Presence of Shiga toxin-producing Escherichia coli O-groups in small and very small beef processing plants and resulting beef products detected by a multiplex polymerase chain reaction assay

Open Access
Author:
Svoboda, Amanda Lyn
Graduate Program:
Food Science
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
May 30, 2012
Committee Members:
  • Catherine Nettles Cutter, Thesis Advisor
  • Edward G Dudley, Thesis Advisor
  • Edward William Mills, Thesis Advisor
  • Chitrita Debroy, Thesis Advisor
Keywords:
  • E. coli
  • non-O157
  • beef processing
  • small plants
  • very small plants
  • Pennsylvania
Abstract:
Shiga toxin-producing Escherichia coli (STEC) are pathogens attributed to numerous foodborne illnesses resulting in gastrointestinal disease of varying severity, including hemolytic uremic syndrome (HUS) in humans. Cattle, and consequently beef products, are considered a major source of STEC. E. coli O157:H7 has been regulated as an adulterant in ground beef since 1994. The USDA-Food Safety and Inspection Service (USDA-FSIS) has indicated that 6 additional STEC (O145, O121, O111, O103, O45, and O26) will be regulated as adulterants in raw beef trim and ground beef, beginning in June 2012. However, the overall incidence of non-O157 STEC is difficult to estimate because routine screening of these serogroups is not performed, due to a lack of rapid molecular methods and/or standard culture methods. A standard detection protocol for non-O157 STEC is not currently established, largely due to the broad range of both phenotypic and genotypic characteristics this group of pathogens display. While some methods have been employed to detect STEC in large beef processing environments and/or products, little is known about the presence of STEC in small and very small beef processing plants or the resulting beef products. The first objective of this study evaluated the effectiveness of three different enrichment media (tryptic soy broth (TSB) + novobiocin (TSBn); modified E. coli broth + novobiocin (mECn); and modified TSB (mTSB) containing 8 mg/L novobiocin, 16 mg/L vancomycin, 2 mg/L rifampicin, 1.5g/L bile salts, and 1 mg/L potassium tellurite; and described by Possé et al. (2008) to isolate a non-O157:H7 STEC. Carcass, environmental, fecal, or ground beef samples were artificially inoculated with approximately 10² CFU/mL of E. coli O145 and enriched for 24 hr at 42°C with each of the selected enrichment media. After enrichments, O145 was confirmed using a multiplex PCR assay (DebRoy et al., 2011) which had been optimized for the detection of STEC serogroups O157, O145, O121, O113, O111, O103, O45, and O26. All three enrichment media were effective for the detection of E. coli O145. Interestingly, fewer background microorganisms were detected on agar plates when subjected to enrichment with mTSB. The second objective of this study was to determine if small and very small beef processing plants are a potential source of STEC, utilizing the enrichment and detection methods optimized in the first objective. In this survey, environmental swabs, carcass swabs, hide swabs, fecal samples, and ground beef from small and very small beef processing plants were obtained to determine the presence of STEC. The previously optimized multiplex PCR assay was used to detect the presence of STEC O-groups and the presence of Shiga toxin (stx) and intimin (eae) genes were determined using PCR primers described by Paton and Paton (1998). Results demonstrated that 55.5% (151/272) of the environmental samples, 36.9% (75/203) of the carcass samples, 85.2% (23/27) of the hide samples, 37.5% (12/32) of the fecal, and 18.6% (22/118) of the ground beef samples tested positive for one or more of the serogroups. However, only 7.7% (21/272) of the environmental samples, 5.9% (12/203) of the carcass samples, 0% (0/27) of the hide samples, 0% (0/32) of the fecal, and 0% (0/118) ground beef samples tested positive for stx1 and/or stx2 genes. In addition, 13.6% (37/272) of the environmental samples, 7.9% (16/203) of the carcass samples, 0% (0/27) of the hide samples, 0% (0/32) of the fecal samples, and 0.8% (1/118) of the ground beef samples tested positive for eae gene. A second survey was completed to evaluate the effectiveness of three different sampling methods (sponge swab, hide clipping, and a novel M-Vac sampling method (Microbial Vac Systems, Inc., Bluffdale, Utah) for detecting STEC on cattle, swine, and sheep hides at one abattoir. Samples were assayed for eight STEC serogroups, (O157, O145, O121, O113, O111, O103, O45, and O26) using a multiplex PCR assay (DebRoy et al., 2011). The presence of stx genes in samples were identified using an additional multiplex PCR assay (Paton and Paton, 1998). Results demonstrated that 92% (24/26) of cattle hides tested positive for one or more STEC O-groups using the sponge swab and hair clipping methods, while 88% (23/26) of cattle hides tested positive for one or more STEC O-groups using the M-Vac sampling method. Swine hides tested positive for one or more STEC O-group in 93% (28/30) of samples collected with the sponge swab method, as compared with 80% (24/30) of samples collected using the hair clipping method, while 97% (29/30) of samples collected were positive for the STEC O-group using the M-Vac sampling method. Sheep hides were positive for one or more STEC O-group in all (11/11) of the samples collected with the sponge swab and M-Vac methods, while 82% (9/11) of samples collected were positive with the hair clipping method. However, only 3.8% (1/26) of cattle hides, 40% (12/30) of swine hides, and 27.3% (3/11) of sheep hides tested positive for stx1 and/or stx2 genes, when all sampling methods were considered. The data resulting from the beef processing plant survey may establish a baseline for the presence of non-O157 STEC in small and very small processing establishments in Pennsylvania and the resulting beef products. Results from the hide study may serve as an indication of the presence of STEC in small and very small beef slaughter facilities, which also may process multiple species. Collectively, these studies may be useful to regulatory officials, researchers, and public health personnel who are interested in determining the presence of these pathogens in the meat supply. Future studies focusing on specific locations in the beef processing environment or in beef trim, as well as resulting ground beef products, may result in a better understanding the path(s) that these pathogens take to reach the final product. Understanding pathways of transmission in a beef processing environment may help researchers and processors determine interventions that can be employed to control these pathogens, resulting in a safer food supply. Funding for this project was provided by the USDA-NIFA grant 2009-03611.