAL CASE STUDY
A.D. Hitchins.

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 200 C
Street SW, Washington DC, USA 20204-0001

Problem: To accurately estimate the public health risk of Listeria monocytogenes (Lm), data
are needed to estimate both the density and prevalence of Lm in the food supply. While
prevalence data of Lm are available for many commodities, density (cfu/g food) data are
sparse. This sparseness complicates the modeling of density frequency distributions of Lm
for food commodities.

Methods: These distributions are used in risk assessments. In an effort to create density
distributions for foods, pooling of all known density data was tried.
Results: The log 10 of the percentage of samples with densities >100 cfu/g was directly
proportional to the corresponding log 10 percentage of the prevalence (r2 = 0.68). This
generalized relationship accounted for 90% of the variability for foods with Lm densities >100
cfu/g. Remaining variation was partly due to the food commodity. Therefore density
frequency distributions can be constructed that predict within ± 0.5 of a log10 unit, the percent
density >100 cfu/g. Furthermore, the density values >10 cfu/g can also be modeled using the
generalized relationship.

Conclusion: Use of the generalized relationship allows a rational way to incorporate
prevalence data into a density frequency distribution when density data are sparse or
absent.




                                              163
EPIDEMIOLOGICAL ANALYSIS OF LISTERIA
MONOCYTOGENES RECOVERED FROM FOOD AND
ENVIRONMENT PROCESSING PLANT
KEROUANTON A.a, BRISABOIS.Aa., MARAULT M.a and PICARD B.b
a
 Unite d'Epidémiologie Bactérienne, Agence Francaise de Sécurité Sanitaire des Aliments,
39-41 rue du 11 novembre 1918, 94700 Maisons-Alfort, France. b Laboratoire de
Microbiologie, Faculté de Médecine de Brest, 21 av. Camilles Desmoulins, 29200 Brest,
France
Problem: The purpose of this study is to characterize Listeria monocytogenes strains which
have been isolated in 12 food processing plants of different sort and their environment in
order to establish a correlation between isolates and origin of the samples. During the last
past year, 2,272 samples have been performed in these processing plants, including 1,081
food samples (532 raw products, 192 making products and 357 finished products) and 1,189
environment samples (floor, wall, process equipment).

Results: L. monocytogenes was detected in 345 isolates, mainly found in raw products of
food samples and on the floor concerning environment samples. During this period, a total of
864 strains have been collected, in which 175 were isolated from 6 dairy products plants and
their environment and 689 were isolated from 5 pork and poultry processing plants and their
environment. All these strains have been serotyped using Japanese antisera (Eurobio,
France). Serovars 1/2a, 1/2c, 4b, 1/2b, 3b, 3a and 3b were respectively shared by 65.4,
13%, 8%, 7.2%, 3.5%, 2.4% and 0.5% of strains.
Serovar 4b has been found in a pork processing plant and in a dairy product processing
plant. Serovar 1/2a and/or Serovar 1/2b were found in 11 of the 12 processing plants.
Subtyping study of the strains were performed by the Pulsed-Field Gel Electrophoresis
method using a low-frequency cleavage restriction endonuclease (apaI). Electrophoresis
was realized with CHEF DRIII apparatus (Bio-Rad S.A., France). Ninety-five pulsotypes were
defined among the 864 strains analyzed. Serovar 4b strains were differentiated in 14
pulsotypes whereas Serovar 1/2a were highly polymorphic in 51 different pulsotypes. The
most frequently pulsotype was observed in 11% of all isolates and was found in serotype
1/2a (96%) and 3a strains.

Conclusion: Comparison of data about isolates with PFGE results by statistical analysis
show a differentiation of these strains according to the origin of the plant in a perspective of
risk assessment. (This work was supported by a grant from industrial association UNIR)




                                              164
EPIDEMIOLOGY OF LISTERIOSIS IN FINLAND
Susanna Lukinmaa1. Outi Lyytikainen2 and Anja Siitonen1

Laboratory of Enteric Pathogens' and Department of Infectious Disease Epidemiology,
National Public Health Institute (KTL), Mannerheimintie 166, FIN-00300 Helsinki, Finland

Since 1995, physicians have notified the National Infectious Disease Registry of culture-
confirmed cases of listeriosis. Microbiological laboratories have done the same for the
isolations of Listeria monocytogenes from blood, cerebrospinal fluid (CSF), genital tract,
deep puncture, and surgical specimens, as well as from newborns. The incidence of
listeriosis was 6-10 cases per million inhabitants. From 1995 to 1999 the annual number of
cases varied between 29 and 53.
In 1998-1999, further information was collected from 92 patients, including patients of an
outbreak caused by serotype 3a in butter. Of the isolates, 76 were from blood, 12 from CSF,
and four from other sources. The median age of the patients was 63 years (range, 12-92),
and 51% were male. One third of the cases were treated in Helsinki Metropolitan area.
Twenty-one (23%) of the patients had hematological and nine (10%) solid malignancy. Four
cases were related to pregnancy, including one abortion. Three non-pregnant persons did
not have any predisposing underlying condition. Mortality within a week of the positive listeria
culture was 19% and within a month 24%. The deceased persons were significantly older
(median 77 versus 60 years, p=0.001), all had at least one underlying disease and half had
malignancy. All 92 strains of L. monocytogenes from 1998 to 1999 were studied by
serotyping of 0 and H antigens and by pulsed-field gel electrophoresis (PFGE). The running
conditions for PFGE were 1 to 28 s for 10 h at 210 V, 28 to 30 s for 10 h at 210 V on 1.0%
agarose gel when digested with enzyme AscI.
The strains were divided into three serotypes: 1/2a (50%), 3a (33%) and 4b (17%).
Altogether 24 PFGE subtypes were observed among these strains. Serotype 1/2a was
divided into 17, 3a into one and 4b into six PFGE subtypes. Most of the cases were sporadic
excluding the strains that were connected to the outbreak caused by the isolates of serotype
3a in butter.




                                              165
EXPOSURE OF LISTERIA MONOCYTOGENES IN AN OUTBREAK
CAUSED BY BUTTER
Riitta Maijala1, Outi Lyytikäinen2 and Tuula Johansson3

Risk analysis1 and Department of Food Microbiology3, National Veterinary and Food
Research Institute, P.O. Box 368, FIN-00231 Helsinki; Department of Infectious Disease
Epidemiology2, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki,
Finland

Problem: The data on the levels of bacteria and the amounts of food consumed from
outbreak investigations allows us to study the effects of exposure of L.monocytogenes. In
1998-1999, an outbreak caused by L. monocytogenes serotype 3a in butter occurred in
Finland. Most of the cases (15/25) were immunocompromized and hospitalized at the
Helsinki University Central Hospital (HUCH) where 7 g butter packages produced by the
implicated dairy were used as an only butter brand.
Method: Based on the data on hospital stay, butter consumption as well as the qualitative
and quantitative analyses of L. monocytogenes in butter, attack rates and exposure were
estimated.

Results: The incubation studies on naturally contaminated butter showed that the levels of
L. monocytogenes found in 7 g butter packages at the time of outbreak detection could
reliably be used for the estimations. The attack rate among HUCH patients varied from 70 to
117 cases per 1000 patient at risk depending on the contamination level of butter (100-60%)
used for the estimations. The highest single dose (7.7x104 CFU per one meal) could have
been enough to cause the listeriosis cases at HUCH. However, this data also supports
another hypothesis, according to which these listeriosis cases were caused by a prolonged
daily consumption of contaminated butter during hospital stay. The estimated daily dose,
based on the hospital kitchen data or the highest detected level in a wholesale sample
(11x103 CFU/g), varied from 1.4x101 to 2.2x103 CFU/day or from 2.2x104 to 3.1 x105
CFU/day, respectively.

Conclusion: The choice of the hypothesis has a crucial impact on the interpretation of this
data for the dose-response estimations. If a foodstuff is typically used for a longer period by
the population at risk, even the low levels of L. monocytogenes can be hazardous for health.




                                             166
VACUUM PACKED GRAVAD OR COLD-SMOKED RAINBOW
TROUT/SALMON ONCE MORE THE CAUSE OF AN OUTBREAK
OF LISTERIOSIS?
Tham W., H. Ericsson, S. Helmersson, T. Netterby, H. Unnerstad, M-L Danielsson-Tham.

Department of Food Hygiene, P.O. Box 7007, Faculty of Veterinary Medicine, SE-750 07
Uppsala, Sweden

During 2000 there has been a notable increase in cases of listeriosis in Sweden. The normal
yearly number of reported cases is 30-35 but by late November we have already 47 cases.
We have also noted an increased occurrence of L. mono cy to gene s in vacuum-packed
gravad or cold-smoked rainbow trout (Oncorhynchus mykiss) and salmon (Salmo salar).
Some fish samples harboured more than 1000 c.f.u. L. monocytogenes per gram. Those
findings have been made in fish sent to different laboratories for routine microbiological
control. Furthermore, the three most common clonal types (REA/PFGE) of L.
monocytogenes isolated from humans during 2000 (by late November) have also been found
in those food products on several occasions. It should be kept in mind that the first rainbow
trout-borne outbreak of listeriosis ever reported took place in Sweden during 1994/95. This
outbreak involved at least 8 individuals.




                                            167
OCCURRENCE OF LISTERIA SPP. AND L. MONOCYTOGENES IN
PORTUGUESE PRODUCTION LINES OF FRESH AND COLD-
SMOKED FISH
Manuela Vaz-Velho..1,3, Gabriela Duarte, G.1 Paul Gibbs1,2
1
 Escola Superior de Biotecnologia, Universidade Catolica Portuguesa, Porto, Portugal;
2
 Leatherhead Food Research Association, Surrey, England; 3Escola Superior de Tecnologia
e Gestao, Institute Politecnico, Viana do Castelo, Portugal

Problem: The overall occurrence of Listeria spp. and L. monocytogenes in processing lines
of three Portuguese cold-smoking plants and their fresh fish suppliers was ascertained.
Results: The overall frequency (n=215) of Listeria spp. in fresh fish and environmental
samples of trout farms (trout farms 1 and 2) and salmon importer was 7.4%. L.
monocytogenes was found in 5 (2.3%) samples. The overall frequencies of Listeria spp. in
environmental sites (n=74) and fish samples (n=141) were respectively 2.7% and 9.9%. L.
monocytogenes was recovered from one sample of the dam water and from two samples of
salmon-trout from trout farm 2 but never from trout farm I. The overall frequencies of Listeria
spp. and L. monocytogenes in salmon samples (n=60) were respectively 6.7% and 3.4%. A
total of 407 samples were collected in the three surveyed factories (Plants, A, B and C). The
overall frequency (n=407) of Listeria spp. was 22.4%. L. monocytogenes was found in 55
(13.5%) samples. The overall frequencies of Listeria spp. in environmental (n=132) and fish
(n=275) samples were respectively 6.1% and 30.2%. L. monocytogenes was present in 8
(6.1%) environmental and 47 (17.1%) fish samples, respectively. The overall frequencies of
Listeria spp. in samples from Plant A (n=178). Plant B (n=134) and Plant C (n=95) were
respectively 27.0%, 17.9% and 20.0%. L. monocytogenes was present in 26 (14.6%), 13
(9.7%) and 14 (14.7%) samples from Plants A, B and C, respectively. Counts of Listeria
spp., determined by Most Probable Number (MPN) technique in 58 samples of cold-smoked
salmon and salmon-trout, ranged from 0.16 to 44 MPN of microorganisms per gram of fish.




                                             168
LISTERIA MONOCYTOGENES ON A DAIRY FARM
Elisabet Waaka, Wilhelm Thamb and Marie-Louise Danielsson-Thamb

a
 Arla Foods IoM, S-105 46 Stockholm, Sweden, b Department of Food Hygiene, P.O Box
7007, Faculty of Veterinary Medicine, SE-750 07 Uppsala, Sweden.

Problem: Due to a high incidence of Listeria monocytogenes in silo milk at a Swedish dairy
an investigation of possible sources were investigated. After thorough detective work one
farm was identified as delivering milk often contaminated with Listeria. The purpose with this
investigation was to study to what extent this farm was contaminated with L. monocytogenes
and to characterise and compare the isolates by use of a strain discriminating method.

Methods: The investigated farm had about 40 dairy cows. A total of 173 faecal samples, 144
pooled samples of milk (576 quarter samples), 14 samples of silage and 5 samples of other
feed such as sugar beet pulp and feed grain were taken at the farm. Milk and faecal samples
were sampled from the cows on five occasions from 1998 to 1999, during the outdoor
season in May, June and September and during the indoor season in November and March.
Samples of silage and other feed were taken four times in June, September November and
March. Milk samples were taken aseptically from the teats of each cow. The sampled volume
from each teat was approximately 10 mL. Faecal samples of approximately 100 g were
collected via rectal retrieval. The grass silage used as a feed was sampled at the same time
as the sampling of the cows. Silage was taken from different parts of the bunker silos, from
the centre as well as from the top and edges. Listeria was analysed both qualitatively and
quantitatively in all samples. Restriction enzyme analysis (REA) and Pulsed field gel
electrophoresis (PFGE) was performed on all isolates of L. monocytogenes.

Results: L. monocytogenes was found in samples of silage, faeces and milk. The
occurrence varied depending on the season, with the highest incidence in spring. Two cows
had a subclinical mastitis. One cow shed L. monocytogenes at a level of log 4 CFU mL-1, the
other cow shed L. innocua at a level of log 5 CFU mL-1. Other milk samples were only
positive by qualitative analysis. Several different clonal types of L. monocytogenes were
identified at the farm. Identical clonal types were isolated in silage and faeces samples.
Identical clonal types were also found in milk samples from the same cow during several
months.

Conclusion: It might be hypothesised that a minority of dairy farms contributes to the
majority of Listeria contamination in silomilk.




                                             169
TWO CONSECUTIVE NATIONWIDE OUTBREAKS OF
LISTERIOSIS, FRANCE, OCTOBER 1999 - FEBRUARY 2000
Veronique Vaillant 1, Henriette de Valk 1, Jocelyne Rocourt 2, Frederic Stainer 3, Olivier
Pierre 4, Veronique Goulet1.

1
 Institut de Veille Sanitaire, St Maurice ; 2 Centre National de Reference des Listeria, Institut
Pasteur, Paris ;3 Direction Generale de l'Alimentation, Paris;4 Direction Generale de la
Concurrence, de la Consommation, et de la Repression des Fraudes, Paris

Problem : In France, listeriosis surveillance is based on the routine molecular typing of
isolates and mandatory notification of all culture confirmed cases with routine collection of
the food history. In January 2000 a small nationwide outbreak of listeriosis involving 10
cases received extensive media attention when the incriminated product "rillettes" was
withdrawn from the market. In the aftermath of the recall, a second nationwide outbreak of
listeriosis occurred caused by a Listeria strain different from the strain of the first outbreak.
Methods :To identify risk factors for infection we carried out a case control study. A case
was defined as a patient, resident in France, from whom the outbreak strain was isolated
from a usually sterile site, between November 1, 1999 and March 1, 2000. Controls were
sporadic cases of listeriosis infected with a strain other than the strains involved in the
present and previous outbreak, diagnosed in France from December 1,1999 to March 1,
2000. At the moment of their illness, all cases and controls had been asked about underlying
medical conditions and consumption of 74 specific foods in the 2 months prior to their illness,
as part of the routine enhanced surveillance of listeriosis.
Results :A total of 32 cases were identified between November 12, 1999, to February 28,
2000. The case control study showed that the consumption of pork tongue in jelly was
strongly associated with infection with the outbreak strain ( OR: 75.5; 95 % CI: 4.7, 1216.0).
However, trace back results did not allow to incriminate a single manufacturer of pork tongue
in jelly and the outbreak strain has not been isolated from the incriminated food, nor from any
production sites. The consumption of pork tongue in jelly was discouraged on
epidemiological evidence alone.
Conclusion : The consecutive occurrence of these two outbreaks confirms the epidemic
potential of listeriosis even in a context of decreasing incidence and underlines the
importance of timely case reporting and systematic typing of human L. monocytogenes
strains allowing early detection and separate investigation of different clusters.




                                              170
THE PREVALENCE AND THE BIODIVERSITY OF LISTERIA
MONOCYTOGENES IN BIRD FECES IN FINLAND
S. Hellström, T. Autio, J. Lunden, H. Korkeala

Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, P.O. Box
57, FIN-00014 Helsinki University, Finland

Problem: Listeria monocytogenes is a common bacterium in the nature and in the feces of a
wide variety of wild animals. Birds carrying L. monocytogenes may disseminate the
bacterium in our environment. The aim of this study was to examine the prevalence and the
biodiversity of L. monocytogenes in Finnish birds.
Methods: Fecal samples of 112 birds were examined. A total of 84 samples were collected
from urban sites and 28 samples from the municipial solid waste landfill site. The samples
were analyzed according to ISO 11290 method and additional cold enrichement was
performed. Identification was made both by gram staining, catalase reaction and using API
Listeria and also by multiplex-PCR. The primers used in the PCR were specific for Listeria
16S rRNA sequence and L. monocytogenes listeriolysin O sequence. L. monocytogenes
isolates were characterised by pulsed-field gel electrophoresis (PFGE) using enzymes AscI
and ApaI.
Results: L. monocytogenes was recovered from 21 (23 %) samples. 16 of these samples
were collected from landfill site and 7 from urban sites. Prevalence of L. monocytogenes in
bird feces in landfill site was 57 % and in urban sites 8 %.
The PFGE-analysis of 89 isolates revealed 27 genotypes. Four samples revealed more than
one PFGE-type. Samples collected from landfill site and from urban sites had two identical
PFGE-types.
Conclusion: L. monocytogenes is commonly found in the feces of the Finnish birds. The
prevalence is higher in landfill site than in urban sites. Biodiversity of.L monocytogenes
among Finnish birds is high.




                                             171
A POINT-SOURCE OUTBREAK OF LISTERIA MONOCYTOGENES
LINKED TO WHIPPING CREAM.
J.M. Farber1, F.J. Pagotto1, E. Daley1, S. Kopil1, A. Hughes1, J. Drew2, J. Wylie2,
S. Giercke2, D. Nowicki2, S. Harlos3, G. Hammond4, and J. Kettner5.

1
 Bureau of Microbial Hazards, Food Directorate, Health Canada, Ottawa, Canada, K1A 0L2;
2
 Food Protection, Manitoba Health, Manitoba, Canada; 3Winnipeg Regional Health Authority,
Manitoba, Canada 4Public Health Branch, Manitoba, Canada; 5Chief Medical Officer of
Health, Manitoba, Canada.

Problem: A point source outbreak of gastrointestinal and generalized illness caused by
Listeria monocytogenes was associated with a church gathering where the food history
indicated canned whipping cream as the implicated food. An investigation into the growth
and survival of L. monocytogenes in whipping cream was undertaken.
Method: Molecular typing of isolates from blood, throat and stool samples revealed
indistinguishable pulsed-field gel electrophoresis (PFGE) and random amplified
polymorphic DNA (RAPD) patterns. L. monocytogenes isolated from a can of whipping
cream had identical PFGE patterns as the case isolates. Challenge studies were
undertaken, using pressurized (50-55 psi) versus non-pressurized cans of whipping cream.
Containers were inoculated with 102 CFU per gram of L. monocytogenes, and samples
were then incubated at 8 and 220C for 4 weeks.
Results: L. monocytogenes was able to grow in whipping cream under pressurized and
non-pressurized conditions at 8 and 220C, respectively. There did not appear to be any
difference in the growth rate of L. monocytogenes in the non-pressurized or pressurized
whipping cream, regardless of storage temperature. However, L. monocytogenes did grow
faster in whipping cream stored at 220C, as compared to 80C storage.
Conclusions: This represents the first report of a listeriosis outbreak linked to the
consumption of whipping cream. This points out that any food vehicle which can support
the growth of the organism is capable of causing foodborne listeriosis.




                                          172
CRITICAL POINTS FOR LISTERIA MONOCYTOGENES
CONTAMINATION IN SALMON SLAUGHTERHOUSES AND
FILLETING PLANTS
Liv Marit Rorvik 1 2, Heidi Kjos 2, Ragna Heggebo2

1
 National Veterinary Institute, Pb. 8156 Dep., 0033 Oslo, Norway Norwegian School of
Veterinary Science, Pb 8146 Dep., 0033 Oslo, Norway


Ready-to-eat fish products like smoked salmon, as well as raw materials like slaughtered
and filleted salmon, are sporadically contaminated with Listeria monocytogenes. Several
investigations have shown that products are contaminated with the bacteria during
processing, and that certain strains of L. monocytogenes may colonize the processing
environment for years. The sources for L. monocytogenes to the processing environment
have not been determined. The aim of this study was to examine possible sources and
critical points for L. monocytogenes colonization on the processing line in salmon
slaughterhouses and filleting plants.
Samples of sea-water outside the plants and from the transport boats, ice, and living salmon
were examined for L. monocytogenes. Processing equipment and environment, as we\\ as
products of 24 slaughterhouses or filleting plants were also investigated. Conclusions: Sea-
water and living fish do not appear to be sources for L. monocytogenes to the processing
environment, which is consistent with earlier studies. Cooling vats, storing vessels and
conveyer belts are critical points for L. monocytogenes contamination in the processing
plants.




                                            173
law, regulations, prevention




             174
U.S. FDA/FSIS DRAFT ASSESSMENT OF THE RELATIVE RISK
TO PUBLIC HEALTH FROM FOODBORNE LISTERIA
MONOCYTOGENES AMONG SELECTED CATEGORIES OF
READY-TO-EAT FOODS.
R. E. Brackett and R. L. Buchanan

U.S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
200 C-Street, SW
Washington, DC, USA 20204


As part of its ongoing efforts to decrease the incidence of foodborne listeriosis in the United
States, the U.S. Food and Drug and Administration in collaboration with the USDA Food
Safety and Inspection Service conducted a quantitative microbial risk assessment on the
public health impact that Listeria monocytogenes contamination of ready-to-eat foods. The
relative public health risk of twenty categories of ready-to-eat foods was assessed following
the framework for the conduct of microbial risk assessments outlined by Codex Alimentarius.
The risk assessment was conducted using advanced mathematical modeling techniques so
that probabilistic models for both the exposure of consumers to foodborne L. monocytogenes
and the likely public health impact of those exposures could be estimated. The exposure
assessment was based on L. monocytogenes levels at retail that were subsequently
corrected for home storage in order to estimate pathogen level at the time of consumption.
The results of the exposure assessment served as the input of the hazard characterization
that considered the dose-response relationship, including differences in susceptibility among
the general population, prenatal and neonatal children, and the elderly. Differences in the
virulence of different L. monocytogenes isolates were also considered. The quantitative
results of the assessment were presented in three formats; relative risk per 100,000,000
servings, relative risk ranking per serving, and relative risk ranking per annum. The risk
assessment has been made available in a draft form in order to acquire additional data and
suggestions on how the assessment can be improved. The risk assessment is available at
http://www.foodsafety.gov/~dms/lmrisk.html.

CD-Homepagefile:




                                               175
RISK COMMUNICATION
Helga Odden Reksnes,

Director of Communication
Norwegian Food Research Institute

Risk communication and food safety are closely connected. For many consumers the
decision-making at the food counter is not based up on actual findings in food laboratories or
decisions made by local or global bureaucrats – it is a story beginning and ending with their
empathy for and trust related to the product, the producers, the authorities and the
manufactures. And this is again related to the different organisations’ willingness and ability
to practice communication, in particular risk communication. Within the international scientific
framework of risk analysis, risk communication has the recent years been acknowledged as
playing an important part, often decisive with regards to the outcome or effect of risk
assessment and risk management on public opinion and behaviour. Both WHO/FAO, the
Codex System and EU and other multinational bodies have given priority to the elaboration
of models within risk analysis – all more or less recognising the importance of risk
communication as an integrated part of the process. But what does this mean – how can risk
communication become an important tool in risk analysis, moving from statements in various
documents to significant contribution to an appropriate end result. Becoming more than open
and transparent distribution of scientific complex documents for all interested and affected
parties to comment on? And involving reliable and relevant stakeholders in the risk
assessment and risk management processes. The focus so far has been mainly on the
communication of risk not on risk communication. It is time to also concentrate on what
happens within the risk analysis process; the interaction between assessors and managers
and how the different interested/affected parties are actually involved in the stages of risk
analysis. The process of communicating the risk is but a tiny part at the end of the risk
analysis process.

I will focus upon the following themes:
1. What is risk and how does risk materialise in the food sector?
2. What is communication and how can we become excellent communicators?
3. Why risk communication?
4. Risk communication in risk society
5. A risk communication methodologyRisk, (of Greek Rhiza; clip or rock) is originally a
maritime expression. It dates back to the 16th century and has to do with navigating ships in
dangerous waters. Risk in today’s natural science food safety world is defined as ”a function
of the probability of an adverse health effect and the magnitude of that effect, consequential
to a hazard in food”. Somewhat different from the original meaning but nevertheless still
expressing what is typical of all risk issues: There is no 0-risk. There’s always an element of
insecurity, the need to define the probability and the magnitude. However, facing the
challenges of food safety in modern society, this technical definition of risk needs modifying;
we must introduce another factor caused by individual risk perception based on personal
values, social background etc. and perhaps caused by mistrust to science and governmental
bodies. Let’s call it the human outrage factor. Another challenge is the globalisation of risk.
International trade with food travelling fast, viruses spread with people travelling fast,
demand global approaches and solutions regarding risk assessment and risk management.
                                              176
2. What is communication and how can we become excellent communicators?
The word communication is derived from Latin ”communicare”, meaning common, to share,
indicating a process having joint action as its purpose. To communicate with somebody is a
complex process where many factors influence the outcome. To communicate means
sharing visions, objectives, attitudes, knowledge, information and opinions. The
communication process involves a transmitter or a sender, a receiver, a message and a
channel. If communication is successful, the receiver gives feedback to the
transmitter/sender in order to acknowledge that the message is received, interpreted and
understood.. Who says what to whom through which channels have become very
complicated issues in our information society. The amount of information forces most people
to spend a lot of time just sorting out what issues to concentrate on, what sources to survey
and trust and which channels to use. Just imagine yourself – how you pick up fragments of
the morning paper sorting through your email while checking today’s appointments,
attending a meeting while reflecting on what to stress in the next, who to call in the coffee-
break etc. How many of you are actually concentrating on what I’m saying just now? One
person trying to communicate with 50 persons at the same time is indeed a challenge…
In order to become excellent communicators, we need to concentrate on the message, the
medium and the audience. We must communicate on the receiver’s terms. The slogan of
consumerism – “the consumer is the queen of the ball” – applies also when is comes to
communicating with the consumer. She makes her choices at the food counter every day
based on a number of fragments of information from many different sources. The most
important objective for all serious players in the food field must be to assist the consumer in
her risk assessing and risk managing process, optimising her choice according to her values,
quality awareness, personal taste and at the same time securing that the food meets the
food standards and regulations. And following global rules and regulations…

3. Why risk communication?
     It’s a consumer right! A democratic right to know about the facts of food and food
       production (scientific process, numbers, estimates, basis for decisions etc.)
     Better health – the preventive effect. A contribution to the possibility for the consumer
       to make sensible and healthy food choices.
     Gain trust. To ensure legitimacy for the professional bodies involved in risk analysis
       nationally and globally (politicians, food safety administration and scientific bodies)
       through transparency and openness.
     Prevent outrage. By opening closed arenas and hidden processes, visualising what
       could otherwise create anxiety or give room for speculations and making harmless
       “the harmful”, the unknown.
     Find better solutions. Handling risks by involvement-strategies results in increased
       understanding and insight, shared responsibility and participation in decision making.
     Obtain understanding and support. Getting feedback through joint arenas, exchanging
       views and opinions and getting acquainted with each other’s points of views, learning
       to respect the other interested parties.
Communication is vital for obtaining goals, in order to gain trust and ensure that different
measures have the intended impact. Without communication, joint action is impossible. Joint
action is an absolute must within the risk analysis framework. In these situations, actors are
present on different levels and represent many sectors and interest. Make up a list of
stakeholders relating to the Listeria-issue and I can assure you that the list will be incredibly
long. Without strategic and planned communication, it’s impossible to involve those who are
affected by the risk analysis.
                                              177
4. Risk communication in risk society
The German sociologist Ulrich Beck coined the term risk society in his book
Risikogesellschaft; Auf dem Weg in eine andere Moderne, 1986. In short, what we know as
the modern industrial society, Beck claims, has arrived at a new stage, not bound for a post-
modern society as was to be expected, but for a second edition of the modern industrial
society. The characteristic of this second stage is risk, hence the concept risk society. In this
society, shaping values is systematically followed by shaping risks. Beck is especially
occupied with the role of experts and how they view the public and he states that “science is
one of the causes, the medium of definition and the source of solutions.” Defining acceptable
risks on the basis of mathematical or technical motivations is impossible according to Beck.
– Who’s to say who’s right, asks Beck and underlines that participation in the risk
assessment process is very important. He states that handling risk could lead to
democratisation of democracy. And here we are right in the middle of the risk analysis plate.

No matter what issues are taken with Beck’s rather radical views on experts and
technologists, there’s no getting away from the fact of Beck very aptly describing and
explaining a social development for all to see, and leaving its mark also in the food sector.
Man-made risks associated with food have become every day issues, the mad cows and
genetically modifies food are both good examples. The recent Foot and Mouth disease-
outbreak in Europe has demonstrated the vulnerability of modern society, the enormous
costs related to handling the disaster and the social implications for those affected and the
community as a whole. The public has developed a sound scepticism to expertise and
science and barriers between the unlearned and learned have become more and more
evident. Aside from voicing the concerns of the public, media has entered the scene as an
unbiased social debater. Science and expertise have taken on a new role in social
development, the meaning of which they are unable to convey. All of this having an impact
on public authority and all other actors taking part in the risk analysis process.
Communication may be the essential tool to succeed in risk society and risk analysis, in
particular risk communication.

1. A risk communication methodology
Risk communication has often been regarded as an adjunct to risk assessment and risk
management. Like a three step rocket; first the experts assess the risk, than the
administration manage the risk and at the end somebody, usually not very clearly defined,
tries to communicate the result. Therefore, risk communication work has aimed at educating
the general public. The belief has been that if the public is informed properly, it will act
rationally. Experience and practise teach us that this way of defining risk communication has
not given the wanted outcome. Furthermore, it is rather the experts, administration and
business sector that are in need of training in communication. Risk communication must
provide a continuous contact between all parties concerned with assessing and managing
the risk. Risk communication must also involve all interested parties, help improve
transparency of decisions and increase the potential level of the acceptance of the outcome.
The Norwegian Food Safety Risk Communication Programme developed a model to
illustrate how risk communication functions as contact film between risk assessors and risk
managers and opening the risk analysis process to public participation and evaluation.

An FAO/WHO expert consultation in Rome 1998, defined risk communication putting the
phenomenon into the risk analysis perspective and identified factors influencing risk
perception, principles for risk communication, barriers and strategies. According to the
                                              178
experts risk communication can be defined as follows: Risk communication is the exchange
of information and opinions concerning risk and risk-related factors among risk assessors,
risk managers, consumers and other interested parties. This 1998-report has been quoted
on many occasions and many of you are probably tired of hearing this message. As I
mentioned initially, there’s a strong need for concrete measures and initiatives. Within the
framework of the Norwegian Food Safety Risk Communication Programme, here are some
ideas presently being developed in collaboration with relevant national and international
bodies.

1. Establishing an interdisciplinary forum to develop risk communication as a proper
   scientific discipline, especially with regards to the risk analysis process. This means
   establishing binding contact between social sciences and natural sciences nationally and
   internationally to ensure mutual understanding and knowledge sharing between the
   disciplines in order to create the best possible professional platform for risk
   communication. Such a forum should evaluate and develop interaction/communication
   methodology between risk assessors and risk managers, with a view of presenting such
   interactions to a broader public (consumers and other interested parties). The forum
   should meet regularly and develop risk communication models related to specific risk
   topics and processes.

2. Establish a system for environmental scanning and mapping and analysing existing
   relevant material nationally and internationally regarding risk communication and
   consumer perceptions, initiate new investigations when needed. This means adopting the
   environmental scanning tool as a means to identify scientifically important risk topics
   (emerging diseases, food borne crisis etc) and/or perceived risk topics within the
   administration, in the scientific community or among consumers, mapping needs and
   desires within the same target groups, developing and implementing suitable tools for risk
   communication. This could also imply setting up a specific databased web to present
   data/surveys to ensure knowledge transmitting and better utilisation of data.


3. As a result point 1 and 2, it would be possible to develop and test different approaches
   and tools for excellent risk communication. This means developing

    programmes for educating government administration and scientific bodies, possibly
       also the private food sector,
    web-based communication arenas to cover important target groups need for
       communication related to risk and food safety,
    methodology for assessing the effect of risk communication efforts, both in the
       developing and the developed world
    suggestions for clear and simple communication of risk assessments.
There should be a potential for development of applying risk communication methodology to
present international food safety initiatives (including FAO/WHO Codex Alimentarius
Commission work) to a broader public. There is also a potential for crossover learning effects
from food safety approaches to other risk topics.

Starting with Listeria?
The last two years, risk analysis initiatives related to Listeria have been prioritised both within
the EU-system, the Codex Committee on Food Hygiene with a solid knowledge base from
expert consultations organised by FAO/WHO. An initiative was taken by WHO Food Safety
                                               179
Programme in 2000 to investigate the nature of the interaction between assessors and
managers of Microbiological hazards in Food within the risk analysis framework. This
represents a new approach and understanding of risk communication - From communicating
risks to risk communication. The assessment of Listeria monocytogenes is in progress:
invitation for all interested parties to review the relevant reports on hazard identification and
hazard characterization and exposure assessment of Listeria monocytogenes in ready-to-eat
foods are published on the website of the WHO Food Safety Programme. Principles and
guidelines for microbiological risk management are climbing the steps in the Codex system
pushed forward by members of the Codex Committee on Food Hygiene. The same
committee are developing guidelines for the control of Listeria monocytogenes using the risk
analysis structure. Now is the timing perfect for a risk communication initiative, a case study
to be implemented along with the ongoing joint risk assessment/risk management process
related to Listeria monocytogenes.

Closing remarks
The food world is complex. The many various players and stakeholders on the scene result
in different statements, attitudes and moves. They frequently appear to clash due to being
based on various institution roles. Sometimes the messages actually clash, expressing
conflicts of interest, disagreement over scientific matters etc. Mass media are, and must be,
criticising of public authorities and business, also concerning food. Conflicts are good news.
Attempts at withholding information from being published, better still. The unbiased
information given by one public specialist is no longer sufficient. Anti-expertise is frequently
provided, the media compounding and reinforcing the conflicts. Yesterday’s truth becomes
tomorrow’s lie. Zero risk no longer exists. Food safety has proved a flexible concept. The
whole responsible food sector has to produce confidence with the consumer by putting their
cards on the table and entering into a dialogue with both each other, the media and the
consumer. Differing views by various players should be presented in a factual way, without
antagonists getting their hackles up.

Risk society has changed the role of the authorities and affects the roles of all the players in
the food field. Efficient solutions demand joint action. The significance of good relations and
effective networks between the parties concerned require knowledge about communication
as an agent and the ability to communicate. Knowledge about target groups, attitudes and
behaviour is decisive. Risk contains much more than just mathematically measurable
quantities and should not be left to experts alone to define. The public are conducting their
own risk analysis when buying or preparing food every day. An able risk communicator will
take this into consideration when judging risk from his or her scientific, administrative or
business position. The globalisation of risk means that the main playing field no longer is on
a national level. The international institutions must develop a sound framework for risk
analysis in food safety making sure that all stakeholders on a global level are involved in the
process. Risk communication should be an important tool in this framework.




                                              180
Literature
The most important sources of this paper are:

   1. Different papers from the project The Norwegian Food Safety Risk Communication
      Programme 1997-1999 (D.H. Reksnes, Educate the administration – not the
      consumers, Risk communication; H.Reksnes, Genetically modified food – much more
      than sheer science).
   2. Nordic Council of Ministers; Communication as a challenge to Food Control
      Authorities, TemaNord no. 585, 1998.
   3. Lowe Hedman; Trygga konsumenter, trovärdige producenter och tilförlitliga medier.
      En utvärdering av Kommunikasjonsprogrammet Trygg mat (Evaluation of the
      Norwegian Food Safety Risk Communication Programme), Uppsala University,
      Information Science, Media and Communication Section, april 1998 (Swedish only)
   4. FAO/WHO Expert Consultation on the Application of Risk Communication to Food
      Standards and Safety matters, Rome, February 1998
   5. Ulrich Beck, Risk Society; towards a New Modernity, Sage Publications, 1992




                                           181
LISTERIA MONOCYTOGENES IN DOMESTIC ENVIRONMENTS:
RECOMMENDATIONS OF THE INTERNATIONAL FORUM ON
HOME HYGIENE
RR Beumer1,2, SF Bloomfield1, M Exner, GM Fara1, KJ Nath1 and E Scott1.

1 International Forum on Home Hygiene, PO Box 1106, 1211 Geneva, Switzerland
2 Laboratory of Food Microbiology, Wageningen University, PO Box 8129, 6700 EV
Wageningen, The Netherlands.

Introduction
The International Forum on Home Hygiene (IFH), a non-profit organisation, comprises
scientists and hygiene professionals active in hygiene policy and scientific research. It was
formed because these experts believe there is an unmet need to raise awareness of the role
of domestic hygiene in the prevention of infections acquired in the home.

Listeria monocytogenes
Since Listeria spp. are commonly found in the environment, the presence of these organisms
in the domestic environment is not surprising. L. monocytogenes was found in dish cloths,
bathrooms, kitchen sinks, washing up brushes, tooth brushes and refrigerators (1). Humans
are exposed to this pathogen on a regular basis, because of its ubiquity in food products and
the (domestic) environment. However, until now there was no evidence that domestic sources
of L. monocytogenes play an important role in the contamination of people and their foods.

Recommendations for suitable hygiene procedures
As far as dish cloths are concerned it is considerd that disposable cloths should be used
wherever possible. For reusable cloths, detergent and water washing alone is not considered
sufficient to achieve a hygienic state. As a routine, all non-disposable dish cloths and sponges
should be made hygienic with one of the following procedures: rinsing with detergent and hot
water followed by 1) immersion in hot water (2 min 90ºC); 2) a dishwash cycle (60ºC); 3)
laundering at 60ºC or 4) application of a disinfectant (see: guidelines on the selection and use
of disinfectant products) (2).

References
1 Beumer RR, Te Giffel MC, Spoorenberg E and Rombouts FM (1996) Listeria species in
   domestic environments.
 Epidemiology and Infection 117 437-442.
2 Anonymous (1991) British standard document BS 7152. Guide to choice of chemical
   disinfectants. London: BSI
 Standards.




                                              182
CONVENTIONAL AND NOVEL STRATEGIES FOR INHIBITING
LISTERIA IN FOODS
Elliot T. Ryser, Department of Food Science and Human Nutrition, 2108 S. Anthony Hall,
Michigan Slate University, East Lansing, MI 48824-1225, USA

Food manufacturers rely on a wide variety of preservation and novel processing techniques
to ensure production of a safe and wholesome product with a suitable shelf-life. Various
physical (e.g. heating, cooling, freezing, irradiation), chemical (e.g. salt, organic acids, fatty
acids, nitrite, liquid smoke, herbs/spices, sanitizers) and biological (e.g. fermentation,
bacteriocins) food preservation strategies have been developed over many years to
minimize growth and survival of Listeria as well as other pathogenic and spoilage organisms
in foods. These methods are most effective when applied simultaneously or sequentially
which is the basis for the widely recognized "hurdle concept" in food microbiology. Given the
all-to-frequent occurrence of Listeria monocytogenes as a post-processing contaminant in
ready-to-eat foods such as processed meats, various thermal and non-thermal post-
pasteurization strategies have been recently developed to enhance the safety of these
products. Emphasis also has been given to new packaging strategies (e-g- modified
atmosphere, vacuum packaging, antimicrobial films) that will minimize growth and survival of
Listeria in ready-to-eat food products during extended refrigerated storage In addition to
these conventional preservation packaging methods, a growing number of novel non-thermal
processing techniques are also being investigated to meet consumer demand for minimally
processed foods with fresh-like taste- Some of these novel techniques include high pressure
processing, pulsed electric field, pulsed light and oscillating magnetic fields.




                                               183
INACTIVATION OF LISTERIA MONOCYTOGENES CA AND OHIO2
IN ORANGE JUICE AND MILK BY HIGH HYDROSTATIC
PRESSURE (HHP)
Hami Alpas, Faruk Bozoglu

Food Engineering Department, Middle East Technical University, Ankara, 06531, TURKEY

Problem: Inactivation of pressure resistant strains of Listeria monocylogenes in orange juice
and milk by using High Hydrostatic Pressure (HHP).

Method: Our previous work* has shown that among nine different L. monocytogenes strains,
CA and Ohio2 were more resistant to pressure at 345 MPa for 5 min at 25°C. The food
samples were inoculated with cells of both strains at early stationary phase to obtain about
108 cfu/ml food sample. Pressurization of food samples were carried out at 345 MPa at 50°C
for 5 min excluding pressure increase and release time. Pressurized milk and orange juice
were first stored at 4°C for 24 h to see the effect of chilled storage and then at 37°C for an
additional 48 h to satisfy the optimum growth condition for Listeria. After pressure treatment
duplicate 0.1 ml volumes were surface plated onto Tryptic Soy Agar (TSA) plates. The
number of surviving organisms was enumerated as c.f.u after incubation for 48 h at 37°C.
For shelf-life studies, pressurized samples were streak plated on duplicate plates on
prepoured non-selective (TSA) and selective (Modified Oxford, MOX) agar media plates. The
plates were incubated at 37°C for 48 h and examined for presence or absence of pathogens.

Results: The viability of both pressure-resistant Listeria strains was reduced by more than 8
log cycles in orange juice and milk respectively right after pressurization. In addition, no
growth was seen for these species on MOX agar plates after storage at 4°C for 24 h and
even at 37°C for an additional 48 h in orange juice. It is evident that HHP processing of
orange juice to inactivate Listeria strains has the added advantage that it sensitizes the cells
to high acid conditions, which results in more that 8 log cycle reduction in viability. In
pressurized milk, no growth was seen after storage at 4°C for 24 h however after storage at
37°C for and additional 48 h both strains grew on selective media plates. This shows the
recovery of the injured cells of Listeria at high temperature.

Conclusion: From the results of this study it can be concluded that high hydrostatic
pressure pasteurization (345 MPa, 50°C, 5 min) applied to high acid foods and for low acid
foods can be successfully applied to obtain high levels of destruction (more than 8 log
cycles) of Listeria monocytogenes Ca and Ohio2. The recommended shelf-life of these
products can also be extended even at elevated storage temperatures.

* Alpas et al. "Variation in resistance to hydrostatic pressure among strains of food-borne
pathogens". Applied and Environmental Microbiology, 65, 4248-4251,1999.




                                              184
RESULTS SINCE THE INTRODUCTION OF LISTERIA
SURVEILLANCE IN HUNGARY
R. Kiss1, K. Krisztalovics2. M. Füzi3:


1. National Center of Public Health, National Institute of Food Hygiene and Nutrition,
   Departement of Microbiology
2. National Center of Epidemiology, Departement of Epodemiology
3. National Center of Epidemiology, Departement of Microbiology


        Oure surveillance activity, functioning since several decades in Hungary got
expanded - among others - with the compulsory reporting of Listeria. Data are handled by
National Center of Epidemiology, Departement of Epodemiology and are reported weekly in
the publication EPINFO.
        Listeria monocytogenes strains isolated in the course of listeriosis cases are sent by
the laboratories to the Laboratory of National Center of Epidemiology, Departement of
Microbiology for confirmation and sero-typing. Listeria monocytogenes examinations from
food - which are performed in cooperation with some Public Health Laboratory Service Food-
microbiological laboratories - in oure laboratory at the National institute of Food Hygiene and
Nutrition, Departement of Microbiology since 1989 - became compulsory according to the
decree of the Ministry of Health in relation of milk products, since 1999.
        Discipline of reporting is not yet satisfactory, but with the compilation of a data-sheet
we wish to give help to professionals for the examination of listeriosis and of L.
monocytogenes contaminated products.
Data-sheets help to study the environment of the patients, to get to know their mutrition
habits, their connection with animals or consumption the contaminated foods, etc. They
include the methodology of the identification and the circumstances of food production.
These data-sheets were presented to some Public Health Laboratory Service laboratories in
the course of reading papers and reporting our results but as in such forums hospital
microbiologists take part some lectures too, so the isolated strains get to us from Public
Health Laboratory Service human- and food- and hospital-microbiological laboratories.

      Listeria monocytogenes strains were isolated by the Public Health Laboratory
Service in the course of supervision of milk factories or when examining samples taken in
shops, in such cases we helped the elimination of contamination with factory, environmental
and product control.
Environment-studies are more difficult in the case of death due to Listeria monocytogenes.

According to our experiences, our work helps successfully the detection of Listeria cases
and food safety. We are waiting for the ordaining of the examination of meat-products,
dishes and ready to eat food, raw form which may complete our knolidge relating to L.
monocytogenes contamination of our foods. As the HACCP system is compulsory on the
place of the production of food in Hungary, several factories have own microbiological
laboratories or make supervision their supplied and delivered products by outside
laboratories, the factory environment and the hygiene of workers also with there the from of
controls to help the elimination of listeriosis gets gradually expanded. Listeriosis cases of the
given period, positive haman, food and environmental samples, the serotype distribution of
the examined sterains are presented in tables.

                                              185
Year       No. of listeriosis       letality
1998       25                       0.05
1999       14                       0.04
2000       4                        0.04
2001       2

Ages     1998         1999
Newborne 6            2

6-9          1        1
20-49        7        2
50-          12       9

listeria    pos. blood          vagina         androl    tisu   other   liquor   all
sample
1998              7             6              24        2      10      13       62
1999              5             1                        1      11      4        22

other: pus, throat swab

Human serotypes: 1/2a, 1/2b, a, 4b, 4ab


Food samples:
row milk, pasteurised milk, pasteurised cocoa, cheese, salami, vegeteble
serotypes: 1/2a, 1/2b, 1/2c, 4a, 4b

The serotyping was performed by the use of DENKA-SEIKEN sera




                                                        186
MICROBIOLOGICAL CRITERIA FOR LISTERIA
MONOCYTOGENES IN FOODS.
Birgit Nørrung, DVM, PhD,

Institute of Food Safety and Toxicology, Danish Veterinary and Food Administration Mørkhøj
Bygade 19-DK 2860 Søborg.

Introduction: Listeria monocytogenes is a bacterial pathogen causing serious illness as
septicaemia and meningitis in humans. Although listeriosis occurs infrequently at an
incidence below 10 cases per million the fatality rate is rather high, up to 75% in highly
susceptible individuals such as immunocompromised individuals suffering from cancer,
AIDS, etc. In contrast the fatality rate is often low in persons without predisposing factors. In
general the fatality rate is often referred to as approximately 20 %. Although other modes of
transmission exist, foods have been clearly identified as a primary source of infection. The
high prevalence of L. monocytogenes in foods in general, together with a high mortality rate
of listeriosis suggest that L. monocytogenes represents an important hazard to human
health. Consequently, the occurrence, spread, growth and survival of L. monocytogenes in
foods and food environments have to be controlled.
Microbiological criteria for L. monocytogenes in foods: From available data on risk
assessment, it is concluded that the level of L. monocytogenes consumed is an important
factor affecting the incidence of listeriosis. Foods that do not support growth of L.
monocytogenes are less likely to be a source of listeriosis, whereas foods that support
growth to high levels, should be the target of risk management efforts. Based on current
epidemiological information from several countries, a concentration of L. monocytogenes not
exceeding 100cfu/g of food at the time of consumption seems to be of low risk to the
consumer. In order to evaluate the impact on human illness of different numbers (from
absence in 25g to 1000/g) of L. monocytogenes in ready-to-eat foods, the Codex Committe
on Food Hygiene (CCFH) has asked the FAO/WHO to initiate risk assessment work in this
area. This work is just about to be finalized at a Joint Expert Meeting on Microbiological Risk
Assessment to be held in Rome from the 30th of April to the 4th of May. The outcome of this
work will hopefully be useful in the preparation of future microbiological criteria for L.
monocytogenes in foods.

In several countries, criteria or guidelines for tolerable levels of L. monocytogenes in ready-
to-eat foods have been established (Gravani 1999). Some countries like USA and Italy
require absence of L. monocytogenes in 25 g of foods (zero tolerance) while other European
countries (e.g. Germany, the Netherlands, and France) have a tolerance of below 100 cfu L.
monocytogenes per g at the time of consumption. Finally some countries e.g. Canada and
Denmark, have a tolerance of below 100/g for some foods and a zero tolerance for others,
especially those which are supportive of growth and with extended shelf-lives (Farber et al.
1996, Nørrung et al. 1999). Several countries have concluded that a complete absence of L.
monocytogenes for certain ready-to-eat foods is an unrealistic and unattainable requirement,
that limits trade without having a positive impact on public health and consequently might
detract resources form other potentially more efficient measures against L. monocytogenes.
Therefore there is a need for international microbiological criteria for L. monocytogenes in
foods, under special consideration of risk assessment approaches. At present such criteria
are in preparation in both the CCFH and in the Commission of the European Communities.

                                              187
Details in the present legislation within the EU and the ongoing discussions and principles
within the documents under preparation in the CCFH and the Commission of the European
Communities will be presented.

References
Farber, J.M., Harwig, J. (1996) The Canadian position on Listeria monocytogenes in ready-
to-eat foods. Food Control, 7, 253-258.

Gravani (1999) “Listeria in Food-Processing Facilities- Status of L. monocytogenes in Foods”
in Listeria, Listeriosis and Food Safety, edited by E.T. Ryser and E. H. Marth 1999. Marcel
Dekker, Inc. New York.

Nørrung, B., Andersen, J.K., Schlundt, J. (1999) Incidence and control of Listeria
monocytogenes in foods in Denmark. Int. J. Food Microbiol., 53, 195-203.




                                            188
EPIDEMIOLOGICAL INVESTIGATIONS AND TYPING OF
LISTERIA MONOCYTOGENES (L.M.) ISOLATES FROM
PASTEURIZED CHEESE AND HUMAN LISTERIOSIS CASES
DURING A SUSPECTED OUTBREAK IN GERMANY
Stefan Brockmann1, A. Sabrowski2, R. Oehme1 E, Gutwein1, D, Maslo G. Ptaff1, P. Kimmig1

1
 State Health Office Baden-Wuerttemberg (President Prof. Dr. V. Hingst) Wiederholdstrasse
15, 70372 Stuttgart, Germany,
2
 Chemical and Veterinary Medicine State Laboratory, Stuttgart, Germany

Problem: A milk-processing plant produced and sold pasteurized redsmear-cheese which
showed the growth of Lm. during routine self control. The local food safety authority was
informed and they examined 123 samples (7 brands with 18 different cheeses) produced
between Jan. and Feb, 2000, 101 (82.1%) samples contained L. m., 40 (32.5%) were highly
contaminated between 1x104 - 6x106 CFU/g. A public health warning was issued and recall
of the cheeses was initiated.

Method: The State Health Office contacted via mail laboratories and local public health
authorities in the State, with the call for data record, sending stool specimens or isolated
strains to the department. Stool specimens were examined by routine procedure, Isolated
strains by serotyping and PFGE (Smal, Acsl). The strains were further examined by RAPD
PCR at the National Reference Center.

Results: From Jan. to Feb., 80 tons of cheese were produced, from which 80-90% were
recalled, Only six persons in the state, suffering from diarrhea after consumption of
suspicious cheese, followed the public health warning to contact local authorities. All of them
recovered during a few days, From stool specimens of these persons L. m. was isolated in
one case. One case of neonatal listeriosis and one case of meninigitis were reported by
hospitals as the result of the warning, No other cases of Listeriosis were reported in the state
during the time from 1s1 February to 30th June 2000. Serotyping of the isolated listeria
strains from cheeses showed identical serogroup (1/2b), the same RAPD PCR and similar
PFGE pattern. The stool isolate and the isolate from the neotatal listeriosis showed
serogroup 1/2b and 4, and different RAPD types as well as PFGE pattern, compared with
the cheese isolates, However; the strain of the patient suffering from meningitis showed
identity with the cheese strains in Serotyp, PFGE and RAPD, Interviewing of the patients
family led to the assumption that contaminated redsmear-cheese was consumed,

Conclusion: Despite massive consumer exposition of Listeria-contaminated cheese only
one case of Listeriosis could be linked to the consumption of the pasteurized redsmear-
cheese, Molecular Subtyping by RAPD PCR and PFGE greatly enhance surveillance and
outbreak investigation. However, since no active surveillance net is established and clinical
laboratories, hospitals and local public health authorities do not support submission of
Isolates and flow of Information, the detection of outbreaks will remain a difficult issue and
the number of cases will still be severly underreported. Therefore we highly appreciate all
effort to improve and establish food borne disease surveillance systems.


                                              189

				
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