Confirmation of VTEC

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Confirmation of VTEC

• Enteric Reference Laboratory
• ESR NCBID Wallaceville

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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 O157H7H7
Motility Test Motile H7 Non-Motile H7
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VTEC PCR

• hlyA
• eaeA
• Stx2
• Stx1
• 16S

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Comparison of PFGE Patterns of Human Isolates of
E. coli O157H7
ID Number
Health District
Phage Type
PFGE Pattern
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Comparison of PFGE Patterns of Human Isolates of
E. coli O157H7 Phage Type 21
ID Number
Health District
Phage Type
PFGE Pattern
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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

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MLVA Analysis of E. coli O157H7 in NZ

• Assessed all 80 E. coli O157H7 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.

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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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