Detection and typing of STEC at the NRL NL

Transcription

1 Detection and typing of STEC at the NRL NL Annet Heuvelink Food and Consumer Product Safety Authority (VWA), Zutphen, The Netherlands NRL E. coli NL

2 Detection samples Animal faeces Foods Environmental samples NRL E. coli The Netherlands A.E. Heuvelink 2

3 Detection methods for STEC O157 Cultural methods Immunomagnetic separation and concentration (IMS) techniques VIDAS system NRL E. coli The Netherlands A.E. Heuvelink 3

4 Isolation method E. coli O157 (ISO 16654:2001(E)) Selective enrichment: modified tryptone soy broth + novobiocin (mtsb+n) (6 h and 1824 h at 41.5 C) Immunomagnetic separation and concentration (IMS) Isolation: Confirmation: cefixime tellurit sorbitol MacConkey (CTSMAC) agar (50 µl) (1824 h at 37 C) 2nd selective agar of own choice (50 µl) indole production / biochemical identification kit agglutination with E. coli O157 antiserum NRL E. coli The Netherlands A.E. Heuvelink 4

5 VIDAS immunoconcentration & VIDAS UP E. coli O157:H7 (biomérieux) NRL E. coli The Netherlands A.E. Heuvelink 5

6 Recirculating immunomagnetic separation (RIMS) Pathatrix (Matrix Microscience) NRL E. coli The Netherlands A.E. Heuvelink 6

7 Test options Same Day When would you like your result? Next Day Days What size sample? 25g 375g Colourimetric or PCR? Colourimetric or PCR? Colonies, Colourimetric or PCR? Colourimetric PCR Pooling Colourimetric PCR Pooling Colonies Colourimetric PCR Pooling Colonies CE50SD PE50SD CE50SD PE50SD PE50SD CE50 PE50 PE50 4.5hrs static 30 min PX 15 min CX 4hrs static 30min PX PCR 5.5hrs static 60min PX 15min CX 4hrs static 60min PX PCR 3 hr PX Plate O/N static 30 min PX 15min CX O/N static 30 min PX PCR O/N static 30 min PX Plate PE250SDP 5hrs static 60min PX PCR PE250SDP 5hrs static 60min PX PCR CE250P O/N static 30min PX 15min CX or PE250SDP O/N static 30min PX PCR / plate NRL E. coli The Netherlands A.E. Heuvelink 7

8 Typing of STEC O157 PCR : stx 1, stx 2, eae, hly EHEC PFGE : XbaI digestion of genomic DNA (PulseNet protocol, CDC, Atlanta, US) pattern analysis with BioNumerics software (Applied Maths, Kortrijk, Belgium) Phage typing (Laboratory for Enteric Pathogens of the Central Public Health Laboratory in London) stx subtyping, MLVA, MLST, SNP typing NRL E. coli The Netherlands A.E. Heuvelink 8

9 Detection methods for VTEC nono157 Molecular methods (realtime) PCR Cultural methods NRL E. coli The Netherlands A.E. Heuvelink 9

10 Realtime PCR GeneDisc VTEC/screening FAM Flic H7 rfbe O157 Negative Ctrl ROX E. coli stx E. coli eae Inhibition Ctrl NRL E. coli The Netherlands A.E. Heuvelink 10

11 Realtime PCR GeneDisc 3 Packs for VTEC O157 & STEC Screening GeneDisc Pack: stx1 & stx2, eae, O157, ehx EHEC Identification GeneDisc Pack: O26, O103, O111, O145, H7 Salmonella, O157 & STEC Screening GeneDisc Pack: stx1 & stx2, eae, O157, Salmonella NRL E. coli The Netherlands A.E. Heuvelink 11

12 Realtime PCR GeneDisc Our study design Samples: animal faeces & raw meats Enrichment: mtsb+n (41.5 C) & BPW (37 C) PCR screening DNA extraction: Chelexbased stx1 & stx2, eae, O157, and Salmonella (GeneDisc & Light Cycler) O26, O103, O111, O145, and H7 (GeneDisc & Light Cycler) Cultural method VTEC O157: VIDASICE and plating onto selective agar Salmonella: selective enrichment using MRSV and plating onto selective agar NRL E. coli The Netherlands A.E. Heuvelink 12

13 Realtime PCR GeneDisc Results animal faeces raw meats mtsb+n (41.5 C) BPW (37 C) GeneDisc Light Cycler VIDAS ICE Different target genes NRL E. coli The Netherlands A.E. Heuvelink 13

14 Detection methods for VTEC nono157 Molecular methods (realtime) PCR Cultural methods NRL E. coli The Netherlands A.E. Heuvelink 14

15 New selective differential media for STEC Possé et al., FEMS Letters Microbiol 2008; 282:12431 Differentiation medium (D) Serotype Dsucrose Lsorbose FP% D (range) Confirmation medium (C) Darabinose Lrhamnose dulcitol Draffinose FP% D+C (range) O26 O103 O111 O145 Commensal E. coli % % + 39 (32 47) 45 (38 53) 45 (3853) 8 (0 15) % + 98% % + 86% ( ) 0.3 ( ) 37.6 ( ) ( ) NRL E. coli The Netherlands A.E. Heuvelink 15

16 New selective differential media for STEC Possé et al., FEMS Letters Microbiol 2008; 282:12431 O145 (green cfu) O103 and O111 (blue cfu) O26 (purple cfu) NRL E. coli The Netherlands A.E. Heuvelink 16

17 Thank you for your attention