Comparison of Detection Methods of non-o157 STEC

Transcription

1 Comparison of Detection Methods of non-o157 STEC USDA is an equal opportunity provider and employer Mick Bosilevac, PhD USDA -Agricultural Research Service U.S. Meat Animal Research Center Meat Safety and Quality Research Unit Clay Center, Nebraska 17 June, 2014 Use of product names by USDA implies no approval to the exclusion of others that may also be suitable

2 Shiga Toxin Producing Escherichia coli (O157:H7 and non-o157) Shiga toxin producing E. coli live in the intestines of ruminant animals such as cattle, goats, and sheep. These E. coli generally do not make the animals sick, and other kinds of animals, like birds and wild pigs can spread the E. coli through the environment to things such as produce and water sources. The major source for human illnesses are attributed to cattle. An infected person may have severe stomach cramps, fever, vomiting, and bloody diarrhea. Most people recover within 5 to 7 days. However some infections can become severe and life-threatening. Young children and the elderly are more likely to develop severe illness and hemolytic uremic syndrome (HUS) than others, but even healthy young adults can become seriously ill.

3 Shiga Toxin Producing Escherichia coli (O157:H7 and non-o157)

4 Shiga Toxin Producing Escherichia coli (O157:H7 and non-o157) The CDC estimates that there are approximately 176,000 foodborne illnesses associated with STEC annually in the U.S. E. coli O157:H7 is responsible for approximately 63,000 (36%) of the foodborne STEC illnesses. The remainder of the illnesses associated with STEC (113,000 or 64%) are caused by non-o157 STEC. 70 to 80 percent of confirmed non-o157 STEC illnesses are caused by six STEC serogroups O26, O45, O103, O111, O121, and O145. These illnesses can be as sever to those caused by E. coli O157:H7. In the U.S, at least one outbreak and several sporadic illnesses from non- O157 STEC serogroupshave been associated with ground beef products.

5 Shiga Toxin Producing Escherichia coli (O157:H7 and non-o157) Because of the public health concern regarding the non-o157 STEC serogroups, in 2011, FSIS announced its intent to declare the six non- O157 STECs (O26, O45, O103, O111, O121, and O145) adulterants in non-intact raw beef products and product components. On June 4, 2012, FSIS began testing beef trimmings for the six non- O157 STEC in addition to E. coli O157:H7. At that time there were no commercial methods for non-o157 STEC that had been AOAC approved, so the FSIS granted Letters of No Objection to testing methods on a case-by-case basis so beef processors had supporting documentation regarding the reliability of non-o157 STEC verification testing results.

6 No-Objection Letters Issued for Non-O157 STEC Test Methods Log Number 12-SMP-0848-N-A 12-SMP-0849-N-A 12-SMP SMP N-A 12-SMP-0854-N-A Company Name Biocontrol Systems Biocontrol Systems IEH Laboratories Life Technologies Method Name Assurance GDS Top 7 STEC (eae) method Assurance GDS MPX Top 7 STEC method IEH Non-O157 STEC detection and identification method RapidFinderSTEC Screening and Confirmation Assays for Beef Products 12-SMP-0855-N-A 12-SMP-0858-N-A 12-SMP-0860-N-A 12-SMP-0914-N-A 12-SMP-0926-N-A Neogen Corporation Bio-Rad Laboratories DuPont Qualicon NeoSeek Approach to STEC Detection Identification (confirmatory test) iq Check VirX and iq Check SerO STEC test methods BAX System Real-Time PCR STEC Suite Vivione Biosciences, Inc. Rapid-B non-0157 STEC test kit Pall Corporation GeneDisc Top 6 STEC test kit

7 Foodborne Pathogen Test Kits Validated by Independent Organizations Method Name GeneDisc STEC GeneDisc STEC Top 6 iq-check STEC VirX and iq-check STEC SerO GeneDisc STEC & Salmonella IEH E. coli O157 and STEC with Intimin Test System BAX System Real-Time PCR Suite for detecting non-o157:h7 STEC Assurance GDS MPX Top 7 STEC Atlas STEC EG2 Combo Detection Assay GeneDisc Plate STEC Top 7 Manufacturer Pall GeneDisc Technologies Pall GeneDisc Technologies Bio-Rad Laboratories Pall GeneDisc Technologies IEH Laboratories DuPont Nutrition & Health Diagnostics BioControl Systems Roka Bioscience, Inc Pall GeneDisc Technologies External Validation AOAC-RI # AOAC-RI # AOAC-RI # AOAC-RI # AOAC-RI # AOAC-RI # AOAC-RI # AOAC-RI # AOAC-RI #

8 Detection methods for non-o157 STEC STEC: Targets for detection The FSIS MLG method targets stx, eaeand O-group (wzx, wzy) genes using PCR Genes for Shiga toxins (stx stx), intimin (eae eae), and O- group (wzx wzx, wzy) ) are easy targets for PCR detection that can indicate the presence of an EHEC. PCR = stx +, eae +, O26 + "Escherichia coli (E. coli) with Pili and Flagella" Mr Ang Li. National University of Singapore (Singapore)

9 Detection methods for non-o157 STEC STEC: Targets for detection The FSIS MLG method targets stx, eaeand O-group (wzx, wzy) genes using PCR PCR = stx +, eae +, O26 + or these targets can indicate the presence of two or three separate E. coli thatare are not adulterant EHEC, because PCR cannot discriminate cells that contain all three targets from cells that do not. "Escherichia coli (E. coli) with Pili and Flagella" Mr Ang Li. National University of Singapore (Singapore)

10 Detection methods for non-o157 STEC STEC: Targets for detection The FSIS MLG method targets stx, eaeand O-group (wzx, wzy) genes using PCR Potential positive = stx+ eae+ any Top6 O-group

11 Detection methods for non-o157 STEC STEC: Targets for detection The FSIS MLG method targets stx, eaeand O-group (wzx, wzy) genes using PCR n=437 stx1/stx2 + n=959 * ** n=232 * * n=415 n=107 ** n=183 n=139 n=261 eae + n=815 n=324 n = 3,972 O-group + n=753 * = Top6 EHEC isolate

12 STEC Detection Methods I Detection Methods Commercial Kits - BAX - BioControl GDS - Pall GeneDisc Official Methods - FSIS MLG - US MARC confirmation Comparison using inoculated materials - Individual Methods as per manufacturer - One enrichment tested by all methods Comparison using 500 natural enrichments Referral Lab - IEH - NeoSeek

13 STEC Detection Methods I Objective: To identify strengths and weaknesses of commercial Shiga toxin-producing E colidetection methods and kits in a side by side fashion. FSIS MLG 5B STEC Detection DuPont Qualicon BAX System STEC Suite BioControl Systems Assurance GDS Top STEC Pall GeneDisc System for non-o157 STEC IEH Laboratories Neogen NeoSEEK US Meat Animal Research Center STEC detection and isolation protocols

14 STEC Detection Methods I Three part evaluation Two inoculation studies 1. Each method/system used according to its protocol (medias, incubation temperatures & times, material) 2. Each method/system used to test the same TSB enrichments (ground beef varying weight/volume/time) One set of natural samples 3. Each method/system used to on 500 enrichments from a regional service lab

15 STEC Detection Methods I Part 1 -Inoculation studies Each method/system used according to recommended guidelines. Either package insert, AOAC validation document, or personal communication with manufacturer. Three of each Top6 EHEC used (in samples <200g), or Two of each Top6 EHEC used (in samples >200g) all inoculated at low CFU/sample, plus one negative (non-inoculated) for each set examined. All enrichments confirmed to be inoculated by Culture Isolation. Results are presented as number of Culture Isolation results (positive and negative) properly identified. Media BPW mtsb mtbsnovo 8 mtsbnovo 2 mehec prewarmedto 42C or 46C Materials Trim (TRM) 150g, 375g Ground Beef (GB) 25g, 325g Incubation temperature & time 41C or 42C 9h to 20h O stx eae Ave CFU , ,

16 MLG STEC Detection Methods I Sample 375g TRM Part 1 -Inoculation studies Medium (42C) Time Temp 1L mtsbnovo 8 18h 42C Results correct No. % n BAX 325g GB 1L mtsbnovo 2 (46C) 12h 20h 41C 41C GDS 325g GB 375g TRM 1L mehec 1L mehec 10h 18h 10h 18h 42C 42C 42C 42C Pall 25g GB 375g TRM 225mL BPW 18h 1.5L BPW 18h 42C 42C IEH 150g TRM 150mL IEH 9h 42C NeoSEEK 325g GB 1L mtsb 16h 42C

17 STEC Detection Methods I Part 2 -Inoculation studies Each method/system used to detect Top6 EHEC from same ground beef enrichment. Enrichments varied in sample size, volume of mtsbused and time enriched at 42C. Three of each Top6 EHEC used at low CFU/sample, plus one negative (non- inoculated) for each set examined. Repeated using two of each in 325g samples due to FedEx shipping failure. A group of 34 challenge E. coli previously isolated from ground beef (non-top6 EHEC/Top6 non-ehec/other) inoculated at higher CFU into 25g sample conditions. All enrichments confirmed to be inoculated by Culture Isolation and comparison back to the initial strain that was used. Results are presented as number of Culture Isolation results properly identified. Sample Medium (42C) Time Temp 25g GB 225mL mtsb 8h 42C 65g GB 585mL mtsb 12h 42C 325g GB 1L mtsb 16h 42C

18 STEC Detection Methods I Part 2 -Inoculation studies Isolates used in second round of inoculation studies. Top6 EHEC O stx eae Ave CFU , , Challenge strains O stx eae CFU O stx eae CFU , , ,2-21 unt ,2-33 unt unt unt unt unt unt 1, ,2-21 unt

19 STEC Detection Methods I Part 2 -Inoculation studies Top6 EHEC Sample Set n MLG 25g GB 19 65g GB g GB 19 Challenge Strains 13 25g GB (58%) 17 (89%) 14 (74%) 10 (77%) 34 (97%) BAX 19 (100%) 19 (100%) 14 (74%) 7 (54%) 35 (100%) GDS 18 (95%) 18 (95%) 16 (84%) 5 (38%) 30 (86%) Pall 19 (100%) 19 (100%) 19 (100%) 13 (100%) 29 (83%) IEH 18 (95%) 18 (95%) nr 13 (100%) 35 (100%) NeoSEEK 19 (100%) 19 (100%) nr 13 (100%) 35 (100%) over all percent correct:

20 STEC Detection Methods I Part 3 Natural Samples Each method/system used on 500 enrichments collected from a regional service lab beef trim, rapid enrichment media, 42C, 8-10h 10mL poured off for our use and analyzed (or the DNA lysis/extract prepared) the same day Any sample found positive for stx and eae by one or more methods taken into culture confirmation One EHEC-O26 confirmed in the 500 enrichments Another 73 enrichments contained a mixtures of stx + E. coli, eae + E.coli and E. coli of Top6 O groups (stx stx -, eae - )

21 STEC Detection Methods I Part 3 Natural Samples Number of Methods that identified a sample as Potential Positive n=135 EHEC Culture Isolation STEC (stx + ) EPEC (eae + ) Top6 O (stx +,eae + )

22 STEC Detection Methods I Number of Natural samples found Reactive and Potential Positive by each method with types of E. coli of isolated from Potential Positive samples. Screen/Reactive Confirm/ Potential positive Part 3 Natural Samples MLG BAX GDS Pall IEH NeoSeek USMARC EHEC STEC EPEC Top6 O no isolate Analysis software improved

23 STEC Detection Methods I Conclusions All methods examined detect Top6 EHEC as well as, or better than the reference MLG method Inoculation studies Each system should be run as described by its manufacturer Changes to media showed limitations in some methods Natural samples Show agreement on true positives Considerable disagreement on negative or non-confirmable samples The number of Potential Positive samples identified that appear to be false positive ranges from 86-98%

24 Improved targets for STEC detection Intimin subtype : serotype Particular serogroups or O- groups only carry particular intimin (eae) subtypes. EHEC O-group eae Ο26 β Ο145, Ο157 γ Ο45, Ο103, Ο121 ε Ο111 θ(γ 2 ) "Escherichia coli (E. coli) with Pili and Flagella" Mr Ang Li. National University of Singapore (Singapore)

25 Improved targets for STEC detection "Escherichia coli (E. coli) with Pili and Flagella" Mr Ang Li. National University of Singapore (Singapore) O Group gene SNP s : EHEC Sequencing of E. coli genomes has identified single nucleotide polymorphisms (SNP s) that correlate with EHEC lineage. These SNP s are detected by PCR and Mass Spec analysis of the PCR product. Thus EHEC and non-ehec of the same O-group can be distinguished from one another.

26 Improved targets for STEC detection NeoSEEK analysis uses the relationship between eae subtype and serogroup with EHEC O-group SNP to provide a molecular culture confirmation. Example: in a sample with stx + eae β + O26 STEC SNP = POS + eae ε+ + O45 nonstec SNP = NEG "Escherichia coli (E. coli) with Pili and Flagella" Mr Ang Li. National University of Singapore (Singapore)

27 Correlating eaesubtype and O group reduces the number of potential positive samples (stx +, eae +, O group + ) n = stx + = stx + eae + = stx + eae + O group + = 500 (100%) 500 (100%) 165 (33.0%) 197 (39.4%) 83 (16.6%) 103 (20.6%) 67 (13.4%) 76 (15.2%) stx + eae subtype:o group = 31 (6.2%) 49 (9.8%) O group subdivisions = 7 (1.4%) The natural sample containing the EHEC-O26 was identified by both methods.

28 Improved targets for STEC detection Evolution of Enterohemorrhagic Escherichia coli Hemolysin Plasmids and the Locus for Enterocyte Effacement in Shiga Toxin-Producing E. coli Boerlin et al., I&I 66:2553

29 STEC Detection Methods II Natural samples from across the U.S. 400 ground beef and 150 beef trim enrichments FSIS MLG BAX Biorad Pall GeneDisc NeoSeek Roka 325g samples enriched in 1L mtsb+novo8. All samples positive for eae and stx by one or more methods (n=27) were subjected to thorough rounds of culture isolation that identified in 14 of the samples: 2 EHEC (O26 & O157) O157) 10 STEC 5 EPEC (eae+, stx-) 3 E. coli of Top6 O group

30 STEC Detection Methods II Number of 400 ground beef and 150 beef trim samples found Reactive and Potential Positive by each method with types of E. coli of isolated from Potential Positive samples. Screen/Reactive Confirm/ Potential positive MLG BAX Bio-Rad Pall NeoSeek Roka (9) EHEC (2) STEC (10) EPEC(5) Top6 O (3) no isolate

31 STEC Detection Methods III Analyses of FSIS Beef Broths FSIS has been providing ARS enrichment broths from their Microbiological Testing Program for Escherichia coli O157:H7 and non-o157 Shiga toxin-producing Escherichia coli (STEC) - O157:H7 screen positive -STEC screen positive (stx+/ +/eae+) O-group- -STEC screen negative (stx+/ +/eae-) (stx-/eae+) -Matched negative broth for each of the above Assigned an identifier & data recorded Aliquoted(x8) Various DNA lysis prepared 365 have been fully processed

32 STEC Detection Methods III Analyses of FSIS Beef Broths BAX--STEC BAX Other STEC detection methods IMS & Culture isolation Each FSIS broth is tested with BAXBAX-O157 screen + STEC screen/confirmation. Other STEC detection methods are also performed for comparison. GDS Top7 STEC, GeneDisc STEC, BioRad VirX VirX,, etc Any FSIS broths that are Potential Positive go forward to culture isolation. Suspect colonies are screened and isolates are serotyped and characterized for virulence factors.

33 STEC Detection Methods III Analyses of FSIS Beef Broths 365 FSIS Broths processed. = MLG 5B is now equivalent to the BAX real time STEC suite Bio Bio--Rad iq check Genedisc System Neogen ANSR BioControl GDS LifeTechnologies RapidFinder Roka ATLAS

34 STEC Detection Methods III Analyses of FSIS Beef Broths 365 FSIS Broths processed. 161 found reactive (stx + /eae + ) by one or more methods. 144 identified as Potential Positive by one or more methods. Culture isolation has identified 78 samples that contain: EHEC, STEC, EPEC and E. coli of target serogroups. 28 contain EHEC-O157:H7 1 contains EHEC-O157:H7 and EHEC-O45 3 contain EHEC-O103 1 contains EHEC-O26 2 contain EHEC of other serogroups(o5 and O74) The remainder of the samples (n=43) contain: 27 STEC, 12 EPEC and 32 E. coli of target serogroups.

35 STEC Detection Methods III Analyses of FSIS Beef Broths Screen/Reactive Confirm/ Potential positive BAX Bio-Rad ANSR LifeTech GDS Pall Roka nd EHEC (35,29 O157/7 non-o157) STEC (27) EPEC(12) Top6 O (32) no isolate 20/ / / / / / /

36 STEC Detection Methods II & III Conclusions Continued side-by-side comparison of STEC detection methods does not result in a clear winner. Methods that rely solely on the presence of stx, eaeand and O- group identify an unacceptable number of Potential Positive samples. Of concern is the large number of False Negatives observed in the comparison performed within the FSIS broths. - No method managed to identify all 5 Top-6 EHEC samples. Methods that use multiple targets or targets uniquely characteristic of EHEC may offer the best option, but more reliable targets are needed. As are methods that can more accurately detect the cells of interest.

37 STEC: Improved targets for detection Outer circle shows the distribution of islands. Blue: shared co-linear backbone. Red: position of EDL933-specific sequences (O-islands). Green: MG1655-specific sequences (K-islands). Tan: O-islands and K-islands at the same locations in the backbone. Purple: Hypervariable regions. Sequencing the genome of E. coli O157:H7 identified genomic islands unique to E. coli O157:H7 compared to E. coli K12. Termed O-islands (OI) these portions of the genome turned out to posses a number of virulence related genes. OI-57, OI-71, and OI-122 in particular contain genes that correlate with EHEC. - nle genes; nleb, nlef,, etc - Z2098, Z espk Nature 409, (25 January 2001)

38 Prevalence of alternative targets for detection of EHEC in enrichments with and without stx % prevalence in enrichment marker All samples STEC no-stec (n=253) (stx pos) (stx neg) stx stx chua eae ehx esp K fyua G Ibe irp nleb nlec nlef pch D Q suba tra T beef carcass samples were screened using PCR for stx1, stx2, eaeand and 14 different genes associated with increased virulence of EHEC. Then prevalence rates in enrichments with and without stx were examined. Some markers showed varying degrees of correlation with stx. All stx + enrichments were cultured and EHEC, STEC and EPEC were isolated.

39 Number and types of E. coli isolated from beef carcass swab enrichments with increasing number of screening markers Isolates 30 EHEC STEC EPEC n n EPEC STEC EHEC Number of virulence markers present in enrichment Enrichments that contained between 10 and 15 virulence markers yielded increasing proportions of EHEC and decreasing proportions of STEC.

40 Improving on sensitivity for detection of non-o157 STEC? The latest test methods that have come into our lab for evaluation. Recombinant bacteriophage tail fibers from EHEC specific phage may offer increased performance in term of specificity and sensitivity to concentrate the target EHEC before placing in a detection assay or plating for isolation A flow cytometric approach that interrogates each individual cell within an enrichment across parameters of size, shape, internal and external immuno-and molecular targets simultaneously and in real time.

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