Thorough identification of pathogenic Shigatoxin producing Escherichia coli (STEC) in Belgium
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1 Thorough identification of pathogenic Shigatoxin producing Escherichia coli (STEC) in Belgium Dr. Ir. Sarah Denayer NRL VTEC WIV-ISP, Brussels, Belgium 10 th Annual workshop of the NRLs for E. coli in the EU 5-6 November 2015 IDESTEC 2 RT 12/
2 Regulation Regulation EC No. 209/2013 as regards microbiological criteria for sprouts (1/07/2013): Absence of STEC O157, O26, O111, O103, O145 and O104:H4 in 25g
3 IDESTEC 2 12/12 Enrichment Extraction gdna qpcr Screening Isolation if screening positive Confirmation Further characterisation year # samples tested Presumptive Detection VTEC VTEC Detected (9.4%) 16 (0.9%) (12.7%) 31 (1.7%)
4 ILVO Comparison of methods 4 non-o157 STEC strains (O26, O103, O111, O145) ± 10 cfu/25g Efficient enrichment Less efficient enrichment
5 ILVO Isolation or recovery percentage (%) Comparison of methods 4 non-o157 STEC strains (O26, O103, O111, O145) ± 10 cfu/25g Minced beef Carcass swab Sprouted soy Lettuce mix Direct plating Food matrix After acid treatment
6 ILVO Ct > 25: = Less efficient enrichment More interfering background microbiota
7 ILVO Isolation or recovery percentage (%) High selective agar medium (CHROMagar STEC) + Less selective agar medium (ChromID EHEC) Minced beef Carcass swab Sprouted soy Lettuce mix Food matrix CHROMagar STEC ChromID EHEC Both media Combination of both isolation media
8 Naturally contaminated samples Enrichment qpcr Screening if screening positive acidification year qpcr screening and isolation Samples tested Presumptive detection VTEC STEC Detected (12.7%) 31 (1.7%) ph- treatment (N=150 samples) Isolation (without IMS) 14 (9.3%) 11 (7.3%) 7 (4.7%) Confirmation No treatment Acid treatment Verhaege et al. Submitted for publication
9 II. qpcr screening method for VTEC Enrichment Extraction gdna qpcr Screening Isolation if screening positive Confirmation Further characterisation Comparison 2 gdna extraction methods FEP: Food Extraction Pack (Pall ) Quality Quantity Price Time NSFK: Nucleospin food Extraction Kit (Macherey-Nagel ) FEP + x1 1.7 /sample 10min NSFK +++ x /sample 3h qpcr Screening targeting stx1, stx2 and eae genes CoSYPS Path E. coli (WIV-ISP) GeneDisc STEC (Pall ) qpcr Verstraete et al, 2012 (ILVO) Foodproof STEC Screening (BIOTECON Diagnotics)
10 Comparison different qpcr Screening systems qpcr Screening: CoSYPS Path E. coli uida - uida + Not Escherichia coli Unknown sample ipah - Escherichia coli or Shigella ipah + Shigella or EIEC eae - AND aggr + Other Escherichia coli eae + eae - AND aggr - Piednoir et al Submitted for publication EAEC or EASTEC stx 1 - and stx2 - stx 1 + or stx2 + EHEC or EPEC stx 1 - and stx2 - stx 1 + or stx2 + stx 1 + or stx2 + STEC or other E. coli stx 1 - and stx2 - EAEC EASTEC EPEC EHEC STEC Other E. coli
11 III. Molecular characterisation of VTEC Strain collection: 757 strains- 773 gdna Food (268), human (395) and animal (55) origins+ (45 unknown origin) 37 O-groups, 22 H-types 5 pathotypes 19 virulence factors Genetical relation between STEC isolates in Belgium? PFGE, LSPA-6, Tir(A255T) SNP, Q21/Q933, Clade typing, stx2 subtyping Virulence profile of strains circulating in Belgium? Luminex
12 Strain serogroup List of the 42 genes in the VTEC characterization Luminex system. Each column represents an independent multiplex assay. Serogroups-13 H-7 PF-8 DEC-9 Nle-5 wzx O26 flic-h2 eae stx 1 nlea Results of the 13 targets of the serogroup-13 assay wzy O45 flic-h7 O103 O104 ehxa stx 2 nleb wbgn O55 flic-h8 katp eae nlee wzx O91 flic-h11 espp ipah nlef wzx O103 flic-h19 saa LT nleh1&2 wzx O104 flic-h21 suba STh wzx O111 flic-h28 ent/espl2 aggr wzx O113 terb aaic bfpa O26 O45 O55 O91 O111 O113 O118 O121 O128 O145 O157 MFI ratio MFI ratio MFI ratio MFI ratio MFI ratio MFI ratio MFI ratio MFI ratio MFI ratio MFI ratio MFI ratio MFI ratio MFI ratio O O O O O O O O O O O128 74wzy 1 O O wzy O O wzx O128 Pathogenic Blank wzy O145 discrimination wzx O157 Serotyping + Probe coupled to bead Target with green fluorescent label Target hybridized to probe
13 Genetic diversity O157 STEC LSPA-6 Q21/Q933 alleles Stx subtype 100,00% 100,00% 100,00% 75,00% 50,00% 25,00% Lineage I Lineage I/II Lineage II 75,00% 50,00% 25,00% Q21 Q933 Q933+Q21 75,00% 50,00% 25,00% Stx2a Stx2c Stx2a+c Stx2ac-ve 0,00% Food Human 100,00% 75,00% 50,00% Clade 0,00% Clade 1 Clade 2 Clade 3 Food Human 100,00% 75,00% 50,00% 0,00% Tir (A255T) Food Human Food, n=70 Human, n=100 Tir T Tir A 25,00% Other clades ( 4, 5, 6, 7, 9) 25,00% 0,00% Food Human Clade 8 0,00% Food Human
14 Conclusions Isolation method STEC on different matrices optimized ph treatment usefull to reduce high background flora Use of 2 isolation media is recommended IMS not specific enough to reduce background flora 4 qpcr screening methods for STEC validated and evaluated Usefull for ISO/TS revision! Typed strain collection (human, food, environment) available Luminex detection system for 42 genes developed Used for: Rapid detection of new emerging pathogens Outbreak investigation, trace back/forward Determination of reservoirs, transmission routes, virulence factors of STEC circulating in Belgium
15 IDESTEC 2 12/12 was a collaboration of: Prof Dr. Lieven De Zutter (Ugent) Dr. Ir. Sigrid De Keersmaecker (WIV-ISP, PBB) Dr. Marc Heyndrickx (ILVO) Dr Ir Koen De Reu (ILVO) Bavo Verhaegen (Ugent/ILVO) Dr. Katelijne Dierick (WIV-ISP, FoodPathogens) Dr. Elodie Barbau Piednoir (WIV-ISP) Dr. Nadine Botteldoorn (WIV-ISP) Dr. Ir. Sarah Denayer (WIV-ISP, NRL VTEC)
16 Acknowledgements: Dr K Verstraete (ILVO) Dr M Elhadidy (Ugent/ILVO) E Dumoleyn (ILVO) E. Wattijn, W. Boukhouchi, E. De Vits, Z. Boukhouchi, C. Delsaut (WIV, VPA) D. Van Geel (WIV, PBB) NRC pathogenic E. coli Dr. Denis Pierard Klara De Rauw NRL pathogenic E. coli France Dr Estelle Loukiadis FASFC IDESTEC 2 12/12
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