The contribution of typing methods to risk assessment of VTEC/STEC
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Presentation of the EFSA's second scientific conference, held on 14-16 October 2015 in Milan, Italy. MICROBIOLOGICAL RISK ASSESSMENT
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The contribution of typing methods to risk assessment of VTEC/STEC
- 1. The contribution of typing methods to risk assessment - the case of Vero cytotoxin- (Shiga toxin-) producing E. coli (VTEC/STEC) Flemming Scheutz WHO Collaborating Centre for Reference and Research on Escherichia and Klebsiella Foodborne bacterial infections Microbiology & Infection control STATENS SERUM INSTITUT
- 2. Haemolytic uraemic syndrome HUS • Acute renal failure • Microangiopathic haemolytic anaemia (Intravascular haemolysis and destruction of the red blood cells) • Thrombocytopenia (Drop in the blood platelet count)
- 3. Once upon a time….
- 4. Non-O157s now share the scene with O157 from cases of HUS O26:H11 in Italy (stx2a highly virulent) O80:H2 in France (stx2a, stx2a2d, stx2c2d, stx2d, eae ξ) O104:H4 in the Republic of Georgia O73:H18 in Spain O91:H21 in Spain O59:H19 EAEC in Argentina O174:H8, H21 and H28 in Argentina
- 6. Stx subtypes and variants Stx/VT1: 3 subtypes; 13 variants VT2: 7 subtypes; 93 variants 47 vtx1 and vtx2 238 out of 285 valid sequences in GenBank a Stx/VT1 d c VT2 d b g c a e f
- 7. HUS & vtx subtypes
- 8. HUS &
- 9. HUS;
- 10. HUS associated VTEC = HUSEC and to a lesser degree vtx2d in eae negative
- 11. Siphoviridae Podoviridae vtx1 and vtx2 bacteriophages vtx1 vtx2 Courtesy of Maite Muniesa
- 12. Diversity: Morphology 1b 12 13b 16b 1a 4 13a 16a Courtesy of Maite Muniesa
- 13. AEM 2013 62% of 100 fecal samples from 100 healthy individuals showing no enteric symptoms carried Stx phages, with an average value of 2.6 x 104 Stx phages/g Bacteriophages in healthy humans
- 14. Prevalence of VTEC in healthy humans including food handlers or workers in daycare centers during 2010-2012 in Japan Healthy carriers Diarrhoeal cases 84.2 2.1 /100,000 /100,000 Tomoko Morita-Ishihara et al. IASR 2015
- 15. Stx phages in food Minced Beef 0 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36Salad 0 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Minced beef Saladlog10stxGC.25/g log10stxGC.25/g Sample nº At least 50 % of the Stx phages in these samples were infectious 102-105 Stx phages / 25 g minced beef. 100% of the samples 102-103 Stx phages / 25 g fresh salad. 69 % of the samples Food Microbiological criteria EU regulation Imamovic & Muniesa, AEM. 2011 Able to propagate in host strains Bar: qPCR *: + end point PCR Barcelona area has a low incidence of VTEC infections Courtesy of Maite Muniesa
- 16. Courtesy of Stefano Morabito :H10
- 17. Next generation sequencing NGS or Whole genome sequencing WGS
- 18. 18 2324 E. coli genomes – Panseq SNP phylogeny (ML) O157 O121 O113 O145 O26 Courtesy of Chad Laing
- 19. Acknowledgments 19 Application of WGS to outbreak investigation of gastrointestinal pathogens 1,075 strains of STEC O157 from clinical and animal isolates from England, Northern Ireland, Wales and Scotland collected from 1985 to 2014
- 20. Application of WGS to outbreak investigation of gastrointestinal pathogens Maximum clade credibility tree of 530 ∆25 SNP representatives of O157:H7 = Family Tree of O157:H7 using ∆25 SNP addresses SNP = Single Nucleotide Polymorphism Tim Dallman et al. Microbial Genomics 2015
- 22. Application of WGS to outbreak investigation of gastrointestinal pathogens 175 years ~1840 150 years 400 years The ancestral O157:H7 state is stx2c (lineage II) Early rapid diversification Shiga toxin complement and insertion sites are relatively stable Stx2a is relatively recent and has been acquired ~60 years ago on multiple occasions Courtesy of Tim Dallman YEARS
- 23. Courtesy of Tim Dallman O157:H7 has become more pathogenic to humans over its evolution Strong association with stx2a and severe disease YEARS
- 24. 24 Application of WGS to outbreak investigation of gastrointestinal pathogens Uneven Diversity of O157 54 ∆250 Clusters All 3 lineages have one ∆250 cluster that comprises > 50% of isolates of that lineage These 3 ∆250 clusters contains 84% of UK Cattle isolates ∆250 clusters with <5 isolates associated with foreign travel or imported food Supports Global Dispersion then Regional Expansion Courtesy of Tim Dallman
- 25. 25 Courtesy of Tim Dallman
- 26. The implicated O157 strain with the SNP address 18.35.397.765 had a strong NW geographical signal including some possible environmental exposures
- 27. Once upon a time…. These days: Outbreaks: • Watercress Risk factors: • Swimming • Contact with ruminants • Contact with another person with diarrhoea • Visiting farms • Steaks and ground beef • Travel to Africa (Turkey and Marocco) Host factors: • Antaacids • Cardiovascular disease • Gastrointestinal infection • Hispanic ethnicity (in the US) Hamburgers = ground beef
- 28. Microbiological risk assessment (MRA) (i) hazard identification: • vtx/stx subtyping by PCR or WGS (ii) hazard characterization: • WGS of bacteriophages and their hosts; • WGS assignment of host SNP addresses (iii) exposure assessment & (iv) risk characterisation • WGS & EPIDEMIOLOGY
- 29. ACKNOWLEDGEMENTS • Tim Dallman, Public Health England • Chad Laing; Public Health Agency of Canada • Stefano Morabito; Istituto Superiore di Sanità, Rome • Maite Muniesa; University of Barcelona
- 30. THANK you for your attention