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We investigated the phylogeny of biofilm forming (BF) and non-biofilm forming (NBF) Escherichia coli (n = 114) from beef processing environment and genetic elements in their BF and persistence, by comparative genomic analysis. Phylogroup B1 made up the largest proportion of both BF (73.8%) and NBF (50.9%) groups. E. coli from all sources examined had mixed phylogroups except for those recovered from equipment after cleaning which were exclusively phylogroup B1. Both core genome and gene content trees showed a tree-wide spread of BF strains, with clusters including both BF and NBF strains. Genome wide association studies (GWAS) by Scoary or Pyseer did not find any genes/mutations overrepresented in the BF group. Retrospective analysis of phenotypes found significant correlation (P E. coli group. These findings suggest gene function redundancy in E. coli for biofilm formation and additional substrate utilization and iron acquisition in its persistence.

Variable rate fertilization is one of the core technologies of precision agriculture, and it is particularly important to develop a representative field sampling scheme with less workload. A grid s...

The transmissible locus of stress tolerance (tLST) confers resistance to multiple stresses in E. coli. Utilizing 18,959 E. coli genomes available in the NCBI database, we investigated the prevalence, phylogenetic distribution, and configuration patterns of tLST, and correlations between tLST, and virulence and antimicrobial resistance (AMR) genes in E. coli. Four tLST variants were found in 2.7% of E. coli, with the most prevalent (77.1%) variant being tLST1 followed by tLST2 (8.3%), tLST3b (8.3%) and tLST3a (6.3%). The majority (93%) of those tLST were in E. coli belonging to phylogroup A in which the prevalence was 10.4%. tLST was also found in phylogroup B1 (0.5%) and C (0.5%) but not found in B2 or D-G. An additional 1% of the 18,959 E. coli genomes harbored tLST fragments to various extent. Phylogenetic analysis revealed both intra- and interspecies transmission of both chromosomal and plasmid-borne tLST, with E. coli showing a preference of chromosomal over plasmid-borne tLST. The presence of tLST and virulence genes in E. coli was overall negatively correlated, but tLST was found in all genomes of a subgroup of enterotoxigenic E. coli (ST2332). Of note, no Shiga toxin-producing E. coli (n = 3,492) harbored tLST. The prevalence of tLST and AMR genes showed different temporal trends over the period 1985 to 2019. However, a substantial fraction of tLST positive E. coli harbor AMR genes, posing a threat to public health. In conclusion, this study improves our understanding of the genetic characteristics of tLST and E. coli harboring tLST. IMPORTANCE This study, through a large-scale genomic analysis, demonstrated that the genomic island tLST related to multiple stress resistance (such as extreme heat resistance and oxidative stress tolerance) in E. coli is differentially present in subgroups of E. coli and is strongly associated with certain phylogenetic background of the host strain. The study also shows the transmission mechanisms of tLST in E. coli and other bacterial species. The overall negative association of tLST, and virulence genes and antimicrobial (AMR) genes suggest the selective pressures for the acquisition and transmission of these traits likely differ. Even so, the high prevalence of tLST in the enterotoxigenic E. coli clone ST2332 and co-occurrence of tLST and AMR genes in E. coli are concerning. Thus, the findings better our understanding of tLST evolution and provide information for risk assessment of tLST harboring bacteria.

The objective of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in colon contents and on carcasses from pigs slaughtered at provincially licensed abattoirs (PLAs) in Alberta, Canada. In 2017, carcass sponge samples and colon content samples were collected from 504 healthy market hogs at 39 PLAs and analyzed for E. coli O157:H7. Carcass samples were also analyzed for E. coli and aerobic colony count (ACC). Nine (1.8%) of 504 carcass samples were confirmed positive for E. coli O157:H7. Seven (1.4%) of 504 colon content samples were confirmed positive for E. coli O157:H7. These positives were found in 5 (12.8%) of 39 PLAs from hogs originating from eight farms. The E. coli O157:H7 isolates recovered from the positive samples (n = 1 isolate per sample) were clonal, as determined by pulsed-field gel electrophoresis. Six E. coli O157:H7 isolates obtained over 8 months from one PLA that only processed hogs and sourced hogs from one farm had indistinguishable pulsed-field gel electrophoresis patterns. All 16 E. coli O157:H7 isolates harbored eae and ehxA and were of stx2a subtype, suggesting that swine can carry E. coli O157:H7 of importance to human health. All carcass sponge swabs (100%) were positive for ACC. E. coli was present in 72% of carcass swabs. Carcasses from PLAs slaughtering both beef and hogs had a numerically higher ACC mean value but not statistically different compared with the carcasses from PLAs slaughtering only swine (2,799 and 610 CFU/cm2, respectively). E. coli showed a similar trend with a mean value of 0.88 CFU/cm2 in PLAs slaughtering both species and 0.26 CFU/cm2 in PLAs slaughtering only swine (P ≤ 0.05). This study provides evidence that healthy market hogs from different producers and farms in Alberta can carry E. coli O157:H7, and some strains of the organism may be able to establish persistence on some swine farms. HIGHLIGHTS

The goal of this research was to evaluate the efficacy of a novel rechargeable nonleaching polycationic N-halamine coating applied to stainless steel food contact surfaces to reduce Listeria monocytogenes contamination on ready-to-eat (RTE) foods. Four L. monocytogenes strains were inoculated onto the charged (C; chlorine activated) or noncharged (NC) N-halamine-coated steel coupon surfaces that were either intact or scratched. After inoculation, test surfaces were incubated at 2, 10, and 25°C for 0, 48, and 72 h. L. monocytogenes transfer from coated adulterated surfaces to RTE meat (beef sausages and roast beef) was also tested at 2°C. L. monocytogenes on both intact-C and scratched-C surfaces was significantly reduced at all temperatures; however, in the presence of organic material, these coatings were more effective for reducing L. monocytogenes at 2 and 10°C than at 25°C (P < 0.05). In contrast, on NC intact and scratched surfaces, reduction at 25°C increased (P < 0.05), decreasing the difference in L. monocytogenes levels between charged and noncharged intact and scratched surfaces at this temperature. Overall, greater L. monocytogenes reduction was achieved on intact-C and scratched-C (4.1 ± 0.19 log CFU/cm2) than on intact-NC and scratched-NC (2.3 ± 0.19 log CFU/cm2) surfaces at all temperatures (P < 0.05). The combination of surface condition and chlorine with coupons exposed for 2 h at 2°C in the presence of an organic load (50% meat purge) did not significantly affect the bactericidal efficacy of the N-halamine coating. Regarding transfer to RTE meat, an overall 3.7-log reduction in L. monocytogenes was observed in sausages and roast beef. These findings suggest that a novel rechargeable N-halamine coating on stainless steel surfaces can inactivate L. monocytogenes. HIGHLIGHTS

This study investigated the potential causative agents for vacuum-packaged pork that had shown gross package extension during a routine storage life study in a Canadian pork plant using both conventional and culture-independent methods. The spoilage-associated bacteria in purge samples from two packages were enumerated using selective media and profiled using 16S rDNA amplicon analysis. The presence of Clostridium estertheticum was detected using species-specific real-time PCR. An enrichment procedure was used to isolate C. estertheticum from one of the purge samples. The average population density in the two purge samples of total aerobes, lactic acid bacteria (LAB), coliforms and Brochothrix thermosphacta was 9·4, 9·1, 6·0 and 4·6 log CFU per ml respectively, as determined by plating. The estimated numbers of C. estertheticum were >7 log cells per ml. Clostridium estertheticum was recovered although the enrichment condition used for isolation favoured the growth of LAB more than that of Clostridium spp. Based on 16S rDNA amplicon analysis, the microbiota in the two purge samples had 64·7 and 20·7% of Clostridium spp., and 32·5 and 70·1% of LAB respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Blown pack spoilage of vacuum-packaged meat may lead to severe economic losses and is often associated with beef, venison and lamb. This study is the first to report vacuum-packaged chilled pork can also be subject to blown pack spoilage, and data support the conclusion that the causative agent is likely Clostridium estertheticum. The lysozyme-digestion step greatly improved the isolation efficiency for C. estertheticum, a spore-forming anaerobic organism that has been proven to be difficult to recover. This method can be used for isolating spore-forming organisms from food samples.

Despite the importance of biofilm formation in contamination of meat by pathogenic Escherichia coli at slaughter plants, drivers for biofilm have been unclear. To identify selection pressures for biofilm, we evaluated 745 ‘Top 7’ from cattle and 700 generic E. coli from two beef slaughter plants for motility, expression of curli and cellulose, and biofilm-forming potential. Top 7 were also screened for serogroup, stx1, stx2, eae and rpoS. Generic E. coli were compared by source (hide of carcass, hide-off carcass, processing equipment) before and after implementation of antimicrobial hurdles. The proportion of E. coli capable of forming biofilms was lowest (7.1%; P < 0.05) for cattle isolates and highest (87.3%; P < 0.05) from equipment. Only one enterohemorrhagic E. coli (EHEC) was an extremely-strong biofilm-former, in contrast to 73.4% of E. coli from equipment. Isolates from equipment after sanitation had a greater biofilm-forming capacity (P < 0.001) than those before sanitation. Most Top 7 were motile and expressed curli, although these traits along with expression of cellulose and presence of rpoS were not necessary for biofilm formation. In contrast, isolates capable of forming biofilms on equipment were almost exclusively motile and able to express curli. Results of the present study indicate that cattle would rarely carry EHEC capable of making strong biofilms to slaughter plants. However, if biofilm-forming EHEC contaminated equipment, current antimicrobial hurdles would inadvertently perpetuate the most robust biofilm-forming strains. Accordingly, new and effective anti-biofilm hurdles are required for meat-processing equipment, to reduce future instances of food-borne disease.ImportanceAs the majority of enterohemorrhagic E. coli (EHEC) are not capable of forming biofilms, sources were undetermined of the biofilm-forming EHEC isolated from ‘high-event periods’ in beef slaughter plants. This study demonstrated that sanitation procedures used on beef-processing equipment inadvertently select for survival of the most robust biofilm-forming strains of E. coli. Cattle only rarely carry EHEC capable of forming strong biofilms (1/745 isolates evaluated), but sanitation of equipment markedly increased (P < 0.001) biofilm-forming capacity of E. coli. In contrast, chilling carcasses for 3 days at 0°C reduced (P < 0.05) biofilm-forming capacity of E. coli. Consequently, an additional anti-biofilm hurdle for meat-processing equipment, perhaps involving cold exposure, is necessary to further reduce the risk of food-borne disease.

The locus of heat resistance (LHR) can confer heat resistance to Escherichia coli to various extents. This study investigated the phylogenetic relationships and the genomic and phenotypic characteristics of E. coli with or without LHR recovered from beef by direct plating or from enrichment broth at 42°C. LHR-positive E. coli isolates (n = 24) were subjected to whole-genome sequencing by short and long reads. LHR-negative isolates (n = 18) from equivalent sources as LHR-positive isolates were short-read sequenced. All isolates were assessed for decimal reduction time at 60°C (D(60°C)) and susceptibility to the sanitizers E-SAN and Perox-E. Selected isolates were evaluated for growth at 42°C. The LHR-positive and -negative isolates were well separated on the core genome tree, with 22/24 positive isolates clustering into three clades. Isolates within clade 1 and 2, despite their different D(60°C) values, were clonal, as determined by subtyping (multilocus sequence typing [MLST], core genome MLST, and serotyping). Isolates within each clade are of one serotype. The LHR-negative isolates were genetically diverse. The LHR-positive isolates had a larger (P

Carnobacteria have been implicated in food spoilage, but also in protection against pathogenic bacteria. We report the isolation and complete genome sequences of three bacteriophages (phages cd2, cd3, and cd4) that specifically target Carnobacterium divergens . The genome sizes are approximately 57 kbp and have limited homology to known enterococcal and streptococcal phages.

Biofilm formation in food processing plants reduces the efficacy of sanitation. The presence of transmissible locus of stress tolerance (tLST) also enhances resistance of planktonic cells of Escherichia coli to sanitation chemicals but the role of tLST in resistance of biofilm-embedded cells remains unclear. This study investigated the link of tLST to biofilm formation and its contribution to resistance of biofilm-embedded E. coli to sanitation. Biofilms were formed as single-strain and as dual-strain biofilms in association with E. coli, Aeromonas australensis or Carnobacterium maltaromaticum. Biofilms on stainless steel were compared to floating biofilms formed at the air-liquid interface (pellicles). The resistance of biofilm-embedded tLST positive strains of E. coli to chlorine, hydrogen peroxide, and peroxyacetic acid was higher than the resistance of tLST negative strains. Higher biofilm density as measured by crystal violet staining was observed in tLST-positive strains of E. coli when compared to tLST negative strains. Biofilm density positively correlated to resistance to disinfectants. The use of confocal laser scanning microscopy detected more compact structure of pellicles compared to solid surface-attached biofilms, resulting in higher chlorine resistance despite the absence of tLST in strains of E. coli. Collectively, the findings of this study elucidated the impact of tLST in strains of E. coli on biofilm formation and sanitizer resistance. These findings may inform the development of improved sanitization protocols for food facilities.

Three E. coli O157:H7 outbreaks have been attributed to contaminated pork in Alberta, Canada, recently. This study investigates the phylogenetic relatedness of E. coli O157:H7 from pigs, cattle, and pork-production environments for source attribution. Limited strain diversity was observed using five conventional subtyping methods, with most or all strains being in one subgroup. Whole-genome single nucleotide polymorphism analysis confirmed the recent ancestry of the isolates from all three sources. Most environmental isolates clustered closer with pig isolates than cattle isolates. Also, a direct link was observed between 2018-outbreak environmental isolates and isolates collected from a pig farm in 2018. The majority of pig isolates harbor only one Shiga toxin gene, stx2a, while 70% (35/50) of the cattle isolates have both stx1a and stx2a. The results show some E. coli O157:H7 strains could establish persistence on pig farms and as such, pigs can be a significant source of the organism.

We investigated the impact of peroxyacetic acid (PAA; 200 ppm) spray on the microbiota and shelf life of commercial, vacuum-packed beef stored at chiller temperatures. Ribeye cuts (n = 147) were collected from a local beef plant on the day of production for two consecutive days, with one set collected at the start of work with the PAA spray nozzles turned off (control) and during routine production with the PAA spray nozzles turned on (PAA) each day. Packs were stored at 4, 2, and −1°C for up to 34, 104, and 180 days and sampled at appropriate intervals for sensory assessment, microbial enumeration, and microbial profiling by 16S rRNA gene amplicon analysis. Treatment with PAA did not affect the initial meat pH, the initial numbers of total aerobes, lactic acid bacteria, or Enterobacteriaceae (P > 0.05) before storage; however, it delayed the onset of spoilage by 7, 21, and 54 days at 4, 2, and −1°C, respectively. Square-root models of the variation of growth rate with temperature indicated lactic acid bacteria grew faster and Enterobacteriaceae grew slower on PAA-treated than on untreated meat. Negative associations between pH and deterioration of meat during storage were observed for PAA-treated meat. During storage, the microbiota were primarily dominated by Carnobacterium and Lactobacillus/Lactococcus on control meat but by Leuconostoc on PAA-treated meat. Serratia, Yersinia, and Clostridium were identified by linear discriminant effect size analysis as biomarkers for control meat; Clostridium was found in high abundance in samples that had the highest spoilage scores. IMPORTANCE The findings of this study show that PAA solutions applied at low concentrations under commercial settings positively modulated the meat microbiota. It did not have bactericidal effects for beef subprimals with very low microbial loads. However, it differentially impacted the members of the microbiota, which resulted in delayed onset of spoilage of vacuum-packed beef subprimal stored at all three temperatures (4, 2, and −1°C). This differential impact could be through one or a combination of the following factors: favoring the growth of lactic acid bacteria, which may in turn exert a competitive exclusion that might be due to production of antimicrobial compounds such as organic acids and bacteriocins; exerting synergistic antimicrobial effects with low temperatures against members of Enterobacteriaceae; and direct or indirect inhibitory effects against members of the clostridia. These findings not only advance our understanding of the microbial ecology of vacuum-packed meat stored at chiller temperatures but also suggest that bacteriostatic concentrations of antimicrobial interventions can be explored for shelf-life extension.

This study investigated the impact of meat processing surface bacteria (MPB) on biofilm formation by non-O157 Shiga toxin-producing Escherichia coli (STEC), and potential links between biofilm formation by STEC and biofilm-related genes in their genomes. Biofilm development by 50 MPB and 6 STEC strains in mono- and co-cultures was assessed by the crystal violet staining method, and their expression of curli and cellulose was determined using the Congo red agar method. Genes (n = 141) associated with biofilm formation in the STEC strains were profiled. Biofilm formation in general correlated with cellulose and curli expression in both mono- and co-cultures. Most MPB strains had antagonistic effects on the biofilm formation of the STEC strains. Of the genes investigated, 81% were common among the STEC strains and there seems to be a gene-redundancy in biofilm formation. The inability of the O26 strain to form biofilms could be due to mutations in the rpoS gene. Truncation in the mlrA gene in the O145 strain seems not affecting its biofilm formation alone or with MPB. The O45 strain, despite having the greatest number of biofilm-related genes, did not form measurable biofilms. Overall, biofilm formation of STEC was affected by curli-cellulose expression and companion strains.

This study aimed to determine the impact of bacteria from a beef plant conveyor belt on the biofilm formation of Salmonella in dual-species cultures. Beef plant isolates (50) including 18 Gram-negative aerobes (GNA), 8 Gram-positive aerobes (GPA), 5 lactic acid bacteria (LAB), 9 Enterobacteriaceae (EB), and 10 generic Escherichia coli (GEC) were included for developing biofilms in mono- and co-culture with S. Typhimurium at 15 °C for 6 days. Five selected cultures in planktonic form and in biofilms were tested for susceptibility to two commonly used sanitizers (i.e. E-San and Perox-E Plus). In mono-cultures, ≥ 80, 67, 61, 20, and 13% of GEC, EB, GNA, LAB, and GPA, respectively, developed measurable biofilms after 2 days, while all co-culture pairings with S. Typhimurium achieved some level of biofilm production. The predominant effect of EB and only effect of GEC strains on the biofilm formation of S. Typhimurium was antagonistic, while that of Gram-positive bacteria was synergistic, with the effect being more prominent on day 6. The effect was highly variable for the GNA isolates. Six aerobic isolates that formed moderate/strong biofilms by day 2 greatly boosted the co-culture biofilm formation. Seven Gram-negative bacteria were antagonistic against the biofilm formation of the co-cultures. Both sanitizers completely inactivated the selected planktonic cultures, but were largely ineffective against biofilms. In conclusion, all beef plant isolates assessed formed biofilms when paired with S. Typhimurium. Aerobic biofilm formers may create a more favorable condition for Salmonella biofilm formation, while some beef plant isolates have potential as a biocontrol strategy for Salmonella biofilms.

Genome sequences of Escherichia coli O157:H7 originating from pigs are limited in the public databases. We sequenced 104 E. coli O157:H7 isolates from pig and cattle feces and pork production environments in Alberta, Canada. The information will aid studies investigating sources of E. coli O157:H7 contaminating pork and the associated environments.

ObjectivePrevious studies have shown that high glucose (HG) induces endothelial cell (EC) damage via endothelial-to-mesenchymal transition (EndMT). Although the underlying mechanisms are still unclear, recent studies have demonstrated the role of calcium-sensing receptor (CaSR) in mediating EC damage. Therefore, the aim of our study was to investigate whether CaSR mediates HG-induced EndMT and to determine the underlying mechanism.MethodsBioinformatics analysis of microarray profiles (GSE30780) and protein-protein interaction (PPI) analyses were performed to select the hub genes. As for in vitro research, the human aortic ECs (HAECs) were exposed to HG to induce EndMT. The expression of CaSR and β-catenin was determined, as well as their effects on EndMT (endothelial marker CD31, mesenchymal marker FSP1, and α-SMA).ResultsThe bioinformatics analysis indicated CaSR was significantly increased in HG-treated HAECs and was one of the hub genes. The in vitro results showed that HG significantly inhibited the expression of CD31 and increased FSP1 and α-SMA in a concentration- and time-dependent manner. Moreover, CaSR was increased in HAECs after HG treatment. The CaSR antagonist attenuated HG-induced expression of EndMT-related markers. Furthermore, HG treatment increased the nuclear translocation of β-catenin in HAECs. In contrast, blocking the nuclear translocation of β-catenin by DKK1 could attenuate HG-induced EndMT (increased the protein expression of CD31 by 30% and decreased the protein expression of FSP1 by 15% and α-SMA by 25%). CaSR siRNA further inhibited the HG-induced nuclear translocation of β-catenin in HAECs.ConclusionOur research demonstrated that HG-induced EndMT in HAECs might be mediated by CaSR and the downstream nuclear translocation of β-catenin.

Listeria monocytogenes is a deadly intracellular pathogen mostly associated with consumption of ready-to-eat foods. This study investigated the effectiveness of total beef fat (BF-T) from flaxseed-fed cattle and its fractions enriched with monounsaturated fatty acids (BF-MUFA) and polyunsaturated fatty acids (BF-PUFA), along with commercially available long-chain fatty acids (LC-FA), as natural antimicrobials against L. monocytogenes BF-T was ineffective at concentrations up to 6 mg/ml, while L. monocytogenes was susceptible to BF-MUFA and BF-PUFA, with MICs at pH 7 of 0.33 ± 0.21 mg/ml and 0.06 ± 0.03 mg/ml, respectively. The MIC of C14:0 was significantly lower than those of C16:0 and C18:0 (P

Decontamination practices, which often involve thermal treatments, are routinely performed in beef packing plants and have generally improved the safety of meat in North America. We investigated whether Escherichia coli in the beef production chain is becoming more heat resistant due to those treatments. Cattle isolates (n = 750) included seven serogroups (O157, O103, O111, O121, O145, O26, and O45) which were collected between 2002 and 2017. Beef plant isolates (n = 700) from carcasses, fabrication equipment, and beef products were included. Heat resistance was determined in Luria-Bertani broth at 60°C and by PCR screening for the locus of heat resistance (LHR). The decimal reduction for E. coli at 60°C (D(60ºC) values) ranged from 0 to 7.54 min, with 97.2% of the values being 2 min was not significantly different (P > 0.05) among cattle and meat plant isolates. E. coli from equipment before sanitation (median, 1.03 min) was more heat resistant than that after sanitation (median, 0.9 min). No significant difference in D(60ºC) values was observed among E. coli isolates from different years, from carcasses before and after antimicrobial interventions, or from before and during carcass chilling. Of all isolates, 1.97% harbored LHR, and the LHR-positive isolates had greater median D(60ºC) values than the LHR-negative isolates (3.25 versus 0.96 min). No increase in heat resistance in E. coli was observed along the beef production chain or with time. IMPORTANCE The implementation of multiple hurdles in the beef production chain has resulted in substantial improvement in the microbial safety of beef in Canada. In this study, we characterized a large number of Escherichia coli isolates (n = 1,450) from various sources/stages of beef processing to determine whether the commonly used antimicrobial interventions would give rise to heat-resistant E. coli on meat, which in turn may require alternatives to the current control of pathogens and/or modifications to the current cooking recommendations for meat. The findings show that the degree and rate of heat resistance in E. coli did not increase along the production chain or with time. This furthers our understanding of man-made ecological niches that are required for the development of heat resistance in E. coli.

Some Escherichia coli can persist on meat fabrication equipment, thus becoming a recurring source of contamination for meat. This study investigated the potential mechanism by which E. coli persist, by comparing the susceptibility to disinfectants and biofilm forming ability of two populations, recurring and transient, each consisting of 50 E. coli isolated from meat fabrication equipment. Susceptibility to three commercial disinfecting agents, biocide A (a quaternary ammonium compound based sanitizer), B (a sodium hypochlorite based sanitizer), and C (a chlorinated alkaline cleaner) was assessed, by determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Biofilm developed on polystyrene surface for up to 6 days at 15 °C was quantified by crystal violet staining. We found the recurring population did not have higher (P > 0.05) MICs or MBCs of any of the three biocides than the transient population, and the maximum MBCs were well within the respective in-use concentrations of the disinfectants. The development of biofilms was time-dependent for both transient and recurring E. coli populations and was correlated with motility and curli expression. Of the recurring E. coli, 50, 86, and 88% developed strong biofilms by days 2, 4, and 6, respectively. Of the transient E. coli, 58, 82 and 84% did so. The transient group had a higher (P

Carnobacterium maltaromaticum and Carnobacterium divergens are often predominant in the microbiota of vacuum-packaged (VP) meats after prolonged storage at chiller temperatures, and more so in recent studies. We investigated the antibacterial activities of C. maltaromaticum and C. divergens (n = 31) from VP meats by phenotypic characterization and genomic analysis. Five strains showed antibacterial activities against Gram-positive bacteria in a spot-lawn assay, with C. maltaromaticum strains having an intergeneric and C. divergens strains an intrageneric inhibition spectrum. This inhibitory activity is correlated with the production of predicted bacteriocins, including carnobacteriocin B2 and carnolysin for C. maltaromaticum and divergicin A for C. divergens. The supernatants of both species cultured in meat juice medium under anaerobic conditions retarded the growth of most Gram-positive and Gram-negative bacteria in broth assay in a strain-dependent manner. C. maltaromaticum and C. divergens produced formate and acetate but not lactate under VP meat-relevant conditions. The relative inhibitory activity by Carnobacterium strains was significantly correlated (P IMPORTANCE The results of this study demonstrated that both bacteriocins and organic acids are important factors contributing to the antibacterial activities of Carnobacterium from vacuum-packaged (VP) meats. This study demonstrated that formate and acetate are the key organic acids produced by Carnobacterium and demonstrated their association with the inhibitory activity of carnobacteria under VP meat-relevant storage conditions. The role of lactate, on the other hand, may not be as important as previously believed in the antimicrobial activities of Carnobacterium spp. on chilled VP meats. These findings advance our understanding of the physiology of Carnobacterium spp. to better explore their biopreservative properties for chilled VP meats.

The sanitation process commonly used for meat processing facilities includes three major steps: removal of soil (meat debris), cleaning and degreasing with detergent, and sanitization. The microbiological effect of such sanitation practice in commercial setting is largely unknown. Samples were collected from food-contact surface (CS) and non-food-contact surface (NCS) of two conveyor belts (Belt 1 and Belt 2, the latter of which was actively dried with air movers after sanitization) in a commercial beef fabrication facility at five time points: before cleaning (BC), after soil removal (ASR), after cleaning with detergent (ACD), 1.5 h after sanitization (AS), and before work the next day (BW), for enumeration of aerobes, lactic acid bacteria (LAB) and Enterobacteriaceae (EB). Selected isolates from each group of organisms from NCS of Belt 2 were also identified by 16 S rRNA gene sequencing. The mean numbers of aerobes, LAB and EB recovered BC were mostly about 6.0, 3.0 and 2.0 log CFU 1000 cm−2, respectively. None of the individual steps alone resulted in significant changes in numbers of the three groups of microorganisms, with the exception of ACD of both surfaces of Belt 1 and AS of NCS of Belt 2 where ≥1.5 log reduction was observed for aerobes and LAB, respectively. However, the overall sanitation process resulted in significant reductions (p

The N60 method currently mandated by regulatory bodies in North America for routine testing of beef trim for Escherichia coli O157:H7 requires that 60 slices of beef, up to 375 g, be removed from combo bins containing up to 10,000 lb of beef trim. The objective of this study was to design and test a prototype automatic trim sampler that would reduce the resource demands of, and result in more representative sampling than, N60. Ten commercial combo bins were each sampled at five locations of each of four levels, by swabbing an area of 1000 cm2 and by excision of up to five meat pieces with a total area of 100 cm2. The samples were enriched in modified Tryptone Soy Broth supplemented with novobiocin at 20 mg/L, and tested for generic E. coli using real-time PCR. A prototype trim sampler was constructed and tested for recovery of aerobes, coliforms and E. coli and was compared to manual swabbing, using beef trim artificially contaminated with E. coli. Overall, 21.5% of excision samples and 38.0% of swab samples from combo bins were positive for E. coli. Of the 40 levels that were sampled, 50.0% were positive by excision, 70.0% were positive by swabbing. The sampler, designed based on swab sampling, could process trim of ≤1 kg. Of the 12 pairs of manual sampling and automatic sampling compared, ≥ 8 were not significantly different for recovering each of the three groups of indicator organisms (P > 0.05). The findings of this study show that swab sampling can be more sensitive and representative than excision sampling for recovering small numbers of E. coli from beef trim and the prototype sampler can have efficacy comparable to manual swabbing for recovering all three groups of indicator organisms from beef.

Unsaturated fatty acid biohydrogenation products from beef fat and pure fatty acids were subjected to the Ames Salmonella mutagenicity testing, including monounsaturated fatty acids [MUFA: oleic acid, vaccenic acid, elaidic acid; beef fatty acid fractions rich in trans (t)11/t13-t14-18:1 (t11,13,14-Frac), t10-18:1 (t10-Frac)] and dienoic fatty acids [linoleic acid, conjugated linoleic isomers cis (c)9,t11-18:2 and t10,c12-18:2, and a mixed beef dienoic fatty acid fraction high in c9,t13-/t8,c12/t11c15-18:2 (MD)]. Significantly higher anti-mutagenic effects of oleic acid, vaccenic acid, t11, 13, 14-Frac, and t10-Frac against daunomycin were observed at 2.5 mg. All dienoic acids except MD significantly reduced daunomycin mutagenicity at ≥0.25 mg. Anti-mutagenicity of oleic and vaccenic acids against 2-aminoanthracene was found at 2.5 and 0.25 mg, respectively. All dienoic acids significantly reduced 2-aminoanthracene mutagenicity at ≥0.25 mg. Findings of this study show that unsaturated fatty acids, including trans-fatty acids commonly found in beef, can act as strong anti-mutagens.

Enterobacteriaceae comprise food spoilage organisms as well as food-borne pathogens including Escherichia coli. Heat resistance in E. coli was attributed to a genomic island called the locus of heat resistance (LHR). This genomic island is also present in several other genera of Enterobacteriaceae, but its function in the enteric pathogens Salmonella enterica and Enterobacter cloacae is unknown. This study aimed to determine the frequency of the LHR in food isolates of E. coli, and its influence on heat resistance in S. enterica and Enterobacter spp. Cell counts of LHR-positive strains of E. coli, S. enterica and E. cloacae were reduced by less than 1, 1, and 4 log (cfu/mL), respectively, after exposure to 60 °C for 5 min, while cell counts of LHR-negative strains of the same species were reduced by more than 7 log (cfu/mL). Introducing an exogenous copy of the LHR into heat-sensitive enteropathogenic E. coli and S. enterica increased heat resistance to a level that was comparable to LHR-positive wild type strains. Cell counts of LHR-positive S. enterica were reduced by less than 1 log(cfu/mL) after heating to 60 °C for 5 min. Survival of LHR-positive strains was improved by increasing the NaCl concentration from 0 to 4%. Cell counts of LHR-positive strains of E. coli and S. enterica were reduced by less than 2 log (cfu/g) in ground beef patties cooked to an internal core temperature of 71 °C. This study indicates that LHR-positive Enterobacteriaceae pose a risk to food safety.

The aim of this study was to map sources of Escherichia coli on beef in the fabrication facility of a commercial beef plant. At monthly intervals for 9 months, 11 groups of items were sampled at the start of work and after a period of work (approximately 2.5 h after the commencement of work). These items included: tables, chairs and paper towel dispenser in the lounge room where workers take their breaks and band saw (blade and table), knives and mesh gloves used by individual workers, cutting tables, conveyor belt, cuts and trimmings in the cutting rooms. Coliforms and E. coli were enumerated and selected E. coli isolates were genotyped using multiple-locus variable-number tandem repeat analysis (MLVA). Numbers of both groups of organisms were recovered at a higher frequency and often at higher numbers after working than when work started. A total of 970 E. coli isolates were confirmed, consisting of isolates from conveyor belt (45), cutting tables (1), and meat (96) when work started; and from conveyor belt (191), mesh gloves (319), meat (52), knives (7), band saw table (1), tables (3), and cutting tables (255) after working. MLVA of these isolates revealed 189 genotypes, with an overall ratio of isolates to genotypes of 5.1. A small fraction of genotypes (4.8%), though representing 30% of the total isolates, recurred in the samples collected from ≥3 months. Genotypes recovered from knives, band saw table and tables after working were also found on one or more items in the fabrication facility. Highly clustering genotypes were recovered, particularly from mesh gloves, cutting tables and conveyor belt. Of the isolates from conveyor belt before work, 93.3% were of genotypes that were also found among isolates from conveyor belt, mesh gloves and cutting tables after working. Of the E. coli on meat, 62.2% were shared with mesh gloves, conveyor belt and cutting tables. The findings show that lounge room items were not relevant sources of E. coli on beef, while conveyor belt, cutting tables and mesh gloves all contributed significantly to the contamination.