Validation of high-resolution melting assays for the detection of virulent strains of \u3ci\u3eEscherichia coli\u3c/i\u3e O26 and O111 in beef and pork enrichment broths

Extensive validation of diagnostic assays using widely collected surveillance samples is critical for developing pathogen detection assays. The detection of potentially virulent E. coli strains is critical to the red meat industry. We previously developed two high-resolution melting (HRM) assays for detecting potentially virulent and avirulent E. coli O26 and O111 strains. Assays were validated using enriched beef (n = 36) and pork (n = 36) samples collected as part of a U.S. federal regulatory surveillance program. Data from this study showed more than 90% sensitivity and specificity for both the HRM assays, demonstrating suitability for the red meat industry and regulatory agencies

A review of Shiga-toxin producing \u3ci\u3eEscherichia coli\u3c/i\u3e (STEC) contamination in the raw pork production chain

Epidemiological evidence of Shiga toxin-producing Escherichia coli (STEC) infections associated with the consumption of contaminated pork highlight the need for increased awareness of STEC as an emerging pathogen in the pork supply chain. The objective of this review is to contribute to our understanding of raw pork products as potential carriers of STEC into the food supply. We summarize and critically analyze primary literature reporting the prevalence of STEC in the raw pork production chain. The reported prevalence rate of stx-positive E. coli isolates in live swine, slaughtered swine, and retail pork samples around the world ranged from 4.4 % (22/500) to 68.3 % (82/120), 22 % (309/1395) to 86.3 % (69/80), and 0.10 % (1/1167) to 80 % (32/40), respectively, depending upon the sample categories, detection methods, and the hygiene condition of the slaughterhouses and retail markets. In retail pork, serogroup O26 was prevalent in the U.S., Europe, and Africa. Serogroup O121 was only reported in the U.S. Furthermore, serogroup O91 was reported in the U.S., Asia, and South American retail pork samples. The most common virulence gene combination in retail pork around the globe were as follows: the U.S.: serogroup O157 + stx, non-O157 + stx, unknown serogroups+stx + eae; Europe: unknown serogroups+(stx + eae, stx2 + eae, or stx1 + stx2 + eae); Asia: O157 + stx1 + stx2 + ehxA, Unknown+stx1 + eaeA + ehxA, or only eae; Africa: O157 + stx2 + eae + ehxA. STEC strains derived from retail pork in the U.S. fall under low to moderate risk categories capable of causing human disease, thus indicating the need for adequate cooking and prevention of cross contamination to minimize infection risk in humans

Locus of Heat Resistance (LHR) in Meat-Borne \u3ci\u3eEscherichia coli\u3c/i\u3e: Screening and Genetic Characterization

Microbial resistance to processing treatments poses a food safety concern, as treatment tolerant pathogens can emerge. Occasional foodborne outbreaks caused by pathogenic Escherichia coli have led to human and economic losses. Therefore, this study screened for the extreme heat resistance (XHR) phenotype as well as one known genetic marker, the locus of heat resistance (LHR), in 4,123 E. coli isolates from diverse meat animals at different processing stages. The prevalences of XHR and LHR among the meat-borne E. coli were found to be 10.3% and 11.4%, respectively, with 19% agreement between the two. Finished meat products showed the highest LHR prevalence (24.3%) compared to other processing stages (0 to 0.6%). None of the LHR1 E. coli in this study would be considered pathogens based on screening for virulence genes. Four high-quality genomes were generated by whole-genome sequencing of representative LHR+ isolates. Nine horizontally acquired LHRs were identified and characterized, four plasmid-borne and five chromosomal. Nine newly identified LHRs belong to ClpK1 LHR or ClpK2 LHR variants sharing 61 to 68% nucleotide sequence identity, while one LHR appears to be a hybrid. Our observations suggest positive correlation between the number of LHR regions present in isolates and the extent of heat resistance. The isolate exhibiting the highest degree of heat resistance possessed four LHRs belonging to three different variant groups. Maintenance of as many as four LHRs in a single genome emphasizes the benefits of the LHR in bacterial physiology and stress response. IMPORTANCE Currently, a “multiple-hurdle” approach based on a combination of different antimicrobial interventions, including heat, is being utilized during meat processing to control the burden of spoilage and pathogenic bacteria. Our recent study (M. Guragain, G. E. Smith, D. A. King, and J. M. Bosilevac, J Food Prot 83:1438–1443, 2020, https://doi.org/10.4315/JFP-20-103) suggests that U.S. beef cattle harbor Escherichia coli that possess the locus of heat resistance (LHR). LHR seemingly contributes to the global stress tolerance in bacteria and hence poses a food safety concern. Therefore, it is important to understand the distribution of the LHRs among meat-borne bacteria identified at different stages of different meat processing systems. Complete genome sequencing and comparative analysis of selected heat-resistant bacteria provide a clearer understanding of stress and heat resistance mechanisms. Further, sequencing data may offer a platform to gain further insights into the genetic background that provides optimal bacterial tolerance against heat and other processing treatments

Effect of Direct-Fed Microbial Dosage on the Fecal Concentrations of Enterohemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e in Feedlot Cattle

Contamination of beef products by Shiga toxin–producing Escherichia coli is a concern for food safety with a particular subset, the enterohemorrhagic E. coli (EHEC), being the most relevant to human disease. To mitigate food safety risks, preharvest intervention strategies have been implemented with the aim to reduce EHEC in cattle. One class of interventions that has been widely used in feedlots is direct-fed microbials (DFMs), which can contain various dosing rates of probiotic bacteria. Here we compare the use of two different doses of a commercially available DFM on total EHEC load in a commercial feedlot setting. The DFMs used were the standard 109 Propionibacterium freudenreichii and 106 Lactobacillus acidophilus colony forming units (CFUs)/head/day dose of Bovamine (Nutrition Physiology Company, Guymon, OK) and the higher dose, Bovamine Defend (Nutrition Physiology Company), which is dosed at 109 P. freudenreichii and 109 Lactobacillus acidophilus CFUs/head/day. To analyze the total EHEC fecal concentration, 2200 head of cattle were assigned a DFM feed regimen lasting approximately 5 months. At harvest, 480 head of cattle were sampled using rectoanal mucosal swabs. A quantitative polymerase chain reaction assay targeting ecf1 was used to enumerate the total EHEC fecal concentration for 240 head fed the low-dose DFM and 240 head fed the high-dose DFM. No significant difference ( p \u3e 0.05) in the fecal concentration of total EHEC was observed between the two doses. This suggests that using an increased dosage provides no additional reduction in the total EHEC fecal concentration of feedlot cattle compared to the standard dosage

Distribution of \u3ci\u3eEscherichia coli\u3c/i\u3e Passaged through Processing Equipment during Ground Beef Production Using Inoculated Trimmings

The contamination of raw ground beef by Escherichia coli O157:H7 is not only a public health issue but also an economic concern to meat processors. When E. coli O157:H7 is detected in a ground beef sample, the product lots made immediately before and after the lot represented by the positive sample are discarded or diverted to lethality treatment. However, there is little data to base decisions on how much product must be diverted. Therefore, five 2,000-lb (907-kg) combo bins of beef trimmings were processed into 10-lb (4.54-kg) chubs of raw ground beef, wherein the second combo of meat was contaminated with a green fluorescent protein (GFP)–expressing strain of E. coli. This was performed at two different commercial ground beef processing facilities, and at a third establishment where ground beef chubs from the second grinding establishment were mechanically split and repackaged into 3-lb (1.36-kg) loaves in trays. The GFP E. coli was tracked through the production of 10-lb (4.54-kg) chubs and the strain could not be detected after 26.5% more material (500 lb or 227 kg) and 87.8% more material (1,840 lb or 835 kg) followed the contaminated combo at each establishment, respectively. Three-pound (1.36-kg) loaves were no longer positive after just 8.6% more initially noncontaminated material (72 lb or 33 kg) was processed. The GFP strain could not be detected postprocessing in any residual meat or fat collected from the equipment used in the three trials. These results indicate that diversion to a safe end point (lethality or rendering) of the positive lot of ground beef, plus the lot before and lot after should remove contaminated ground beef, and as such provides support for the current industry practice. Further, the distribution and flow of E. coli on beef trimmings through various commercial equipment was different; thus, each establishment needs to consider this data when segregating lots of ground beef and establishing sampling protocols to monitor production

Development and validation of high-resolution melting assays for the detection of potentially virulent strains of \u3ci\u3eEscherichia coli\u3c/i\u3e O103 and O121

Virulent strains of Shiga toxin-producing Escherichia coli (STEC) serogroups O103 and O121 are considered adulterants in beef. Two high-resolution melting (HRM) real-time PCR assays were standardized for the specific detection and discrimination of potentially virulent and avirulent strains of E. coli O103 and O121. The O103 HRM assay offered the possibility to distinguish clearly STEC O103:H2 from STEC O103:H25. The two standardized assays were extensively validated using 215 pure culture strains, laboratory inoculated food samples, and naturally contaminated beef (n = 84) and pork (n = 84) enrichments collected from the red meat surveillance program. Both HRM assays showed 100% inclusivity and exclusivity using pure culture strains and enriched spiked food samples. Data from this study shows the ability of the standardized assays to specifically detect the strains of each target serogroup and, most importantly, to differentiate the strains present into potentially virulent or avirulent groups. The assays standardized in this study can be helpful for food surveillance programs and help mitigate product loss due to the presence of avirulent strains lacking crucial virulence genes (stx and eae)

Impact of mixed biofilm formation with environmental microorganisms on \u3ci\u3eE. coli\u3c/i\u3e O157:H7 survival against sanitization

Biofilm formation by foodborne pathogens is a serious threat to food safety and public health. Meat processing plants may harbor various microorganisms and occasional foodborne pathogens; thus, the environmental microbial community might impact pathogen survival via mixed biofilm formation. We collected floor drain samples from two beef plants with different E. coli O157:H7 prevalence history and investigated the effects of the environmental microorganisms on pathogen sanitizer tolerance. The results showed that biofilm forming ability and bacterial species composition varied considerably based on the plants and drain locations. E. coli O157:H7 cells obtained significantly higher sanitizer tolerance in mixed biofilms by samples from the plant with recurrent E. coli O157:H7 prevalence than those mixed with samples from the other plant. The mixed biofilm that best protected E. coli O157: H7 also had the highest species diversity. The percentages of the species were altered significantly after sanitization, suggesting that the community composition affects the role and tolerance level of each individual species. Therefore, the unique environmental microbial community, their ability to form biofilms on contact surfaces and the interspecies interactions all play roles in E. coli O157:H7 persistence by either enhancing or reducing pathogen survival within the biofilm community

Impact of intense sanitization on environmental biofilm communities and the survival of Salmonella enterica at a beef processing plant

Salmonella enterica is a leading cause of foodborne illness in the U.S. In the meat industry, one action taken to address pathogen contamination incidence is an intense sanitization (IS) of the entire processing plant that many large processors perform annually or semiannually. However, this procedure’s immediate and long-term impact on environment microbial community and pathogen colonization are unknown. Here we investigated the impact of IS procedure on environmental biofilms and the subsequent S. enterica colonization and stress tolerance. Environmental samples were collected from floor drains at various areas 1 week before, 1 week, and 4 weeks after the IS procedure at a beef plant with sporadic S. enterica prevalence. Biofilm formation by microorganisms in the drain samples without S. enterica presence was tested under processing temperature. The ability of the biofilms to recruit and/or protect a co-inoculated S. enterica strain from quaternary ammonium compound (QAC) treatment was determined. The community structure of each drain sample was elucidated through 16S rRNA amplicon community sequencing. Post-IS samples collected from 8 drains formed significantly stronger biofilms than the respective pre-IS samples. S. enterica colonization was not different between the pre- and post-IS biofilms at all drain locations. S. enterica survival in QAC-treated pre- and post-IS mixed biofilms varied depending upon the drain location but a higher survival was associated with a stronger biofilm matrix. The 16S rRNA amplicon gene community sequencing results exhibited a decrease in community diversity 1 week after IS treatment but followed by a significant increase 4 weeks after the treatment. The IS procedure also significantly altered the community composition and the higher presence of certain species in the post-IS community may be associated with the stronger mixed biofilm formation and Salmonella tolerance. Our study suggested that the IS procedure might disrupt the existing environmental microbial community and alter the natural population composition, which might lead to unintended consequences as a result of a lack of competition within the multispecies mixture. The survival and recruitment of species with high colonizing capability to the post-IS community may play crucial roles in shaping the ensuing ecological dynamics

Prevalence and Level of Enterohemorrhagic \u3ci\u3eEscherichia coli\u3c/i\u3e in Culled Dairy Cows at Harvest

The primary objective of this study was to determine the prevalence and level of enterohemorrhagic Escherichia coli (EHEC) O26, O45, O103, O111, O121, and O145 (collectively EHEC-6) plus EHEC O157 in fecal, hide, and preintervention carcass surface samples from culled dairy cows. Matched samples (n=300) were collected from 100 cows at harvest and tested by a culture-based method and two molecular methods: NeoSEEK STEC (NS) and Atlas STEC EG2 Combo. Both the culture and NS methods can be used to discriminate among the seven EHEC types (EHEC-7), from which the cumulative prevalence was inferred, whereas the Atlas method can discriminate only between EHEC O157 and non-O157 EHEC, without discrimination of the serogroup. The EHEC-7 prevalence in feces, hides, and carcass surfaces was 6.5, 15.6, and 1.0%, respectively, with the culture method and 25.9, 64.9, and 7.0%, respectively, with the NS method. With the Atlas method, the prevalence of non-O157 EHEC was 29.1, 38.3, and 28.0% and that of EHEC O157 was 29.1, 57.0, and 3.0% for feces, hides, and carcasses, respectively. Only two samples (a hide sample and a fecal sample) originating from different cows contained quantifiable EHEC. In both samples, the isolates were identified as EHEC O157, with 4.7 CFU/1,000 cm2 in the hide sample and 3.9 log CFU/g in the fecal sample. Moderate agreement was found between culture and NS results for detection of EHEC O26 (k=0.58,

Supplementary data for article: Nastasijevic, I.; Schmidt, J. W.; Boskovic, M.; Glisic, M.; Kalchayanand, N.; Shackelford, S. D.; Wheeler, T. L.; Koohmaraie, M.; Bosilevac, J. M. Seasonal Prevalence of Shiga Toxin-Producing Escherichia Coli on Pork Carcasses for Three Steps of the Harvest Process at Two Commercial Processing Plants in the United States. Appl. Environ. Microbiol. 2020, 87 (1). https://doi.org/10.1128/AEM.01711-20.

Supplementary material for: [https://doi.org/10.1128/AEM.01711-20]Related to published version: [https://vet-erinar.vet.bg.ac.rs/handle/123456789/2029