Enterobacter sakazakii General Microbiology - PowerPoint PPT Presentation

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Enterobacter sakazakii General Microbiology

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Enterobacter sakazakii General Microbiology

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  1. Enterobacter sakazakii General Microbiology Contaminants and Natural Toxicants Subcommittee Meeting March 18, 2003 Maria Nazarowec-White PhD Science Evaluation Unit Canadian Food Inspection Agency

  2. OVERVIEW • History • Ecology • Incidence on Canadian retail market • Growth characteristics • Phenotype characterization • Genotyping • Pathogenicity

  3. History • Name Enterobacter sakazakii proposed in1980 • DNA hybridization studies • Biochemical reactions • Yellow - pigmented colonies

  4. Entrobacter sakazakii colony

  5. Electron micrograph of Entrobactor sakazakii

  6. Surface water Mud Rotting wood Bird dung Rodents Domestic animals Cow’s milk E. sakazakii and the environment

  7. Clinical Strains of E. sakazakii • London 2 • Montreal 1 • Toronto 3 • LCDC 3

  8. Dried-infant formula isolates Company Positive samples (%) A 3/24 (12) B 2/24 (8) C 0/24 (0) D 1/24 (4) E 2/24 (8) total 8/120 (6.7)

  9. Temperature Gradient Incubator • 10 strains • Temperature range 4-50°C • 30 tubes in duplicate

  10. TemperatureGradient Incubator C = Clinical F = Food

  11. Experimental Protocol Growth Study • 3 Different formulae • Cocktails • 5 clinical isolates • 5 food isolates • 4, 10 and 23°C • Direct plating • Generation and lag time

  12. Phenotyping • Biochemical 3 biotypes • Antibiograms 4 antibiograms

  13. Ampicillin Cefotaxime Chloramphenicol Gentimicin Kanamycin Polymixin B Streptomycin Trimethoprim-Sulfamthoxazole Tetracycline Sulfisoxazole Cephalothin Antibiotics used

  14. Antibiotic Resistance Profile Antibiotic Number of strains (18) • Ampicillin 1 • Tetracycline 2 • Chloramphenicol 3 • Cephalothin 17 • Sulfisoxazole 18

  15. Genotyping • Ribotype • Pulsed Field Gel Electrophoresis (PFGE) • Random Amplification of Polymorphic DNA (RAPD)

  16. Genotypic profiles of E. sakazakii

  17. Virulence Factors • Suckling mouse assay • Three cell culture lines • Infectivity • intraperitoneal • oral challenge

  18. Enterotoxin Screening • 4 strains positive for enterotoxin production • 3 clinical and 1 food

  19. Morphological Responses of Cell Lines to Filtrates • 1 clinical strain toxic to 3 cell lines • more pronounced on Y-1 and Vero cells • no effect of boiling on Y-1 and CHO cells • cytopathic effect decreased on Vero cells • 2 clinical strains, no effect on 3 cell lines

  20. Infectivity studies • i.p. injections lethal at 108 CFU per mouse • within 3 days of dosing • per oral route 2 strains caused death • 1 clinical and 1 food

  21. Summary • Incidence in dried infant-formula on the Canadian market found to be 6.7% • Minimum growth temperature 5.5-8.0°C • Generation time at room temperature 40 min • Four of eighteen strains produce enterotoxin • Infectious dose • Heterogeneity among strains

  22. What do we do now??