An Update on Non-0157 Shiga Toxin-Producing E. coli in the Beef Chain

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1 An Update on Non-0157 Shiga Toxin-Producing E. coli MICK BOSILEVAC, Ph.D., U.S. MEAT ANIMAL RESEARCH CENTER USDA is an equal opportunity provider and employer 68 th RECIPROCAL MEAT CONFERENCE

2 Overview In September of 2011 the FSIS announced its intention to consider non-o157 STEC adulterants in beef. This session will focus on what has been learned about non-o157 STEC since that time in regards to their transmission through the beef chain, the efficacy of pre- and post- harvest control measures, and issues surrounding the accuracy of their detection.

3 Acknowledgements Meat Safety and Quality Research Unit Terry Arthur Elaine Berry Jim Bono Dayna Harhay Andy King Norasak Kalchayanand Rong Wang Steven Shackelford Jim Wells Tommy Wheeler Getahun Agga Brandon Luedtke Frank Reno Alberto Alvarado Julie Dyer Bruce Jasch Greg Smith Lawnie Luedtke FSIS Commercial Partners Producers Processors Suppliers Test Kit Companies Service Labs Funding Groups AMIF NCBA NIFA STEC CAP

4 O157 Shiga Toxin-Producing E. 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 items. 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 get better within 5 to 7 days. However some infections can become severe and life-threatening.

5 O157 Shiga Toxin-Producing E. coli (O157:H7 and non-o157) Very young children and the elderly are more likely to develop severe illness and hemolytic uremic syndrome (HUS) than others, but even healthy older children and young adults can become seriously ill. The CDC estimates that there are approximately 176,000 foodborne illnesses associated with STEC annually in the U.S. non-o157 STEC are responsible for approximately 113,000 (64%) of the foodborne STEC illnesses. The remainder of the illnesses associated with STEC (63,000 or 36%) are caused by E. coli O157:H7.

6 O157 Shiga Toxin-Producing E. coli (O157:H7 and non-o157) 70 to 80 percent of confirmed non-o157 STEC illnesses are caused by six STEC serogroups,,,,, and. These illnesses can be as severe to those caused by E. coli O157:H7. In the U.S, at least one outbreak and several sporadic illnesses from non-o157 STEC serogroups have been associated with ground beef products. Because of the public health concern regarding the non-o157 STEC serogroups, FSIS declared the six non-o157 STECs adulterants in non-intact raw beef products and product components, and began testing beef trimmings for the six non-o157 STEC in addition to E. coli O157:H7.

7 Dairy -Cull cows -Veal calves Production -Cull cows Harvest/Processing Further processing Finished products Feedlot

8 non-o157 STEC in Production Do the differences in fed cattle and cull cattle sources affect the prevalence and serogroups present of non-o157 STEC?

9 non-o157 STEC in Production In six regions of the US, a total of 1,042 fed cattle 1,062 cull dairy and 1,019 cull beef cattle were sampled for non-o157 STEC. Samples were collected from numerous production lots of cattle at two processing plants in the western, eastern, and southern regions and three processing plants in the central-north region.

10 O157 non-o157 STEC in Production Culture Positive Fed Dairy Beef n % a 5.5% a 4.7% a Fed n = 92 0% OX 50% 3% 7% 35% 3% 2% Dairy n = 80 7% 9% OX 21% 26% 34% 3% 0% Beef n = 61 2% 8% 21% OX 13% 23% 33% 0%

11 non-o157 STEC in Production OX serogroup (non-,,,,, and ) STEC were identified as O5, O49 O69, O70, O71, O74, O84, O98, O106, O108, O109, O113, O115, O117, O118, O156, O177 and O untypable. Fed n = 70 Ount 21% O49 1% O70 3% O74 19% Dairy n = 40 Ount 13% O5 5% O49 3% O74 8% Beef n = 39 Ount 8% O5 13% O69 8% O177 20% O156 4% O117 1% O115 1% O109 10% O113 4% O84 9% O98 3% O106 1% O108 1% O177 43% O98 15% O84 15% O156 8% O177 31% O118 3% O98 10% O108 3% O84 8% O71 5% O74 5%

12 non-o157 STEC in Production Veal calf hides at harvest Culture Positive Plant 1 Plant 2 Plant 3 n % 63.6% 15.8% Plant 1 Plant 2 n = 48 n = 31 10% Plant 3 n = 9 46% 54% 90% 100%

13 non-o157 STEC in Production Prevalence of non-o157 STEC does not appear to be statistically different between fed, beef and dairy cattle production systems, apart from the distribution of serogroups present. Veal calves (both bob and milk fed) appear to have a higher rates of non-o157 STEC that beef cattle. There are numerous other serogroups (non-,,,,, ) of significant STEC present in the production environments that may need to be assessed as potential public health concerns.

14 Dairy -Cull cows -Veal calves Production -Cull cows Harvest/Processing Further processing Finished products Feedlot

15 non-o157 STEC in Processing Transfer rates of non-o157 STEC from meat animals during Hide/Pelt/Skin removal Percent of non-o157 STEC (by stx PCR) Species Surface a Pre-evis Bovine b Ovine c Swine d a Surface refers cattle hide, pelt,, and pork skin before removal, scalding, or flaming. b Adapted from Barkocy-Gallagher et al., c Adapted from Kalchayanand et al., d Bosilevac, 2011, personal communication.

16 O157 non-o157 STEC in Processing Hides Pre-evis Final % stx positive % STEC isolate Arthur et al., 2002; Barkocy-Gallagher et al., 2003

17 O157 non-o157 STEC in Processing Effectiveness of Existing Antimicrobials for Reducing non-o157 STEC Antimicrobial Treatments and Conditions Peroxyacetic acid; 200 ppm, ph = 2.8 (Inspexx ) Acidified sodium chlorite; 1000 ppm, ph =2.4 (Sanova ) Lactic acid; 4%, ph = 2.3 Hot water; 85 o C at nozzles Citric/phosphoric/hydrochloric acid blend; 2% ph = 1.7 (FreshFX ) Bromine compounds; 300ppm (Bromitize, H2B ) psi for 15 sec

18 non-o157 STEC in Processing Effectiveness of Existing Antimicrobials for Reducing non-o157 STEC Peroxyacetic Acid (PAA) Reduction of STEC Strains O a 0.9 a 1.5 a 1.0 a 1.1 a 1.1 a 1.2 a

19 non-o157 STEC in Processing Effectiveness of Existing Antimicrobials for Reducing non-o157 STEC Acidified Sodium Chlorite (ASC) Reduction of STEC Strains O a 1.9 a 1.1 a 1.2 a 2.0 a 1.0 a 1.4 a

20 non-o157 STEC in Processing Effectiveness of Existing Antimicrobials for Reducing non-o157 STEC Lactic Acid (LA) Reduction of STEC Strains O a 2.6 b 2.0 a 1.9 a 2.7 b 2.2 a 1.9 a

21 non-o157 STEC in Processing Effectiveness of Existing Antimicrobials for Reducing non-o157 STEC Hot Water (HW) Reduction of STEC Strains O a 3.6 a 3.5 a 4.0 a 3.2 a 4.2 a 3.9 a

22 Dairy -Cull cows -Veal calves Production -Cull cows Harvest/Processing Further processing Finished products Feedlot

23 Beef Veal O157 non-o157 STEC in Finished Products Raw Ground Beef and Raw Ground Beef Components Analyzed for E. coli O157:H7 and non-o157 STEC, Calendar Year 2012 Serotype Trim Verification Serotype Trim Verification O157:H7 Total non- O157 STECs 0.53% (12/2,263) 0.91% (14/1,533) O157:H7 7.89% (3/38) Total non- O157 STECs 13.04% (3/23)

24 Beef Veal O157 non-o157 STEC in Finished Products Testing of Raw Ground Beef Component (RGBC) Samples for E. coli O157:H7 and non-o157 Shiga toxin-producing E. coli (STEC): Year-to-Date Totals Serotype Trim Verification Serotype Trim Verification As of May 31, 2015 O157:H7 Total non- O157 STECs 0.00% (0/1,199) 0.44% (5/1,148) O157:H7 0.00% (0/25) Total non- O157 STECs 9.09% (2/22)

25 non-o157 STEC in Processing and Finished Products Non-O157 STEC contamination from hides to carcasses is occurring during processing. Existing interventions used at processing plants are effective at reducing non-o157 STEC. The currently used antimicrobials are as effective on non-o157 STEC as they are on E. coli O157:H7. Non-O157 STEC are isolated from finished beef trim more often than E. coli O157:H7, and the rates of non-o157 STEC in veal is greater than in beef.

26 Dairy -Cull cows -Veal calves Production -Cull cows Harvest/Processing Further processing Finished products Feedlot Detection Technologies

27 non-o157 STEC Detection Genes for Shiga toxins (stx), intimin (eae), and O- group (wzx, wzy) are easy targets for PCR detection that can indicate the presence of an STEC. PCR = stx +, eae +, +

28 non-o157 STEC Detection PCR = stx +, eae +, + or these targets can indicate the presence of two or three separate E. coli that are not adulterant non-o157 STEC, because PCR cannot discriminate cells that contain all three targets from cells that do not.

29 non-o157 STEC Detection The FSIS MLG method targets stx, eae and O-group (wzx, wzy) genes using PCR. Many commercial test kits use the same approach. DuPont BAX Pall GeneDisk Bio-Rad iq-check LifeTech RapidFinder These methods have the user prepare a DNA extract that is then screened using real time PCR for stx and eae. If both targets are present then separate assays for O-group identification are preformed.

30 O157 non-o157 STEC Detection Analyses of Beef Enrichment Broths (n=365) BAX Bio-Rad Pall LifeTech Screen (stx+eae) Potential positive (O-group) STEC (35,29 O157/7 non-o157) 20/5 22/5 17/3 11/1 stx + E. coli (27) eae + E. coli (12) Serogroup + E. coli (32) no isolate

31 non-o157 STEC Detection Accuracy of STEC detection can be improved by antigen capture methods that immune concentrate O-groups before PCR detection of stx and eae, BioControl GDS biomerieux VIDAS or by using additional or alternative targets for detection. intimin (eae) gene subtypes STEC O-group snp s plasmid borne marker found only in eae positive STEC

32 non-o157 STEC Detection - Methods using alternative targets Intimin (eae) subtypes : serotype Particular STEC serogroups (O-groups) only carry particular intimin (eae) subtypes. STEC O-group eae b, O157 g,, e q (g 2 ) Also found in,, BioControl GDS Pall GeneDisk

33 O157 non-o157 STEC Detection - Methods using alternative targets STEC O Group gene SNP s Sequencing of E. coli genomes has identified single nucleotide polymorphisms (SNP s) that correlate with STEC lineages. These SNP s are detected by PCR and Mass Spec analysis of the PCR product (GeneSeek). Thus STEC and non-stec of the same O-group can be distinguished from one another.

34 non-o157 STEC Detection - Methods using alternative targets Plasmid borne marker found only in eae positive STEC Evolution of Enterohemorrhagic Escherichia coli Hemolysin Plasmids and the Locus for Enterocyte Effacement in Shiga Toxin-Producing E. coli Boerlin et al Infection and Immunity 66:2553

35 O157 non-o157 STEC Detection - Methods using alternative targets Analyses of Beef Enrichment Broths (n=450) BAX Pall-VF GeneSeek Roka stx+eae O-group Alternate Target(s) STEC (2) stx + E. coli (10) eae + E. coli (5) Serogroup + E. coli (3) no isolate

36 O157 non-o157 STEC Detection Non-O157 are difficult to accurately identify in contaminated products using stx, eae and O-group genes as the targets. Alternative targets provide some improvement in accuracy. More work is needed in detection target development. E. coli genomes contain islands of virulence related genes that are more frequent in STEC. In particular, Islands -57, -71, and -122 contain genes that correlate with the most virulent and outbreak related non-o157 STEC. nle genes, Z2098, Z2099, and espk are examples.

37 Dairy -Cull cows -Veal calves Production -Cull cows Harvest/Processing Further processing Finished products Feedlot Detection Technologies

38 68 th RECIPROCAL MEAT CONFERENCE