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Confirmation of VTEC

Confirmation of VTEC. Enteric Reference Laboratory ESR –NCBID Wallaceville. Screen positive enrichment broths. Immunomagnetic separation (Dynal). CT-SMAC agar. EHEC agar. ONLY sorbitol +ve colonies Negative 0157. Sorbitol –ve colonies. Haemolytic colonies. ONLY non-haemolytic colonies

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Confirmation of VTEC

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  1. Confirmation of VTEC Enteric Reference Laboratory ESR –NCBID Wallaceville

  2. Screen positive enrichment broths Immunomagnetic separation (Dynal) CT-SMAC agar EHEC agar ONLY sorbitol +ve colonies Negative 0157 Sorbitol –ve colonies Haemolytic colonies ONLY non-haemolytic colonies Negative 0157 6 Colonies TSI, LIA, TSA agar plate Typical reactions on TSI & LIA O157 antigen latex kit (Pro-Lab) O157 positive O157 titration (MAST-Assure) O157 positive BHI Broth H7 PCR (Invitrogen Life Technologies) Fields P et al 1997 Wang G et al 2002 H7 positive Verocell assay for verocytotoxins Toxin positive Multiplex PCR for Stx1 & Stx2 toxingenes, eaeA gene and Intimin (hly) gene (Invitrogen Life Technologies) Paton & Paton 1998 All positive Biochemical id as E. coli Positive E. coli O157:H7[H7] Motility Test Motile = H7 Non-Motile = [H7]

  3. hlyA eaeA Stx2 Stx1 16S VTEC PCR

  4. Comparison of PFGE Patterns of Human Isolates of E. coli O157:H7 PFGE Pattern ID Number Health District Phage Type

  5. Comparison of PFGE Patterns of Human Isolates of E. coli O157:H7 Phage Type 21 PFGE Pattern ID Number Health District Phage Type

  6. MLVA • Multilocus variable number tandem repeat analysis • PCR-based typing method • Potential for use within the PulseNet platform • Advantages over PFGE • Rapid results • Automation – high throughput • Standardised results - easily compared between laboratories

  7. MLVA Analysis of E. coli O157:H7 in NZ • Assessed all 80 E. coli O157:H7 isolates identified in New Zealand in 2006 • Analysed by PFGE and MLVA • Good correlation between results • Included isolates from 8 family outbreaks identified epidemiologically • Similar PFGE profiles • Identical MLVA profiles (8 loci assessed) • Isolates with PFGE patterns that clustered with a family outbreak but were not epidemiologically related had different MLVA profiles.

  8. Acknowledgements Routine confirmation of VTEC was performed under the service contract with the Ministry of Health MLVA testing was done using ESR CF funding My thanks to the Enteric Reference team Dave Duncan, Shevaun Paine and Emily Motion. Thanks also to Chris Pope for help with dendrograms and Lisa McCallum for flow charts

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