J. Med. Microbiol. - Vol. 46 (1997), 1047-1057
0 1997 The Pathological Society of Great Britain and Ireland
PROCEEDINGS OF THE PATHOLOGICAL SOCIETY
OF GREAT BRITAIN AND IRELAND
The 175th Meeting of the Society was held at the University of Shefield, 2-4 July 1997
ABSTRACTS OF MICROBIOLOGICAL INTEREST
SYMPOSIUM: INVESTIGATIONS INTO BACTERIAL ADHERENCE MECHANISMS
Chairmen: R. C. Read and C. I. Douglas
METHODS FOR THE STUDY OF BACTERIAL ADHERENCE ATTACHMENT AND EFFACEMENT: THE MOLECULAR BASIS
AND ITS CONSEQUENCES OF “GRIPING IN THE GUTS”
R. C. Read l? H. Williams
Department of Medical Microbiology, University of Department of Microbiology and Immunology, University of
Shefield Medical School, Beech Hill Road, Shefield, Leicestel: Leicester LEI 9HN
SIO 2 R x
Enteropathogenic Escherichiu coli (EPEC) are a hetero-
Bacterial adherence to host surfaces is an important step geneous group of bacteria responsible for severe persistent
in the pathogenesis of disease. The consequences of diarrhoea in infants. Parish records from the 17th and 18*
bacterial adherence include the establishment of a protected centuries show high infant mortality, especially in hot summers,
niche for the micro-organism, transcription of new proteins in rural and urban slum communities in England and Wales due
by the organism as part of adaptation to its new to a condition recorded as “griping in the guts”, what we now
environment, and receptor-mediated responses by the host know to be EPEC diarrhoea. Today, EPEC are a major health
cell including cytoskeletal rearrangement, and downstream problem in developing countries, accounting for 117 million
events. Methods of studying bacterial adherence have been cases of diarrhoea annually world-wide (excluding China). The
applied to whole organisms, to whole tissues in organ primary histopathological effect of EPEC infection is the so-
culture, to primary cultures of isolated cells, to explant- called attaching and effacing lesion, involving localised
derived monolayers, to monolayers of established cell lines, destruction of intestinal brush border microvilli and distortion
to fractionised cell membranes separated on Sepharose of the apical enterocyte membrane into an actin-rich “pedestal”
columns, to purified receptors including sugars, and to structure at the point of bacterial contact. Lesion formation is a
immobilised extracellular matrix proteins. Each of these crucial aspect of the pathogenesis of EPEC; loss of absorptive
targets for bacterial adherence has advantages and dis- surface area contributes to disease by reducing NaCl uptake,
advantages in terms of their application for the study of while enterocyte death during prolonged infection inflicts
bacterial adherence. Methods that have been employed to severe mucosal damage and leads to protracted diarrhoea. The
measure adherence of bacteria have included conventional pathogenic mechanism of EPEC comprises three functionally
bacterial strains including Giemsa or Gram’s stain, the use distinct phases whose molecular details are well defined: (i)
of radiolabelled isotopes, epifluorescence of bacteria, viable initial non-intimate attachment to epithelial cells by means of
counting of bacteria from disrupted target tissues or cells, bundle forming pili; (ii) stimulation of host cell signal
transmission and scanning electron microscopy, direct and transduction pathways leading to phosphorylation of several
indirect immunofluorescence, enzyme linked immunosorbent host proteins; (iii) gross reorganisation of the cell cytoskeleton
assay and detection of biochemical markers (e.g., urease, in resulting in localised effacement of microvilli and allowing
the study of Helicobacter pylori). Detailed investigation of intimate bacterial attachment to the cell surface.
interactions between host receptors and bacterial adhesins
has been studied by the use of inhibition by specific CROMPS, FIMBRIAE AND FLAGELLA: THE QUEST FOR THE
antibodies and by competitive antagonism of putative
ADHESINS OF AEROMONAS
receptors or adhesins with purified proteins or surgars.
Responses of bacteria to adherence can be studied by Jonathan G. Shaw
chromatography of released bacteria and by detection of Department of Medical Microbiology, University of Shefield
mRNA. Following bacterial adherence the host may respond Medical School, Beech Hill Road, Shefield S10 2RX
either by morphological change (e.g., production of
pseudopods) or by internalisation of the organism, either Aeromonas are increasinglyimportant gastrointestinalpatho-
by macropinocytosis (e.g., Salmonella) or by receptor- gens which are most prevalent amongst younger age groups.
mediated endocytosis. The latter can be studied by the use However, very little is known about their putative virulence
of inhibitors of clathrin, mediated endocytosis, microtubule factors, especiallythose associated with adherence.A number of
polymerisation and F actin polymerisation. models of aeromonad adherence have been tested, ranging from
1048 ABSTRACTS
agglutination of artificial and natural particles to adhesion to for identifying interactions between host and bacterial proteins,
tissue culture cells and intestinal biopsies. Like other entero- providing clues to novel mechanisms of microbial pathogenesis.
pathogens, multiple adhesive mechanisms are thought to be Indeed, the results suggest a new mechanism in gonococcal
present within the genus and these may vary between strain and pathogenesis, i.e., the direct binding of PK, a host metabolic
species. A number of possible aeromonad adhesins have been enzyme, for the essential acquisition of pyruvate, an intracellu-
isolated such as the carbohydrate-reactive outer membrane lar carbon source required for growth.
proteins (CROMPs) fiom A. hydrophila, which are porin-like
proteins that haemagglutinate (HA) erythrocytes. Also, two
major types of fimbriae have been reported, Short/Rigid (S/R), INTERACTIONS BETWEEN BACTERIA AND PLATELETS
and Long/Wavy (L/W) that have been isolated from various C. W. I. Douglas, D. Cox, A. Wray and C. Rees
strains of A . hydrophila and A. veronii. L/W fimbriae have been Department of Oral Pathology, School of Clinical Dentistry,
demonstrated to be colonisation factors of HEp-2 cells and University of Shefield
intestinal biopsies. Although electrophoreticallyand immunol-
ogically distinct from one another, they share N-terminal Bacteria can interact with platelets directly in suspension,
sequence homology with the type IV fimbrial class. Also, a which usually leads to aggregation of the platelets, or with
LPS/43 kDa protein complex has been linked with HA and HEp- platelets present in thrombi on a surface. Adhesion can involve
2 adhesion. The polar flagellum of A. caviae is important in either direct binding to platelet surface molecules, binding via
adhesion to HEp-2 cells, as their removal by shearing or an intermediate ligand, or interaction with matrix proteins
agglutination by antibody greatly reduced bacterial adhesion associated with the thrombus. For example, Borrelia burgdor-
ability to the cell line. Moreover, non-motile polar flagellum- feri binds directly to GPIIB/IIIa, Staphylococcus aureus
negative Tn5 mutants also showed a decrease in adhesive ability. adheres to platelet GPIIb/IIIa but via a fibrinogen bridge and
However, the situation is more complex as polar flagellum Streptococcus sanguis adhesion is via a protein adhesion and is
expressing wild type cells when treated with chloramphenicol, independent of both GPIIb/IIIa and fibrinogen. Also, some
before interaction with the tissue culture monolayer, demon- strains of S. sanguis have been shown to bind to fibronectin
strated greatly reduced adherence. Furthermore, we have present in platlet-fibrin thrombi. Our work has been largely
isolated motile polar flagellum-positive Tn5 mutants which are directed at understanding the molecular events in S. sanguis
non-adherent. In conclusion the adherence of aeromonads is a adhesion to and aggregation of platelets. We have recognised
complexmultifactoralprocess. Across the genus different strains three groups of S. sanguis strains; Grp I adh+ agg+, Grp I1
use a variety of methods including LPS, fimbriae and outer adh- agg+ and Grp I11 adh- agg-. Adhesion of Grp I strains
membrane proteins. However, for A. caviae the polar flagellum is reduced in the presence of plasma and by trypsin treatment,
and at least one other unidentified factor are required. suggesting masking and removal of an adhesin, respectively.
Platelet aggregation by all bacteria appears to require fibrino-
gen-GPIIb/IIIa interaction. S. sanguis strains require additional
GONOCOCCAL ADHESION AND INTRACELLULAR SURVIVAL
plasma factors. Grp I1 strains require IgG and complement. IgG
J. M. Williams, G.-C. Chen and R. F. Rest interacts with the platelet FcyRII but it is not known how
Department of Microbiology and Immunology, Allegheny complement interacts in this context. Grp I strains do not
University of the Health Sciences, 2900 Queen Lane, require complement for platelet aggregation. One Grp I11 strain
Philadelphia, PA, 19129, USA fails to assemble complement efficiently, which might explain
its agg- phenotype. Thrombin is generated when bacteria are
Neisseria gonorrhoeae opacity-associated (Opa) proteins are incubated in plasma. Some species can trigger coagulation
a family of outer membrane proteins involved in adhesion to directly, whereas S. sanguis produces only low levels of
and entry into human epithelial and endothelial cells and thrombin. This thrombin is thought to be essential for platelet
neutrophils. Using a yeast two-hybrid system, we examined aggregation by both Grp I and I1 strains because aggregation is
whether Opa proteins had acquired functions involved in inhibited by antithrombin agents. Much of the thrombin appears
intracellular survival. The yeast two-hybrid system has been to become cell-associated and it probably activates platelets via
used successfully to identify numerous protein-protein inter- the GPIIb receptor. It is hoped that an understanding of the
actions in several biological systems, but has not been used to molecular mechanisms of bacteria-platelet interactions may
identify epithelial cell proteins that bind membrane proteins of present novel targets for therapy of diseases which involve
pathogenic bacteria. Five Opa-Interacting Proteins (OIPs) from platelet pathophysiology, such as infective endocarditis or
human HeLa (epithelial) cells were identified, two of which are infections leading to disseminated intravascular coagulation.
homologous to known proteins. OIPl is homologous to the
human cytoplasmic protein TRIP6, which binds the nuclear MECHANISMS OF CO-ADHESION OF ORAL MICROBIAL
thyroid hormone receptor p. OIP3 is human pyruvate kinase PAIRS AS STUDIED IN A PARALLEL PLATE FLOW CHAMBER
(PK) subtype M2, the subtype found in human cells infected by
N. gonorrhoeae and N . meningitidis. Quite interestingly, one H. J. Busscher, H. C. van der Mei and R. Bos
action of thyroid hormone is to regulate the expression of Materia Technica, University o Groningen, Bloemsingel 10,
f
human PK. Thus, gonococci appear to have evolved a 9712 KZ Groningen, the Netherlands
mechanism to regulate cellular thyroid hormone responses.
In-vitro binding assays and in-vivo immunofluorescencestudies Interspecies binding is generally recognised as a factor
confirmed that Opa proteins bind PK. PK catalyses the contributing to the development of dental plaque, but has
conversion of phosphoenol pyruvate to pyruvate, with the also been implicated in the formation of aquatic biofilms
production of AT€?Studies with a gonococcal mutant unable to and microbial interactions in the urinary tract. In vitro,
utilise pyruvate or lactate for growth indicated that intracellular interspecies binding is mostly studied between planktonic
pyruvate is required for gonococcal survival. Our studies organisms (“co-aggregation”) but in vivo, interactions
demonstratedthat the yeast two-hybrid system is a valuable tool between adhering, sessile micro-organisms and planktonic
ABSTRACTS 1049
ones are equally likely to occur (“co-adhesion”). Co- pellicle of the tooth surface is mediated by a cell surface
adhesion can be studied in a parallel plate flow chamber by adhesion of 185 kDa variously termed streptococcal antigen I/
first allowing a primer strain to adhere after which the I1 (SA 1/11), antigen B or P1. Immunisation with SA I/II
deposition of another strain in the close vicinity of primer confers protection against caries in animal models. Similarly,
strain organisms is compared with deposition on other parts topical application of monoclonal antibodies raised against SA
of a substratum. Therewith, a continuous, filly quantitative I/II prevents caries in animal models and prevents colonisation
measure of co-adhesion is obtained. Physico-chemically, by S. mutans in man. We have analysed the structure of SA 1/11
interspecies binding is an interplay of Lifshitz-Van der by electron microscopy and FTIR and CD spectroscopy,
Walls, acid-base and electrostatic interactions. Our studies determined an adhesion binding site and mapped T cell and B
in the parallel plate flow chamber have revealed that co- cell epitopes. SA 1/11 is an extended molecule (approximately
adhesion can be described as a critical colloid-chemical 70nm long) in which the N-terminal region adopts an a-
phenomenon strongly influenced by minor changes in the helical coiled coil conformation while the C-terminal portion
electrostatic and acid-base interactions and, consequently, is predominantly P-sheet. Interaction with the salivary receptor
with a critical influence of temperature. is of relatively high affinity (& c. 10 -9 M), involves at least
two non-equivalent sites of SA I/II and may require
STRUCTURAL, AND FUNCTIONAL ANALYSIS OF A
recognition of 0-linked glycans. By use of synthetic peptides
and site-directed mutagenesis, we have identified residues
STREPTOCOCCAL, ADHESIN
1025-1044 as critical for adhesion. This epitope is within an
C. G. Kelly, S. Todryk and T. Lehner immunodominant portion of SA 1/11 and is adjacent to
Department o Immunology, UMDS at Guy’s Hospital,
f promiscuous T cell epitopes. We suggest that these findings
London SEl 9RT provide a basis for the design of subunit vaccines which may
direct immune responses to critical fkctional epitopes or for
Infection with Streptococcus mutans is the main cause of the design of agents which may prevent infection by blocking
dental caries. Initial adhesion of S. mutans to the salivary adhesion of S. mutans.
SYMPOSIUM: THE PATHOGENESIS OF SEXUALLY TRANSMITTED DISEASES
Chairmen: R. Jennings and D. Jeffries
ADVANCES I THE PATHOGENESIS OF HIV
N reduction in CD4 cell number over time (10 years)
Jonathan Weber represents the slight excess of T-cell loss over production.
These data have significant implications for the therapy of
Division of Medicine, Imperial College School o Medicine,
f
HIV infection by long term suppression of viral replication
London through combination anti-retroviral therapy.
Considerable progress in understanding how HIV leads to
AIDS has been achieved in the past two years, mostly from PRIMARY AND RECURRENT GENITAL HERPES-VACCINES
the multi-disciplinary study of the effects of therapy. From
AND CONTROL
1986, it was known that only a small proportion of
peripheral blood CD4+ve T-cells were infected by HIV, R. Jennings
and the mechanism of cell loss was obscure. In 1995, Haase Department of Medical Microbiology, University o Shefield
f
showed convincingly that HIV load in lymph nodes was il
Medical School, Beech Hl Road, Shefield, SlO 2RX
much higher than in peripheral blood. Reproducible and
accurate measurement of HIV viral load by RNA RT-PCR There is a long history of the development of vaccines
became available in 1994. Following phase I trials of potent and immunisation against herpes simplex virus (HSV)
anti-retroviral therapy in 1996, Ho (New York) and Shaw infection, but the advances in modern technology have been
(Alabama), in collaboration with mathematical biologists, brought to bear in this area only in recent years. This has
showed that the rapid reduction of viral load and increase in been accompanied by a more detailed knowledge and
CD4 cells could be modelled to provide insight into the rate understanding of the structure and properties of the herpes
of virus production and CD4 loss. These data showed that simplex viruses and such knowledge is being utilised
lo9 virionslday are produced, with a short half-life; currently in the design of novel HSV vaccines. This has
approximately the same number of mature CD4+ve T-cells led to the development of vaccines consisting of one or
are acutely infected by HIV every day, and these infected more recombinant viral glycoproteins, to the development of
cells have a half-life of 24-36 h. Thus, HIV infection can be genetically engineered attentuated vaccines and to the
seen as a highly dynamic process, and the small number of development of replication defective and virally or bacter-
detectable HIV+ve peripheral T-cells at any one time point ially vectored vaccines. Unfortunately these advances have
hides a large number of infectiondday. Presumably, the bone not been matched by an equally rapid increase in the
marrow is replacing T-cells by peripheral expansion to understanding of the immune defences that underlie, and are
compensate for the HIV-induced destruction, and the slow most relevant to, protection against HSV infection, and
1050 ABSTRACTS
particularly those of most relevance to prevention of the of the Thl type offering a strategy for immunotherapy in
establishment of latent infection, modulating viral reactiva- benign disease but strategies to elicit cytotoxic responses are
tion from the latent state or reducing the frequency or required for malignant HPV-associated lesions. Animal
severity of recurrent infections. In common with a number models suggest that prophylactic immunisation with inacti-
of other sexually-transmitted diseases, genital herpes is on vated virus or synthetic virus-like particles (LPVs) can
the increase and there is an urgent need to produce an protect against subsequent viral challenge and there is
effective vaccine which can significantly minimise or reduce optimism for these approaches in human populations.
primary or recurrent illness, or both. Most strategies for
immunisation are directed against recurrent genital herpes,
PATHOGENESIS OF HEPATITIS B AND C: RECENT ADVANCES
i.e., a therpeutic strategy, and as such will need to increase
levels of immunity, whatever form this takes, particularly at H. C. Thomas
the local site of recurrent infections, and ideally maintain Imperial College School of Medicine, London
such raised levels of immunity over long periods of time.
Because of a lack of knowledge of the immune mechanisms It is now clear that Hepatitis B and C exist as multiple
responsible for protection together with absence of an quasi-species in any isolate. The diversity of the viral
animal model of known relevance to the human disease, population is dependent on the level of replication, the
pre-clinical evaluation of vaccines against genital herpes has duration of infection and the rate of error by the viral
retained something of an empirical flavour. Nevertheless, transcriptase. This population of virus particles will come
following extensive, promising pre-clinical evaluation, a under varying host selection pressure, usually immune, as the
number of recently developed and fully characterised infection continues. In acute HBV infection, there is MHC
vaccines have entered clinical trials. In the majority of class 2 presentation of peptides from HBc protein to CD4
cases, the results of these trials have not fulfilled the lymphocytes of the TH1 phenotype, resulting in a MHC class
promise or the quality of responses predicted by the pre- 1 restricted cytotoxic T-cell response to multiple epitopes
clinical assessment. These disappointments, besides reflect- derived from the proteins of HBV: This results in lysis of
ing the lack of knowledge of the immune mechanisms infected cells, followed by a virus neutralising humoral
involved in protection, may also be due to inherent immune response and recovery. In chronic HBV infection,
difficulties in promoting local immune responses, absence either because of failure in antigen presentation or some other
of (as yet infected) protective antigens in the vaccine abnormality of the induction of the immune response, the
preparations, genetic make-up of the host and variations in patient fails to mount an adequate CD4 and CD8 immune
their existing immune status. The nature of current candidate response. Thus there is failure of recognition and lysis of
HSV vaccines, their performance in pre-clinical evaluation, infected cells and hence viral persistence. From time to time
the problems of immunity to HSV, the target populations for during the chronic infection the patient makes an immune
HSV vaccination and the problems associated with clinical response to variant HBV-encoded proteins and these varient
trailing of this vaccine form the basis of this presentation. epitopes appear to evoke a humoral immune response.
Initially this manifests as an immune response to HBe
PATHOGENESIS AND CONTROL OF HUMAN
antigen which, in the absence of a CTL response, results in
selection of the HBe-negative virus. This virus is pathogenic
PAPILLOMAVIRUS INFECTIONS
and patients progress to cirrhosis and liver cancer. In some
Margaret A. Stanley patients antibody to the epitopes of the “a” determinant
Department of Patholog, Tennis Court Road, Cambridge appear and this selects variants which have lost the “a”
CB2 IQP determinant because of mutations in this region, Several such
mutations have now been described, including the glycine to
Papillomaviruses are small double stranded DNA viruses arginine mutation at position 145 and the arginine-XX
that cause warts on cutaneous and mucosal surfaces. The insertion between amino acids 122/123 and 123/124. These
viruses do not grow in tissue culture and clinical lesions HBs variants are also seen in vaccinees born to HBe antigen
contain few viral particles; experimental studies on these positive carrier mothers. Their significance will be discussed
agents depend, therefore, on recombinant technologies. Of in epidemiological terms. In chronic HCV there also exist
the c. 80 human papillomavirus genotypes which have been multiple quasi-species; their number and genetic diversity
cloned from clinical lesions, those infecting the genital tract increase with duration of infection. There is a CD4 and CD8
have attracted most attention, since infection with a sub-set lymphocyte response to the virus-encoded proteins and yet
of these viruses, particularly types 16 and 18, is the major this fails to control the infection, presumably because of
risk factor for the development of cervial cancer in women. inhibition of these responses by variant inhibitory peptides or
The replication cycle of all the papillomaviruses is tightly other, yet unknown, mechanisms. There is evidence that some
linked to keratinocyte differentiation and this is a strategy of the HCV-encoded proteins inhibit the cells response to
for immune evasion. The virus infects primitive basal cells interferon. The envelope of HCV contains two proteins
but viral replication and viral assembly are confined to containing three variable or hypervariable regions. Studies in
differentiating superficial epithelial cells. Viral replication agammaglobulinaemics demonstrate that this diversity of
and release are confmed, therefore, to cells destined for antigenic structure of the envelope proteins is driven by the
death; they are not associated with inflammation and the humoral immune response. Both persistent HBV and HCV
evidence suggests that the immune system is ignorant of or infection are examples of dynamic infections where, due to
indifferent to the infection. However the natural history of substitutions produced in the viral genome by viral tran-
gential HPV infections and the increased incidence of lesion scriptase errors (these enzymes do not have proof editing
in immunosuppressed patients indicate that host defences capabilities) a population of virus particles is generated,
can respond effectively. Evidence from regressing genital which survive with varying efficiency depending on the
warts suggests that this is a cell-mediated immune response selection pressures exerted by the patient’s immune response.
ABSTRACTS 1051
ORAL PRESENTATIONS
ISOLATION AND CHARACTERISATION OF SIDEROPHORE The M, of these flagellins was calculated to be N 40 kDa.
MUTANTS IN BURKHOLDERIA CEPACIA Some strains appeared to express tow flagellin types of
close Mr. The results so far obtained would indicate that,
K. L. Farmer and M. S. Thomas
on the basis of N-terminal analysis, there may be two types
Department o Medical Microbiology, University o Shefield
f f of flagellin proteins, one of which is 60% homologous with
Medical School, Beech Hill Road, Shefield, SIO 2RX that from Bacillus subtilis, with strains carrying either one
or both types and that the type of flagellin found does not
Burkholderia cepacia is a gram-negative non-sporing appear to depend on serogroup. These results represent the
motile rod widely distributed in the environment. Originally first amino-acid and DNA sequence data on C. dificile
isolated as a phytopathogen, it has recently found status flagella. The identification and sequencing of the genes
as an important opportunist pathogen, particularly in encoding these proteins is now under way.
cystic fibrosis (CF) patients. The virulence factors of this
organism have not been fully determined, though several
candidates such as lipase, protease and siderophores have PATHOGENICITY OF STAPHYLOCOCCUS AUREUS IN MICE
been proposed. Siderophores are low mol.wt iron-chelating LACKING INDUCIBLE NITRIC OXIDE SYNTHASE
compounds produced under iron-depleted conditions. They C. G. Gemmell, I. B. McInnes", B. Leung*, X.-Q. Wei* and
enable bacteria to effectively compete with host iron- F. Y. Liew*
binding proteins thus allowing bacteria to establish and
Departments o Bacteriology and *Immunology, University o
f f
maintain infections. Production of pyochelin, a fluorescent
Glasgow, Glasgow
green-yellow siderophore produced by many B. cepacia CF
isolates, has been directly linked to morbidity and mor-
tality in B. cepacia-infected CF patients. We describe Nitric oxide (NO) produced in large amounts by an
the isolation of pyochelin deficient mutants (Pch-) of a inducible nitric oxide synthase (iNOS) has been recognised
B. cepacia CF isolate following transposon mutagenesis as an important microbicidal and immunomodulatory
with mini-Tn5 derivatives. Conjugation experiments invol- mediator. We have investigated its role in septic arthritis
ving an Escherichia coli donor strain harbouring the mini- spread haematogenously following Staphylococcus aureus
Tn5 transposon promoted efficient mutagenesis of B. infection in iNOS-deficient mice. The incidence, rate of
cepacia. Transconjugants were screened initially for the development and severity of arthntis were greater in iNOS-
absence of fluorescence on iron-limited media. Candidate deficient mice than in heterozygous or wild-type MF1 mice.
Pch- mutants were then screened for siderophore produc- Similarly, the incidence and severity of septicaemia and the
tion on CAS agar and further analysed for pyochelin mortality rate were significantly higher in iNOS-deficient
production by thin layer chromatography (TLC) of culture mice. Increased TNFa synthesis in vivo and in vitro and
supernates. A few Pch- mutants were isolated, among enhanced IFN, as compared to IL-4 production in vitro, in
which one appeared to possess a mutation exerting iNOS-knockout mice demonstrated the development of an
pleiotrophic effects. exaggereated Thl polarisation of the host response. iNOS-
deficient mice were unable to limit bacterial growth despite
the presence of high levels of Thl cell activity and
IDENTIFICATION, ISOLATION AND CHARACTERISATION OF concomitant production of IFN and TNFa. Spleen cells
from iNOS-knockout mice were much more responsive to
FLAGELLINS FROM CLOSTRIDIUM DIFFICILE
stimulation with TSST- 1 or staphylococcal enterotoxin A.
A. P. Dodson, M.-C. Barc", S. J. Hyde and S. P. Borriellot FACS analysis confirmed that the proportions of CD4+ and
Institute o Infection and Immunity, Queens Medical Centre,
f CD8+ T-cell populations were similar in iNOS knockout
Nottingham, *Department de Microbiologie et Immunologie, and control mice.
Faculte de Pharmacie, 5 Rue Jean-Baptiste Clement, 92296
Chatenay Malabry, France and t Central Public Health
Labomtory, 61 Colindale Avenue, London WHOLE BLOOD MODEL OF MENINGOCOCCAL
BACTERAEMIA IN CONVALESCENT CHILDREN
Clostridium dzficile causes pseudomembranous colitis
N. Anwar, M. Cole, M. Levin*, R. Galasshi", C. A. Ison and
(PMC) and antibiotic-associated diarrhoea. It is the most
the Meningococcal Study Group
common cause of hospital acquired diarrhoea. To determine
the role of flagella as a virulence factor in the Departments o Medical Microbiology and *Paediatrics,
f
pathogenicity of C. dzficile, we have been able to identify ICSM at St Mary's, Paddington, London W2 IPG
and isolate flagellin proteins with a flagellin antiserum. We
have shown, using immunogold labelling and electron An ex-vivo model of meningococcal bacteraemia has
microscopy, that the antiserum binds specifically with the been used to investigate the bactericidal activity of blood
prepared flagella and not with any other surface antigens. from children with a history of meningococcal disease.
Flagellin proteins from six different C. dzficile strains, of This model allows the assessment of the complete
varying toxigenicities, virulence and serogroup, have been bactericidal activity of blood. Anticoagulated blood was
isolated and N-terminal analysis of the proteins undertaken. inoculated with mid-log phase organisms (1 O6 cfu/ml),
1052 ABSTRACTS
incubated at 37°C on a rotating platform and samples were three of the 11 patients. Resistance to ciprofloxacin is still
taken for determination of the viable counts at 0 and low but should be monitored to prolong the use of this
90 min. Bactericidal activity was expressed as percentage highly effective antibiotic.
survival at 90 min. Three organisms were tested: the
patient's infecting strain, Neisseria meningitidis MC58
(B: 15:P1.7,16) and i meningitidis NCTC 8554 (C:NT.NT).
V PATHOGENIC POTENTIAL OF CHLAMYDIA TMCHOMATIS
The bactericidal activity of blood from 25 children, aged 6 USING AN IN-VITRO MODEL SYSTEM OP HUMAN
weeks to 19 years, attending the out-patient clinic between REPRODUCTIVE EPITHELIUM
2 and 15 months after infection was determined. Differing
M. Taraktchoglou, A. A. Pacey* and A. Eley
patterns of bactericidal activity were seen: (1) those
exhibiting bactericidal activity against all three strains Departments of Medical Microbiology and *Obstetrics and
tested (70% survival); and (4) those sexually transmitted pathogens that can cause a wide range
showing little bactericidal activity against any strain. of infections from asymptomatic to severe infections of
Patients infected with serogroup B strains had greater the female genital tract. It has been suggested that the two
activity against MC58 and those infected with serogroup C most common serovars, E and F, differ in the severity of
strains had greater activity against NCTC 8554. Of the 25 infection they cause in women. E is considered to cause
children tested, the blood from only three showed > 10% mild asymptomatic infections of the lower genital tract, in
survival against their infecting strain irrespective of age or contrast with F that is thought be account for more severe
time since infection. The use of the whole blood model has infections of the upper genital tract, like PID. Another
shown that children of any age, including 0.12 mg/L and hence could be resistant. The common resistance pattern is ASSuTC, but
considered potentially resistant. Only a single isolate was some strains have shown resistance to trimethoprim and
highly resistant (MIC, 16 mg/L), the remainder exhibiting quinolones. The resistance genes are integrated into the
MICs of 0.25-0.5 mg/L. All isolates exhibited resistance chromosome and the use of genetic techniques to
to penicillin and belonged to serogroup B. A number of differentiate strains has been only partially successful. The
different A / S Classes were seen and all isolates, where multiple resistant phage type has also been isolated from
known, were acquired abroad. Mutations in the quinolone- farm animals in a number of countries, i.e., Denmark,
resistance determining region were found in all isolates Germany, France, Ireland and the USA. The paper
with double mutations present in the highly resistant concluded wt a review of the situation in the UK and
ih
isolates. Therapeutic failure was known to occur in only current investigations.
ABSTRACTS 1053
THE INFLUENCE OF VACCINE FORMULATION ON CYTOKINE subunit HSV-2 anitgen preparations mixed with aluminium
LEVELS AND SUBCLASS ANTIBODY RESPONSES OF HSV-2 hydroxide gel (ALH) or formulated into ISCOMs. Spleens
IMMUNISED MICE collected from vaccinated groups after immunisation were
tested following in-vitro re-stimulation for levels of IFN-y
S. A. Mohamedi, A. W. Heath and R. Jennings
and IL-4 in an enzyme-linked immunosorbent assay
Department of Medical Microbiology, University o Shefield
f (ELISA). Similarly, post-immunisation sera were assayed
Medical School, Beech Hill Road, Shefield SlO 2EX by ELISA for subclass IgGl and IgG2a antibodies.
Subsequent to immunisation, a cohort of animals was
It has been reported that immunisation of mice with an challenged with live HSV-1. Levels of subclass IgGl
HSV-2 subunit vaccine consisting of baculovirus-expressed antibodies in HSV-2 infected mice immunised with HSV-2
herpes simplex virus type-2 (HSV-2) glycoprotein D (bgD2) ISCOM vaccine were lower than those recorded after HSV-
mixed with aluminium phosphate (AlP04) as adjuvant can 2 ALH vaccination, while the reverse was observed for
change a dominant live HSV- 1-induced Thl response, TgG2a antibohes. Levels of IFN-y in in-vitro stimulated
towards a Th2 profile, as determined by subclass antibody spleen cell culture supernates from infected mice immu-
levels in mouse sera and patterns of cytokine secretions nised with HSV-2 ISCOM vaccine were also high, while
from mouse spleen cells (York et al., Vaccine 13: 1706 those from HSV-2 ALH-immunised mice were at baseline
1995). These changes are associated with a reduction in levels. IL-4 levels were greatest in spleen cell culture
HSV-directed cytotoxic T lymphocyte (CTL) activity, indi- supernates from infected mice immunised with the HSV-2
cating that such a reduction is the result of preferential ALH vaccine, but high levels of this cytokine were also
re-stimulation by the AlP04-HSV vaccine of a Th2 found in spleen cell supernates from infected mice given
response profile. The current studies investigate if an HSV-2 ISCOM vaccine. Full protection against HSV-1
HSV-immunostimulating complex (HSV-ISCOM) vaccine challenge was achieved in infected mice immunised with
formulation has similar effects to those described above. HSV-2 ISCOM vaccine, but only partial protection in
HSV-2 or mock (PBS)-infected mice were immunised with infected mice immunised with HGV-2 ALH vaccine.
POSTER PRESENTATIONS
RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) to differentiate between strains of different serovars, but we
FINGERPRINTING OF CHLAMYDIA TRACHOMATIS were unable to show any strain to strain variation within a
serovar.
K. M. Oxley and A. Eley
Department o Medical Microbiology, University o Shefield
f f
Medical School, Shefield DEVELOPMENT OF RT-PCR FOR ANTIMICROBIAL
SUSCEPTIBILITY TESTING OF CHLAMYDIAE
The ability to differentiate between strains belonging to
the same serotype would be a usehl tool in epidemio- N. A. Cross and A. Eley
logical studies of Chlamydia trachomatis infections. RAPD Department o Medical Microbiology, University o Shefield
f f
fingerprinting has been applied successfully as a typing tool Medical School, Shefield
to a number of other organisms and we report our
experiences in using this technique to characterise clinical Chlamydia trachomatis and C. pneumoniae are important
isolates of C. trachomatis. Over 160 different RAPD human pathogens which are responsible for a variety of
primers were screened against target DNA isolated from a diseases. Unlike many other bacteria, however, there is no
bank of C. trachomatis serovar E and F strains grown in universally accepted method for testing antibiotic suscept-
McCoy cell culture. Primers were identified that produced ibilities of chlamydiae. Conventionally, for these organisms,
some specific band patterns with C. trachomatis DNA that a cell culture system has to be used but there are inherent
was distinct from contaminating McCoy cell DNA, but disadvantages to this technique. Recently, an RT-PCR
these results could not be reproduced with any consistency. method was proposed for chlamydiae although we believe
In a different approach, the major outer membrane protein that it was not fully optimised and there were problems in
(MOMP) DNA sequence from each of the C. trachomatis reproducibility. Subsequently, we have developed a similar,
strains was specifically amplified in a polymerase chain but improved RT-PCR method of antimicrobial suscept-
reaction and used as target DNA for RADP fingerprinting. ibility testing by amplification of mRNA. The dnaK and
Sixty primers were screened by this method but no useful 16s rRNA genes, which are both expressed early in the
banding patterns emerged. Finally, four primers were chlamydia1 development cycle, were amplified by both one-
specifically designed that had the potential to produce an step and two-step RT-PCR procedures. These results were
RAPD banding pattern against the MOMP target sequence, compared to conventional immunofluorescence (IF) staining.
using a computer simulation program. Experimentally, these The minimum inhibitory concentrations (MICs) of anti-
primers produced a pattern of PCR products that enables us microbial agents against all Chlamydia strains tested were
1054 ABSTRACTS
2-4 fold higher with RT-PCR as compared to IF staining, standard procedures. Of these, 85% were culture positive
and probably reflects an increase in sensitivity of the RT- for Mycobacterium tuberculosis. Of the 491 patients whose
PCR method. This raises questions as to whether the latter isolates were tested for susceptibility to isoniazid, strepto-
MICs are more clinically meaningful. mycin, rifampicin and ethambutol, 90.8% had hlly sensitive
strains and 9.2% had a strain resistant to one or more
drugs. Of the 445 patients with no history of previous
chemotherapy, 6.3% had a resistant strain. Of the 46
UTILITY OF RAPID CULTURE OF NASOPHARYNGEAL patients with a history of previous chemotherapy, 37% has
ASPIRATE IN THE RAPID DIAGNOSIS OF ADULT INFLUENZA a resistant strain. No resistance to either rifampicin or
M. L. Schmid, G. Kudesia*, S. Wake* and R. C. Read ethambutol was detected. There was a strong association
between previous chemotherapy and resistance. Resistance
North Trent Department o Infection and Tropical Medicine,
f
was not associated with age and sex. High concordance
Royal Hallamshire Hospital, Shefield and * PHLS, Northern
between Kenya's results and those of the Mycobacterium
General Hospital, Shefield
Reference Unit in the UK on both drug sensitive and drug
resistant strains indicates that clinically significant and
Nasopharyngeal aspirate (NPA) is frequently used in
comparable data can be obtained from laboratories employ-
children for the diagnosis of RSV and other upper
ing unsophisticated and inexpensive standard procedures.
respiratory tract infections. Viral throat swab culture is
Rates of initial drug resistance are still low in Kenya. The
the gold standard to diagnose influenza A, and the
increase in acquired resistance to isoniazid requires
preferred investigation in terms of patient comfort. During
monitoring.
an epidemic of influenza A (H3Nz) we performed NPA and
throat swab on 39 consecutive hospitalised patients with a
diagnosis consistent with influenza. All specimens were HAEMALBUMIN FORMATION INCREASES THE CAPACITY
cultured by standard techniques at 33°C and 37°C. Rapid AND AVIDITY OF ITS BINDING TO THE
culture was undertaken after centrifugation onto cover slips
PERIODONTOPATHOGEN PURPHYRUMUA2S GINGIVALIS
and stained with fluorescein-tagged monoclonal antibody to
influenza A after 24 and 48 h. Direct immunofluorescence J. W. Smalley and A. J. Birss
(DIF) of fresh NPA was also performed. Standard cultures Unit of Oral Biology, Department o Clinical Dental
f
were incubated for up to 21 days and diagnosis was Sciences, The University of Liverpool, Liverpool L69 3BX
confirmed if any one of cytopathic effect, positive
immunofluorescence or haemadsorption were present. Sev- Albumin is the most abundant haem-carrying protein in
enteen of 39 NPA specimens were influenza A culture- gingival sulcus fluid. The haem requirement of Porphy-
positive by standard culture both at 33°C and 37°C. romonas gingivalis is satisfied by haemalbumin in vitro.
Thirteen of these NPAs (72.2%) were culture positive on Conformational changes induced by haemalbumin binding
rapid culture. Eight of 17 (47.1%) available throat swabs led to a more stable protein complex and decrease its
were positive for infulenza A on culture. Four of 17 degradation by I? gingivalis. We studied the binding
(23.5%) throat swabs were positive on rapid culture. DIF activity of I? gingivalis W50 for albumin and haemalbu-
was positive in four of 17 (22.2%) of the culture-positive mins with 1:l and 2:l haem-protein molar ratios. Cells
NPAs. The sensitivity of NPA was superior to throat swab. exhibited saturation kinetics for albumin and haemalbumin,
Rapid culture appears to be a useful tool and enables binding a maximum of 250 p g of albumin/107 cells, with
diagnosis within 24 h in most infected patients; its 3- and 6-fold greater capacities for 1:l- and 2:l-
sensitivity is superior to DIE NPA was generally well haemalbumins, respectively. Scatchard analysis revealed
tolerated and easily performed. We recommend the use of monophasic binding for albumin (K, = 5 X lo4 M). Binding
NPA and rapid culture in adults for the diagnosis of of both haemalbumins was triphasic, comprising a non-
influenza A. specific binding and two other higher affinity phases. The
highest & was 3 X lo6 M, for both haemalbumins, with
values of 2 X lo5 and 7 X lo4 M, for the lower binding
ANTI-TUBERCULOSIS DRUG RESISTANCE SURVEILLANCE avidities of 1:1- and 2: 1-haemalbumin. respectively. It is
IN KENYA, 1995 concluded that haemalbumin complexes bind to I? gingiva-
lis with both increased capacity and avidity compared to
W. Githui*$, E. Juma", J van Gorkomt, D. Kibugat, J.
apoalbumin. Such binding behaviour would enable cells to
Odhiambo" and F. Drobniewski
discriminate between albumin and haem-bearing albumin
*Respiratory Diseases Research Unit, Clinical Research molecules as a potential source of haem. This may confer
Centre, Kenya Medical Research Institute, l 0. Box 47855,
? an ecological advantage under conditions of haemin
Nairobi, Kenya, t National Leprosy and Tuberculosis limitation in the peridontal pocket or gingival sulcus.
Programme, Ministry of Health, Nairobi, Kenya and $PHLS
Mycobacterium Reference Laboratory, Dulwich Public Health
Laboratory and Department o Microbiology, King 5. College
f BACTERIA, NITRITE, N-NITROSO COMPOUNDS AND
School of Medicine, Dulwich Hospital, London SE22 8QF
GASTRIC CANCER
The aims of the surveillance were: to determine the S. L. Naylor, M. J. Hill, B. J. Johnston and P. I. Reed
prevalence of initial and acquired drug resistance in newly Lady Sobell Gastrointestinal Unit, Wexham Park Hospital,
diagnosed patients with pulmonary tuberculosis, to deter- Slough, Berkshire, SL2 4HL
mine possible risk factors associated with resistance and to
establish standard routine surveillance of drug resistance. There has been controversy concerning the role of
Sputa from 638 newly diagnosed patients were analysed by bacterial production of N-nitroso compounds (NOC) in
ABSTRACTS 1055
gastric carcinogenesis. Increased gastric pH is associated bone destruction found in patients with localised juvenile
with increased risk of gastric cancer, bacterial counts and periodonitis (LJP). How this bacterium causes bone
nitrite concentration. However, it is only accompanied by destruction has not been identified and given the pathology
increased NOC in some study reports. In this laboratory a of LJP it was felt that factors released by the bacterium
new assay method has been developed that permits rapid may be responsible. Gentle saline extraction of this
assay of samples; it has been applied to over 650 gastric bacterium allowed the harvesting of a surprisingly large
juice samples to assay nitrite and total N-nitroso com- amount of protein (but only minimal carbohydrate) which,
pounds (TNOC). As in previous studies nitrite concentra- when analysed by SDS-PAGE, was found to contain
tions were 100 pm at pH 7-8, following the pH-activity profile of fraction was found to stimulate the breakdown of murine
bacterial nitrate reductase. The TNOC concentration was calvarial bone and the synthesis of pro-inflammatory
100 p m at pH 7-8. Bacteria were rarely progression and connective tissue matrix synthesis by
detectable in samples with pH 107/ml above pH 5 (the composition varying purification techniques it has been shown that the bone
with pH). The results are consistent with the hypothesis resorption was due to a cell surface-associated chaperonin
that TNOC produced by bacteria at neutral pH are (cpn) 60, cytokine synthesis was due to a 2-kDa peptide
important in gastric carcinogenesis and explain why the and inhibition of cell cycle progression (and associated
use of sulphamic acid results in a failure to observe this connective tissue matrix synthesis) was due to a 8-kDa
relationship. protein which we have termed gapstatin, as it blocks cells
in the Gz phase of the cycle. The cytokine-inhibiting
protein had a unique mechanism of action, being able to
ESCHERICHIA COLI CHAPERONIN 60 (GROEL) STIMULATES directly induce the transcription of the gene for interleukin-
PRO-INFLAMMATORY CYTOKINE SYNTHESIS IN A 6. The activity of these proteins is likely to contribute to
CONFORMATION-INDEPENDENT MANNER the pathology of LJP, which demonstrates rapid loss of the
bone supporting the teeth. Purification of these surface-
I? Tabona, S. Nair, K. Reddi, A Miller*, M. Preuss" and B. associated proteins had proved difficult and we are
Henderson
currently using the phoA fusion technique for cloning
Maxillofacial Surgery Research Unit, Eastman Dental exported bacterial proteins in order to clone these bio-active
Institute, University of London, 256 Gray's Inn Road, London, surface-associated proteins. Thus A . actinomycetemcomitans
WCIX 8LD and "Department of Chemistly, Imperial College has a number of interesting putative virulence proteins
London, London SW7 2AY associated with its outer surface, including a molecular
chaperone.
Molecular chaperones play a vital role in protein folding.
The 60-kDa chaperones of some bacteria possess other
activities, inducing cellular adhesion molecule expression
and stimulating bone resorption. Here we describe the GROWTH OF STREPTOCOCCUS ORALIS ON THE HUMAN
monocyte cytokine stimulating activity of the cpn60 of SERUM GLYCOPROTEIN, a -ACID GLYCOPROTEIN
1
Escherichia coli, groEL. This molecule is active at H. L. Byers, E. Tarelli, K. A. Homer and D. Beighton
picomolar concentrations. This activity was shown to be
Joint Microbiology Research Unit, Faculty of Clinical
distinct from LPS and appeared to be downregulated in a
Dentistry, KCSMD, Caldecot Road, Denmark Hill, London,
CD 14-dependant manner in monocytes. Trypsin digestion of
SE5 9RW
groEL did not inhibit its ability to induce cytokine
transcription and translation in monocytes suggesting that
The pathogen Streptococcus oralis is isolated from an
the activity resides either in a tryptic peptide to a low
increasing number of cases of bacteraemia in immunocom-
abundance molecule which co-purifies with the groEL. As
promised patients. In order for this organism to persist in
groEL constitutes a significant fraction of the mass of
vivo it must obtain nutrients from host tissue components.
bacterial protein (some 5-lo%), it may represent a major
Serum glycoproteins may act as a source of fermentable
pro-inflammatory stimulus during infection.
carbohydrate. We have therefore investigated the ability of
S. oralis to utilise the glycans of the human serum
glycoprotein a1 -acid glycoprotein (AGP) when AGP is
ACTINOBACILLUS ACTINOMYCETEMCOMITANS, AN ORAL provided as the sole carbon source. When growth was
OPPORTUNIST PATHOGEN WITH A NUMBER OF SURFACE- complete every constituent monosaccharide with the excep-
ATTACHED IMMUNOMODULATORY PROTEINS INCLUDING tion of 22% of the N-acetylglucosamine (GlcNAc) had been
removed from AGP. All the released monosaccharides were
CHAPERONIN 60
utilised, except fucose, which was detected free in the
J. Fletcher*, S. Nairt, €? Tabona*t, S. Meghjit, M Wilson", supernate. No free oligosaccharides were detected in the
J. WardS, S Poole' and B. Hendersont culture supernate during the growth period, providing no
*Department of Microbiology, tMaxillofacia1 Surgery evidence for the presence of endoglycosidase activity.
Research Unit, Eastman Dental Institute, and $Department of The organism produces an array of exoglycosidases
Biochemistry and Molecular Biology, University College (including a- and P-mannosaidases) that sequentially
London and §Division of Endocrinology4, NIBSC, Potters Bar released the sugar residues of AGP until the only
monosaccharide remaining was the terminal GlcNAc linked
Actinobacillus actinomycetemcomitans is a member of the to asparagine. We describe a detailed mechanism by which
normal oral microflora but is also involved in the rapid pathogenic bacteria are able to utilise glycan chains of a
1056 ABSTRACTS
serum glycoprotein to sustain growth. The ability to utilise PCR amplification indicated the presence of the vanX and
glycans may have implications in the pathogenicity of the van2 genes only and not the vanY gene. By contrast, the
bacteria. chicken isolates possessed vanX, vanY and van2 genes, and
showed different banding patterns from amplification of the
intergenic regions: all amplified vans-vanH (3 11 bp ampli-
con) and vanYvan2 (336 bp amplicon); whilst amplification
GROWTH OF STREPTOCOCCUS ORALZS IN HUMAN SERUM
of vanX-vanY produced a 543-bp product or a novel 1353-
K. A. Homer, F J. Foxall*, J. K. Nicholson*, J. Philpott-
! bp product. Preliminary evidence indicates that gene
Howard and D. Beighton clusters conferring vancomycin resistance in VREF isolated
Joint Microbiology Research Unit, Faculty o Clinical
f from patients at King's College London, and VREF isolated
Dentisty, KCSMD, Cladecot Road, London, SE5 9RW and from chickens over the same time period, are not identical.
*Department o Chemistry, Birkbeck College, University o
f f This suggests human infection with VREF may not be
London, London, WClH OPP caused by the VREF harboured by chickens, or by direct
transmission of VanA from chicken to human isolates of E.
Streptococcus oralis is emerging as a major cause faecium .
of septicaemia in neutropenic and immunocompromised
patients. Little is known regarding the virulence
mechanisms of these organisms but it is clear that they
EFFECT OF LACTOFERRIN ON GROWTH, VIABILITY AND
must be capable of proliferation within the circulation
in order to cause disease. We have investigated the HAEI"-BINDING BY BLACK-PIGMENTEaD ANAEROBES
in-vitro growth of S. oralis in normal human serum in M. T. Andres, J. Heath*, 0. Aguilera, J. F. Fierro and
order to identify serum components that may provide C. W I. Douglas*
nutrient sources for microbial replication. Growth in serum Laboratory o Oral Microbiology, School o Stomatology,
f f
was accompanied by the production of a range of University o Oviedo, Spain and *Department o Oral
f f
glycosidic enzyme activities (including sialidase and N- Pathology, School of Clinical Dentistry, Shefield
acetyl-~-D-g~ucosaminidase) with the potential to degrade
the N-linked glycans of serum glycoproteins and liberate Periodontal diseases are frequently associated with the
fermentable carbohydrates. Proton nuclear magentic res- presence of black pigmented anaerobes, particularly Por-
onance studies demonstrated that microbial growth was phyromonas gingivalis and Prevotella intermedia. These
accompanied by cleavage of the molecularly mobile organisms require haemin as a source of iron for growth.
N-acetylsugars (sialic acid and N-acetylglucosamine) Since, lactoferrin (LF), an iron-binding protein, has been
from serum glycoproteins and their subsequent transport detected at significant levels in periodontal sites
and catabolism. The release of fermentable N-acetyl- (0.5 - 1.5 mg/ml), we have investigated the antibacterial
sugars from the glycans of serum glycoproteins is one effect of this protein on black-pigmented species. I?
mechanism by which S. oralis obtains nutrients for growth gingivalis W50, Pr. intermedia ATCC 25611 and the
and may have relevance to the growth of these organisms closely related species Pr. nigrescens ATCC 25261 and a
in vivo. clinical isolate Pn77 were used. Cells suspended in 10 m~
phosphate buffer containing 0.1 m~ EDDA were treated
with apoLf (2 mg/ml) but viability of the strains was not
PRELIMINARY EVIDENCE FOR THE UNRELATEDNESS OF affected. For growth studies, cultures in broth supplemented
HUMAN VREF INFECTION FROM POULTRY M
with 100 m EDDA and either apoLf or iron-saturated Lf
(FeLf) 2 mg/ml were incubated at 37°C and optical
M. Kirk, H. Y. Chen, R. L. R. Hill, M. W. Casewell and densities were measured at 600nm. ApoLf had no effect
D. Beighton on the growth of Pr. intermedia or Pr. nigrescens but
Joint Microbiology Research Unit, Faculty o Clinical
f significantly inhibited the growth of I? gingivalis W50
Dentistry, KCSMD, Caldecot Road, Denmark Hill, London, (approx. 35%). FeLf was without effect. This inhibition
SE5 9RW could be due to direct damage to cells or to haemin
sequestration by Lf. However, both FeLf and apoLf
Nosocomal infections caused by vancomycin-resistant complexed with haemin and Lf binding to I? gingivalis
Enterococcus faecium (VREF) cause clinical concern was enhanced in the presence of haemin suggesting either
because there may be no effective antimicrobial therapy. the existence of separate binding sites or that cell binding
It has been suggested that poultry may provide a reservoir of haemin-Lf complexes takes place. In contrast, Lf binding
for vancomycin-resistant organisms or the gene cluster to II intermedia and Pr. nigrescens was reduced in the
(vanA) conferring vancomycin resistance, which could then presence of haemin, suggesting competition for the same
be disseminated via the food-chain. We have amplified binding site. Lf induced release of bound haemin from
specific regions of the vanA gene cluster by PCR from 37 cells of all three species in a dose dependent manner and
isolates of VREF from patients and 36 VREF isolated from electron microscopy showed a lower cytoplasmic density in
supermarket poultry. All isolates were distinct by PFGE. cells of some strains, suggesting membrane damage had
Using specific primers, intergenic regions of the vanA occurred. These data indicate (1) that Lf may inhibit
cluster were amplified between the fimctional genes vans growth of I? gingivalis in a periodontal pocket but not
and vanH, vanX and vanx and vanY and van2. The growth of PK intermedia or Pr. nigrescens, (2) that growth
presence of the vanX, vanY and van2 genes were also inhibition is not apparently due to haemin sequestration and
determined with intragenic primers. The 37 patient isolates may be due to membrane damage, and (3) in excess
showed amplification of the region between the vans and haemin (e.g., bleeding events), the effects of Lf are likely
vanH genes only, with an amplicon of 311 bp. Intragenic to be reduced.
ABSTRACTS 1057
ENHANCED BINDING OF MENINGOCOCCI TO INFLUENZA A PHENOTYPIC CHARACTERISTICS OF ANTIBIOTIC-SENSITIVE
INFECTED HEP-2 CELLS AM) ANTIBIOTIC-RESISTANT STRAINS OF M O M E L L A
0. R. E l - h e r , M. W. Raza, M. M. Ogilvie, D. M. Weir and CATARRHALIS
C. C. Blackwell 0. R. El-Ahmer, M. W. Raza, C. C. Blackwell, D. M. Weir
Department o Medical Microbiology, University o
f f and M. M. Ogilvie
Edinburgh Department o Medical Microbiology, University o
f f
Edinburgh
It has been suggested that influenza A infection is a
predisposing factor for meningococcal disease. We found Moraxella catarrhalis is increasingly recognised as a
that HEp-2 cells infected with respiratory syncytial virus pathogen in some clinical conditions. We examined two
(RSV) bound significantly more meningococci of all isolates: one grew on New York City medium containing
serogroups, serotypes and subtypes tested. In this study selective antibiotics (MCl) and the other did not (MC2).
we used flow cytometry to examine binding of Neisseria By flow cytometry, we compared binding of the strain
meningitidis strains labelled with fluorescein isothiocyanate labelled with fluorescein isothiocynate to HEp-2 cells and
to HEp-2 cells and HEp-2 cells infected with a local strain HEp-2 cells infected with respiratory syncytial virus
of influenza A; these included 12 immunotype strains of subgroup A (RSVA) or RSV subgroup B (RSVB).
meningococci (W. D. Zollinger, Walter Reed Army Institute Compared with binding to uninfected cells (mean = loo),
of Research). Staphylococcus aureus was the gram-positive strain MC1 bound in significantly higher numbers to the
control. Each strain bound in significantly greater numbers RSVA-infected cells (mean = 387, p < 0.05 95% CI, 143-
to the virus infected cells then the uninfected controls. 1042) but strain MC2 bound in significantly lower
Enhanced binding of the gram-negative isolates to virus numbers (mean = 51, p < 0.05, CI 40-78). Similar results
infected cells could be partly accounted for by increased were obtained for RSVB infected cells. This was the
expression of molecules that bound monoclonal antibodies first example we found of decreased bacterial binding in
to CD14 and CD18. Pre-treatment of the cells with the this model. MC2 was killed by sera from 14 donors
monoclonal antibodies significantly reduced binding of the but MCI was serum resistant. MC1 lacked bands at 81
gram-negative bacteria but not gram-positive bacteria. kDa and 66 kDa but had a distinct band at 19 kDa
Treatment of cells with neuraminidase enhanced binding which was absent in MC2. Similar results were obtained
of gram-positive bacteria (p < 0.05) but not gram-negative with six other antibiotic-sensitive strains. We predlct M.
bacteria. While it has been suggested that susceptibility to catarrhalis strains with characteristics of MC2 are less
bacterial infection following influenza is associated with likely to contribute to serious disease following RSV
decreased immune responses and damage to epithelial cells, infection. The effect of prolonged antibiotic treatment on
our studies indicated that specific changes in virus infected selection of strains with characteristics similar to MCl
cells might also enhance density of colonisation by needs to be examined in complementary clinical and
potentially pathogenic bacteria. laboratory studies.