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Cronobacter _Enterobacter sakazakii_ research and surveillance in

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					Cronobacter (Enterobacter sakazakii)
 research and surveillance in Korea



              KUNHO SEO



              Seoul, Korea
Where is Korea?
                      Fast Facts

Population: 48.5 m
Size: 98,480 square kilometers (38,023 square miles)
Foods concerned with E. sakazakii
 4 major PIF products
 Other imported PIF
 Baby foods
 Sunsik: dried cereal(grains) foods
Contents
  Enterobacter sakazakii in Korea

No outbreak or case has been reported in
Korea
Commercial powdered infant formula
products were recalled due to the
contamination of E. sakazakii in 2006
Several monitoring studies have been
conducted since 2006
  Enterobacter sakazakii in Korea

Zero tolerance policy has been
implemented for certain baby foods
including PIF
Official methods have been established
Food companies has implemented GMP,
HACCP, and sterile production system for
PIF
       Prevalence of E. sakazakii in PIF
         The Annual Report of KFDA, Vol.9, 407~408. 2005



Methods used
 PIF
   FDA MPN
   Chromogenic E. sakazakii Agar or OK medium
   API 20E + PCR


 cereal infant foods
   25g +225 ml EE broth for E. sakazakii
   25g +225ml BPW for B. cereus
Powdered infant formula
 4 of 85 (5.33%) PIF were positive for E. sakazakii
 Contamination level: 0.36 ~2.86 MPN/100 g of PIF


cereal infant foods
 E. sakazakii and B. cereus were isolated from 6 (6%) and 23
 (23%) of 100 samples, respectively
     Prevalence of E. sakazakii in PIF

• Surveys     of E. sakazakii in powdered infant formula
                                                               16
(Meeting Report, FAO Headquarters. MRA Series 10, Rome, Italy. 16 – 20 January 2006)
Prevalence of E. sakazakii in ingredients of Sunsik
Jae-                                                             pp.000~000
Jae-Won Choi et al : Korean J. FOOD SCI. TECNHNOL. Vol 40, No.1, pp.000~000 (2008)


Total of 23 Sunsik ingredients tested for E. sakazakii
Method used: FDA
  Selective agar
     VRBG
     Chromogenic Enterobacter sakazakii agar
     Enterobacter sakazakii medium
  Confirmation
     API 20E
     PCRs: tDNA, ITS, 16S rRNA
            E. Sakazakii in Sunsik




Jae-Won Choi et al : Korean J. FOOD SCI. TECNHNOL. Vol 40, No.1, pp.000~000 (2008)
          E. Sakazakii in Sunsik

5 out of 32 (16%) samples were finally confirmed as E.
sakazakii
The contamination level was 2.86 MPN/100g of Sunsik
       Prevalence of E. sakazakii in
        ingredients of infant foods
Dried fish samples(n=50): shrimp, anchovy, brown
seaweed
Dried powdered vegetable samples (n=50): soybean,
yam, potato
Sunsik samples (n=36): a mixture of powdered grains
and other ingredients such as glutinous rice, barley,
brown rice, seafood, and fruits.
 consumed as raw
 consumed by infants
 considered as well-being food
Method: US FDA (2002)
Prevalence of Enterobacter sakazakii in various foods
                                                                  122        196-
   Kyumson Kim et al : International Journal of Food Microbiology 122 (2008) 196-203




  Sample                            Sample number                         E. sakazakii positive (%)


  Sunsik                            36                                    47.2


  Dried Fish                        50                                    26


  Vegetable                         50                                    2



           Kyumson Kim et al : International Journal of Food Microbiology 122 (2008) 196-203
NVQRS microbial standards for PIF

      Bacteria             Baby Foods

      Coliforms        n=5, c=2, m=0, M=10


  Total Viable Cells      20,000 CFU/g


    E. sakazakii
                           Negative/100g
    (Oct, 2006)

      B. cereus               100/g
KFDA microbial standards for baby foods

        Bacteria                        Baby Foods

        Coliforms                          Negative

                                      liquid form: 100/ml
    Total Viable Cells
                                  (powdered form 20,000/g)

      E. sakazakii                         Negative
      (Oct, 2006)        (powdered form for infants less than 6 months)

        B. cereus                           100/g
           Industry’
           Industry’s responses

Production in clean room system
Walls and ceiling covered with stainless steel
Employee hygiene
Raw material quality control
Frequent sampling: every 10 min
E. sakazakii as an indicator
 Prevalence of foodborne pathogens in PIF
    after implementing HACCP and GAP

Ji Yeon Hwang et al : Korean J. Food Sci. Ani. Resour.
Vol 28(5) : 555-561 (2008)
Method used: modified FDA
EE broth
Selective agar
 Chromogenic Enterobacter sakazakii agar
 Enterobacter sakazakii medium
TSA: yellow colony
Confirmation: API 20E, PCR (16S rRNA)
     Enterobacter sakazakii




Ji Yeon Hwang et al : Korean J. Food Sci. Ani. Resour. Vol 28(5) : 555-561 (2008)
Prevalence of E. sakazakii after taking active quality
              control by the industries

All samples are negative for E. sakazakii
The strict microbiological standard and implementation
of safe production system could reduced the
contamination of E. sakazakii in PIF
         Surveillance Study in Patients
     Jung Beom Kim et al : Yonsei Med J 49(6) : 1017 – 1022, 2008




1146 stool samples were collected from March to
October, 2006, at Uijeongbu St. Mary’s Hospital, in
Korea
fecal swab in 10 mL of BPW-EE broth-DFI agar(5 blue-
green colonies)-TSA(yellow pigment)-Vitek GNI and 16S
rRNA PCR
4 samples were positive for E. sakazakii
Age Distribution among Patients




    Jung Beom Kim et al : Yonsei Med J 49(6) : 1017 – 1022, 2008
    DOI 10.3349 / ymj.2008.49.6.1017
Enterobacter sakazakii




Jung Beom Kim et al : Yonsei Med J 49(6) : 1017 – 1022, 2008
DOI 10.3349 / ymj.2008.49.6.1017
          Current culture method for E. sakazakii
                    (KFDA and NVQRS)

       100g + 900㎖ sterile warm water
              900㎖

                           36 , 24h               (1st enrich.)

                           ↓10㎖
                            10㎖
                            10


                90㎖ EE broth
                90㎖                               (2nd enrich.)

                           36 , 24h
                            10㎕
                           ↓10㎕

Chromogenic Enterobacter sakazakii agar (CESA)
                                                   (selective
                                    VRBG)
      Violet red bile glucose agar( VRBG)
                                                    media)
             E. sakazakii medium

                           36 , 24h

                   TSA agar

                           35 , 24h

                             test,
     Yellow pigment, oxidase test, API32E
                                                 (confirmation)
                               Real-
(Vitec2 compact GN), PEGE, mms Real-Time PCR
            Real-
            Real-time PCR - E. sakazakii
  Seo and Brackett, Rapid specific detection of Enterobacter sakazakii in infant
                                                                        68:59-
formula using a real - time PCR assay. Journal of Food Protection. 2005 68:59-63.


E. sakazakii target genes

   E. sakazakii partial macromolecular synthesis (MMS) operon gene

   rpsU, dnaG, and rpoD

dnaG-rpoD intergenic sequence differs in length and primary sequence
between species in E. coli studies

Limited area is specific to E. sakazakii among other Enterobacter :
sequence analysis and BLAST analysis
       Real-
       Real-time PCR - E. sakazakii

Inclusivity & exclusivity test
  78 E. sakazakii strains

  18 other Enterobacter strains

  57 non-Enterobacter strains

  Tested positive for 100 % of all E. sakazakii strains

  No false positives were recorded among all non-E. sakazakii
  Enterobacter strains and non-Enterobacter organisms.
          Real-
          Real-time PCR - E. sakazakii

Detection limit
                                     CT Value


    Number of cells (CFU/ml)   PBS              10 % Infant formula

              108              20                       20

              107              24                       24

              106              30                       26

              105              34                       32

              104              39                       34

              103              43                       38

              102              45                       43

              101              ND*                      ND
        Real-
        Real-time PCR - E. sakazakii
Detection in samples artificially inoculated with E. sakazakii


                                Number of positive samples/total number of samples


        Inoculum                             6-
                           culture method in 6-7 days          Real-
                                                               Real-time PCR in 2 days

         6 CFU/g                    12/12                              12/12


        0.6 CFU/g                   12/12                              12/12
         Real-
         Real-time PCR - E. sakazakii
Example of confirmation tool


                                                          real time PCR   Confirmation with API test
     Strain: Enterobacter    Origin   Field Office Test
                                                              Test          (level of certainty, %)


    sakazakii Portagen SP1   food            +                 +             E. sakazakii (98%)

    sakazakii Portagen ST2   food            +                 +             E. sakazakii (98%)

    sakazakii 190518 C1I2    food            +                 +             E. sakazakii (98%)

    sakazakii 187985 C4IA    food            +                  -         Serratia rubidaea (99%)

    sakazakii 165747 C2I4    food            +                 +             E. sakazakii (98%)

    sakazakii 187985 C1IA    food            +                 +             E. sakazakii (98%)

    sakazakii 165747 C2I1    food            +                 +             E. sakazakii (98%)

    sakazakii 165747 C1I1    food            +                 +             E. sakazakii (98%)

    sakazakii 165747 C3I1    food            +                 +             E. sakazakii (98%)

    sakazakii 165747 C2I2    food            +                 +             E. sakazakii (98%)
         Real-
         Real-time PCR - E. sakazakii

Example of screening tool



                                                        confirmed by 2ndAPItest
     Strains identified by API   Real-time PCR test
                                 Real-
                                                          (level of certainty,%)

                                         -                         NI*

                                         -                    Erwinia spp.

         Ps. Fuo/putida                  -                    Erwinia spp.

       Flavii oryzihabitans              +            Enterobacter sakazakii (98%)
   E. sakazakii detection methods

Future direction
 More selective media for environmental samples or other foods
 than PIF such as fresh produce etc.

 Immunoassay: Immunomagnetic beads

 Active use of molecular techniques for presumptive screening or
 confirmation tool

 Harmonization of international detection methods
   Real-
   Real-time PCR –Current research

Simultaneous detection of IAC, E. sakazakii, Salmonella spp.
   E. sakazakii and other microorganisms in powdered infant formula: meeting
   report, MRA Series 6, 2004

   Enterobacter Sakazakii and Salmonella in Powdered Infant Formula. Meeting
   Report, FAO Headquarters. MRA Series 10, Rome, Italy. 16 – 20 January 2006
Microbial hazards in PIF
        FAO/WHO, 2004
         Real-
 Triplex Real-time PCR – IAC, Salmonella &
                 E. sakazakii
Plasmid DNA construction for IAC production




                               Cloning for IAC mass production



                                                                 Construction of primers and probes for
                                                                 simultaneous detection of IAC, E.
                                                                 sakazakii, Salmonella spp.
Acknowledgment
                    Dr. Robert E. Brackett
                    Dr. Keith Lampell
                    Dr. Michael L.. Kotewicz
     JY Hyun        Dr. Ben Tall
     JH Lee                      Edelson-
                    Lie. Sharon Edelson-Mammel
     CH Chun        Dr. Kwangyoung Song
     SL Hahn
     JY Park
     CH Sung

     Dr. KS Kwon
     Dr. IK Hwang
     Dr. HS Kwack
     Dr. JS Park
     Dr. S. Heo
Public Health Lab at KKU, Seoul, Korea
Lab members