Socializing at the Air-Liquid Interface: a Functional Genomic Analysis on Biofilm-Related Genes during Pellicle Formation by Escherichia coli and Its Interaction with Aeromonas australiensis.

Pellicles are biofilms that form at the air-liquid interface. We demonstrated that specific strains of Escherichia coli formed pellicles in single cultures when cocultured with Carnobacterium maltaromaticum and E. coli O157:H7 but not with Aeromonas australiensis. Therefore, a combination of comparative genomic, mutational, and transcriptome analyses were applied to identify the unique genes in pellicle formation and investigate gene regulation under different growth phases. Here, we report that pellicle-forming strains do not harbor unique genes relative to non-pellicle-forming strains; however, the expression level of biofilm-related genes differed, especially for the genes encoding curli. Further, the regulatory region of curli biosynthesis is phylogenetically different among pellicle- and non-pellicle-forming strains. The disruption on modified cellulose and regulatory region of curli biosynthesis abolished pellicle formation in strains of E. coli. Besides, the addition of quorum sensing molecules (C4-homoserine lactones [C4-HSL]), synthesized by Aeromonas species, to pellicle formers abolished pellicle formation and implied a role of quorum sensing on pellicle formation. The deletion of autoinducer receptor sdiA in E. coli did not restore pellicle formation when cocultured with A. australiensis but modulated expression level of genes for curli and cellulose biosynthesis, resulting in a thinner layer of pellicle. Taken together, this study identified genetic determinants for pellicle formation and characterized the switching between pellicle to surface-associated biofilm in a dual-species environment, facilitating better understanding of the mechanisms for pellicle formation in E. coli and related organisms. IMPORTANCE To date, most attention has focused on biofilm formation on solid surfaces. By comparison, the knowledge on pellicle formation at the air-liquid interface is more limited and few studies document how bacteria decide on whether to form biofilms on solid surfaces or pellicles at the air-liquid interface to the surface-associated biofilms at the bottom. In this report, we characterized the regulation of biofilm-related genes during pellicle formation and document that interspecies communication via quorum sensing contributes to regulating the switch from pellicle to surface-associated biofilm. The discoveries expand the current view of regulatory cascades associated with pellicle formation.

Rice glycosyltransferase gene UGT2 functions in salt stress tolerance under the regulation of bZIP23 transcription factor.

KEY MESSAGE: Rice glycosyltransferase gene UGT2 was identified to play a crucial role in salt tolerance. The transcription factor OsbZIP23 was demonstrated to regulate the UGT2 expression under stress conditions. UDP-glycosyltransferases (UGTs) play key roles in modulating plant responses to environmental challenges. In this study, we characterized a novel glycosyltransferase, UGT2, which plays an important role in salt stress responses in rice (Oryza sativa L). We found that seedlings overexpressing UGT2 exhibited better growth than wild type in shoot and root under hydroponic culture with salt stress treatments, while ugt2ko mutant lines suffered much more growth inhibition. When the soil-grown UGT2 transgenic plants were subjected to salt stress, we also found that ugt2ko mutant lines were severely withered and most of them died, while the overexpression lines grew well and had higher survival rate. Compared with wild-type plants, UGT2 overexpression greatly increased the expression levels of the reactive oxygen species scavenging genes and stress-responsive genes. Furthermore, the upstream regulatory mechanism of the UGT2 gene was identified and we found that a bZIP transcription factor, OsbZIP23, can bind to the UGT2 promoter and enhance the UGT2 transcription levels. This work reveals that OsbZIP23-UGT2 module may play a major role in regulating the salt stress tolerance in rice.

Association of resistance to quaternary ammonium compounds and organic acids with genetic markers and their relationship to Escherichia coli serogroup.

Sanitizer resistance is being extensively investigated due to the potential for bacterial survival and cross-resistance with other antimicrobials. Similarly, organic acids are being used due to their microbial inactivation potential as well as being generally recognized as safe (GRAS). However, little is known about associations of genetic and phenotypic factors in Escherichia coli related to resistance to sanitizers and organic acids as well as differences between "Top 7" serogroups. Therefore, we investigated 746 E. coli isolates for resistance to lactic acid and two commercial sanitizers based on quaternary ammonium and peracetic acid. Furthermore, we correlated resistance to several genetic markers and investigated 44 isolates using Whole Genome Sequencing. Results indicate that factors related to motility, biofilm formation, and Locus of Heat Resistance played a role in resistance to sanitizers and lactic acid. In addition, Top 7 serogroups significantly differed in sanitizer and acid resistance, with O157 being the most consistently resistant to all treatments. Finally, mutations in rpoA, rpoC, and rpoS genes were observed, in addition to presence of a Gad gene with alpha-toxin formation in all O121 and O145 isolates, which may be related to increased resistance of these serogroups to the acids used in the present study.

Efficacy of Quaternary Ammonium Compounds for Control of Individual and Mixed Cultures of Escherichia coli with High- and Low-Quaternary Ammonium Compounds Resistance.

Escherichia coli is a well-characterized micro-organism in scientific literature. Similarly, quaternary ammonium compounds (QACs) are historical sanitizers in food processing. However, the use of QACs has been questioned due to bacterial resistance in some studies. Therefore, this study aimed to compare effects of single and mixed cultures of E. coli strains of different serogroups with either high (six strains) or low (five strains) resistance to QACs. Twenty-five combinations of strains with either high (H)- or low (L)-QAC resistance were analyzed (H + H vs. L + L). After exposure to QAC, combinations with statistical differences (p < 0.05) compared with individuals were selected and an inactivation model determined using GInaFit®. Only one combination of two strains (C23 and C20) with low-QAC resistance (mixture T18) had greater resistance (p < 0.05) than the individual isolates. The combination T18 and individual strain C23 presented a Weibull model, whereas the other isolated strain (C20) presented a biphasic inactivation model with a shoulder. Whole genome sequencing determined that unlike C20, C23 carried yehW, which may have led to Weibull inactivation. Possibly, very rapid interaction of C20 with the QAC favored increased survival of C23 and overall persistence of the T18 mixture. Consequently, our results indicate that individual E. coli with low-QAC resistance can synergistically interfere with QAC inactivation.

Genetic Characteristics of the Transmissible Locus of Stress Tolerance (tLST) and tLST Harboring Escherichia coli as Revealed by Large-Scale Genomic Analysis.

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.

Biofilm formation by Non-O157 Shiga toxin-producing Escherichia coli in monocultures and co-cultures with meat processing surface bacteria.

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.

Genomic and Phenotypic Analysis of Heat and Sanitizer Resistance in Escherichia coli from Beef in Relation to the Locus of Heat Resistance.

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 (D60°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 D60°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 < 0.001) median genome size by 0.3 Mbp (5.0 versus 4.7 Mbp) and overrepresentation of genes related to plasmid maintenance, stress response, and cryptic prophages but underrepresentation of genes involved in epithelial attachment and virulence. All LHR-positive isolates harbored a chromosomal copy of LHR, and all clade 2 isolates had an additional partial copy of LHR on conjugative plasmids. The growth rates at 42°C were 0.71 ± 0.02 and 0.65 ± 0.02 log(OD) h-1 for LHR-positive and -negative isolates, respectively. No meaningful difference in sanitizer susceptibility was noted between LHR-positive and -negative isolates. IMPORTANCE Resistant bacteria are serious food safety and public health concerns. Heat resistance conferred by the LHR varies largely among different strains of E. coli. The findings in this study show that genomic background and composition of LHR, in addition to the presence of LHR, play an important role in the degree of heat resistance in E. coli and that strains with certain genetic backgrounds are more likely to acquire and maintain the LHR. Also, caution should be exercised when recovering E. coli at elevated temperatures, as the presence of LHR may confer growth advantages to some strains. Interestingly, the LHR-harboring strains seem to have evolved further from their primary animal host to adapt to their secondary habitat, as reflected by fewer genes involved in virulence and epithelial attachment. The phylogenetic relationships among the isolates point toward multiple mechanisms for acquisition of LHR by E. coli, likely prior to its being deposited on meat.

Biofilm-Forming Capacity of Escherichia coli Isolated from Cattle and Beef Packing Plants: Relation to Virulence Attributes, Stage of Processing, Antimicrobial Interventions, and Heat Tolerance.

Despite the importance of biofilm formation in the contamination of meat by pathogenic Escherichia coli at slaughter plants, drivers for biofilm remain unclear. To identify selection pressures for biofilm, we evaluated 745 isolates from cattle and 700 generic E. coli isolates from two beef slaughter plants for motility, the expression of curli and cellulose, and biofilm-forming potential. Cattle isolates were also screened for serogroup, stx1, stx2, eae, and rpoS. Generic E. coli isolates were compared by source (hide of carcass, hide-off carcass, and processing equipment) before and after the implementation of antimicrobial hurdles. The proportion of E. coli isolates 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) isolate was an extremely strong biofilm former, in contrast to 73.4% of E. coli isolates from equipment. Isolates from equipment after sanitation had a greater biofilm-forming capacity (P < 0.001) than those before sanitation. Most cattle isolates were motile and expressed curli, although these traits along with the expression of cellulose and the detection 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. The results of the present study indicate that cattle rarely carry EHEC capable of making strong biofilms in slaughter plants. However, if biofilm-forming EHEC contaminates equipment, current sanitation procedures may not eliminate the most robust biofilm-forming strains. Accordingly, new and effective antibiofilm hurdles for meat-processing equipment are required to reduce future instances of foodborne disease. IMPORTANCE As the majority of enterohemorrhagic E. coli (EHEC) isolates are not capable of forming biofilms, sources were undetermined for biofilm-forming EHEC isolated from "high-event periods" in beef slaughter plants. This study demonstrated that sanitation procedures used on beef-processing equipment may inadvertently lead to the survival of robust biofilm-forming strains of E. coli. Cattle only rarely carry EHEC capable of forming strong biofilms (1/745 isolates evaluated), but isolates with greater biofilm-forming capacity were more likely (P < 0.001) to survive equipment sanitation. In contrast, chilling carcasses for 3 days at 0°C reduced (P < 0.05) the proportion of biofilm-forming E. coli. Consequently, an additional antibiofilm hurdle for meat-processing equipment, perhaps involving cold exposure, is necessary to further reduce the risk of foodborne disease.

Effects of Peroxyacetic Acid Spray and Storage Temperature on the Microbiota and Sensory Properties of Vacuum-Packed Subprimal Cuts of Meat.

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.

Transcriptome Analysis of Listeria monocytogenes Exposed to Beef Fat Reveals Antimicrobial and Pathogenicity Attenuation Mechanisms.

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 < 0.05). Fatty acids c9-C16:1, C18:2n-6, and C18:3n-3 showed stronger inhibitory activity than c9-C18:1 and conjugated C18:2, with MICs of <1 mg/ml. Furthermore, global transcriptional analysis by transcriptome sequencing (RNA-seq) was performed to characterize the response of L. monocytogenes to selected fatty acids. Functional analysis indicated that antimicrobial LC-UFA repressed the expression of genes associated with nutrient transmembrane transport, energy generation, and oxidative stress resistance. On the other hand, upregulation of ribosome assembly and translation process is possibly associated with adaptive and repair mechanisms activated in response to LC-UFA. Virulence genes and genes involved in bile, acid, and osmotic stresses were largely downregulated, and more so for c9-C16:1, C18:2n-6, and C18:3n-3, likely through interaction with the master virulence regulator PrfA and the alternative sigma factor σBIMPORTANCEListeria monocytogenes is a bacterial pathogen known for its ability to survive and thrive under adverse environments and, as such, its control poses a significant challenge, especially with the trend of minimally processed and ready-to-eat foods. This work investigated the effectiveness of fatty acids from various sources as natural antimicrobials against L. monocytogenes and evaluated their potential role in L. monocytogenes pathogenicity modulation, using the strain ATCC 19111. The findings show that long-chain unsaturated fatty acids (LC-UFA), including unsaturated beef fat fractions from flaxseed-fed cattle, could have the potential to be used as effective antimicrobials for L. monocytogenes through controlling growth as well as virulence attenuation. This not only advances our understanding of the mode of action of LC-UFA against L. monocytogenes but also suggests the potential for use of beef fat or its fractions as natural antimicrobials for controlling foodborne pathogens.

Are Antimicrobial Interventions Associated with Heat-Resistant Escherichia coli on Meat?

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 (D60ºC values) ranged from 0 to 7.54 min, with 97.2% of the values being <2 min. The prevalence of E. coli with D60ºC values of >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 D60ºC 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 D60ºC 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.

Complementary Antibacterial Effects of Bacteriocins and Organic Acids as Revealed by Comparative Analysis of Carnobacterium spp. from Meat.

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 < 0.05) to the production of both acids. Genomic analysis revealed the presence of genes required for respiration in both species. In addition, two clusters of C. divergens have an average nucleotide identity below the cutoff value for species delineation and thus should be considered to be two subspecies. In conclusion, both bacteriocins and organic acids are factors contributing significantly to the antibacterial activity of C. maltaromaticum and C. divergens under VP meat-relevant conditions. A few Carnobacterium strains can be explored as protective cultures to extend the shelf life and improve the safety of VP meats.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.

Facultative Anaerobes Shape Multispecies Biofilms Composed of Meat Processing Surface Bacteria and Escherichia coli O157:H7 or Salmonella enterica Serovar Typhimurium.

This study investigated the microbial dynamics in multispecies biofilms of Escherichia coli O157:H7 strain 1934 (O157) or Salmonella enterica serovar Typhimurium ATCC 14028 (ST) and 40 strains of meat processing surface bacteria (MPB). Biofilms of O157 or ST with/without MPB were developed on stainless steel coupons at 15°C for up to 6 days. Bacteria in suspensions (inoculum, days 2 and 6) and biofilms (days 2 and 6) were enumerated by plating. The composition of multispecies cultures was determined by 16S rRNA gene sequencing. In suspensions, levels of O157 and ST were ∼2 log higher in single-species than in multispecies cultures on both sampling days. ST was 3 log higher in single-species than in multispecies biofilms. A similar trend, though to a lesser extent, was observed for O157 in biofilms on day 2 but not on day 6. No difference (P > 0.05) in bacterial counts was noted for the two MPB-pathogen cocultures at any time during incubation. Bacterial diversity in multispecies cultures decreased with incubation time, irrespective of the pathogen or culture type. The changes in the relative abundance of MPB were similar for the two MPB-pathogen cocultures, though different interbacterial interactions were noted. Respective fractions of ST and O157 were 2.1% and 0.97% initially and then 0.10% and 0.07% on day 2, and 0.60% and 0.04% on day 6. The relative proportions of facultative anaerobes in both multispecies cultures were greater in both suspensions and biofilms than in the inoculum. Citrobacter, Hafnia, Aeromonas, and Carnobacterium predominated in biofilms but not always in the planktonic cultures.IMPORTANCE Results of this study demonstrate that Salmonella enterica serovar Typhimurium and E. coli O157:H7 can integrate into biofilms when cocultured with bacteria from meat plant processing surfaces. However, the degree of biofilm formation for both pathogens was substantially reduced in the presence of the competing microbiota, with S. Typhimurium more greatly affected than E. coli O157:H7. The expression of extracellular determinants such as curli and cellulose appears to be less important for biofilm formation of the pathogens in multispecies cultures than in monoculture. In contrast to previous reports regarding food processing surface bacteria, data collected here also demonstrate that facultative anaerobes may have a competitive edge over strict aerobes in establishing multispecies biofilms. It would be important to take into account the presence of background bacteria when evaluating the potential persistence of a pathogen in food processing facilities.

Dynamics of Biofilm Formation by Salmonella Typhimurium and Beef Processing Plant Bacteria in Mono- and Dual-Species Cultures.

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.

Comparative assessment of qPCR enumeration methods that discriminate between live and dead Escherichia coli O157:H7 on beef.

Quantitative Polymerase Chain Reaction (qPCR) is a molecular method commonly used to detect and quantify bacterial DNA on food but is limited by its inability to distinguish between live and dead cell DNA. To overcome this obstacle, propidium monoazide (PMA) alone or with deoxycholate (DC) was used to prevent dead cell detection in qPCR. qPCR methods were used to detect strains of Escherichia coli O157, which can cause infection in humans with an infectious dose of less than 10 cells. A 5 strain E. coli O157:H7 cocktail was inoculated onto beef steaks and treated with interventions used in meat facilities (lactic acid (5%), peroxyacetic acid (200 ppm) or hot water (80 °C for 10 s)). Treatment of PMA or PMA + DC was applied to samples followed by DNA extraction and quantification in qPCR. RNA was also quantified in addition to conventional plating. For lactic acid intervention, qPCR DNA quantification of E. coli O157:H7 yielded 6.59 ±â€¯0.21 and 6.30 ±â€¯0.11 log gene copy #/cm2 for control and lactic acid samples, respectively and after treatment with PMA or PMA + DC this was further reduced to 6.31 ± 0.21 and 5.58 ± 0.38, respectively. This trend was also observed for peroxyacetic acid and hot water interventions. In comparison, RNA quantification yielded 7.65 ± 0.13 and 7.02 ± 0.38 log reverse transcript/cm2 for rRNA control and lactic acid samples, respectively, and for plating (LB), 7.51 ±â€¯0.06 and 6.86 ±â€¯0.32 log CFU/cm2, respectively. Our research determined that treatment of PMA + DC in conjunction with qPCR prevented dead cell DNA detection. However, it also killed cells injured from intervention that may have otherwise recovered. RNA quantification was more laborious and results had higher variability. Overall, quantification with conventional plating proved to be the most robust and reliable method for live EHEC detection on beef.

The locus of heat resistance (LHR) mediates heat resistance in Salmonella enterica, Escherichia coli and Enterobacter cloacae.

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.

Effects of meat pH on growth of 11 species of psychrotolerant clostridia on vacuum packaged beef and blown pack spoilage of the product.

The aim of the study was to determine the effects of meat pH on the abilities of 11 psychrotolerant Clostridium spp. to grow on, and to possibly cause blown pack spoilage of vacuum packaged beef. Beef steaks of pH 5.4-5.6, 5.7-5.9 or ≥6.0, i.e. of normal, intermediate or high pH were prepared and vacuum packaged. Groups of 3 steaks of the same pH range were inoculated with log phase cultures of Clostridium algoriphilum, Clostridium algidixylanolyticum, Clostridium bowmanii, Clostridium estertheticum, Clostridium frigoris, Clostridium frigidicarnis, Clostridium gasigenes, Clostridium lacusfryxellense, Clostridium psychrophilum, Clostridium tagluense or Clostridium vincentii. Each pack was resealed immediately after the steak was inoculated, and pack volumes were determined by water displacement, immediately after resealing and at intervals during storage at 2 °C for 56 days. All of the clostridia grew in packs of high pH beef but none caused pack swelling. Packs of intermediate pH beef inoculated with C. estertheticum began to swell after 14 days, with a mean rate of increase of pack volumes of 6.80 ml/day. One pack of intermediate pH beef inoculated with C. frigoris was swollen after 37 days. Packs of normal pH beef that had been inoculated with C. estertheticum began swelling after 14 days with a mean rate of increase of pack volumes of 7.70 ml/day. Packs of normal or intermediate pH beef inoculated with other clostridia did not swell. After storage, the numbers of most Clostridium spp., as determined by real-time PCR were greater on beef of high pH than of lower pH values, but the numbers of C. frigidicarnis and C. lacusfryxellense were highest on intermediate pH meat, the numbers of C. estertheticum were higher on meat of lower than of high pH, and the numbers of C. tagluense were the same on meat of all pH values. With high pH meat, glucose was reduced to very low level in rinse fluids from packs that had been inoculated with any Clostridium sp. With intermediate and normal pH meat, glucose was reduced to very low concentrations in only rinse fluids from beef that had been inoculated with C. estertheticum.

Storage life at 2 °C or -1.5 °C of vacuum-packaged boneless and bone-in cuts from decontaminated beef carcasses.

BACKGROUND: The microbiological condition of beef produced at North American plants has been improved as a result of the use of effective carcass-decontaminating treatments. The effect of these treatments on the storage life of beef has not been established. In this study, beef primal cuts in vacuum packs stored at -1.5 or 2 °C for up to 160 days were assessed for their microbiological and organoleptic properties. RESULTS: The odours of boneless cuts were acceptable after storage at either temperature for ≤160 days; and the flavours of steaks from boneless cuts stored at 2 or -1.5 °C for ≤70 or ≤120 days, respectively, were acceptable. The storage life of bone-in cuts stored at 2 or -1.5 °C was, respectively, shorter or the same as that of boneless cuts stored at the same temperature. More than 20 microbial species that were mostly obligate aerobes were present on both types of cuts before storage. After storage for ≥30 days, the microflora was dominated by carnobacteria and Enterobacteriaceae were present in the flora from early storage times. CONCLUSIONS: A storage life of 120-140 days was attained by vacuum-packaged beef primals from decontaminated carcasses stored at -1.5 °C. The bone-in cuts stored at 2 °C were spoiled at earlier times, probably by Enterobacteriaceae.

Relationship between Desiccation Tolerance and Biofilm Formation in Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is a major concern in the food industry and requires effective control measures to prevent foodborne illnesses. Previous studies have demonstrated increased difficulty in the control of biofilm-forming STEC. Desiccation, achieved through osmotic stress and water removal, has emerged as a potential antimicrobial hurdle. This study focused on 254 genetically diverse E. coli strains collected from cattle, carcass hides, hide-off carcasses, and processing equipment. Of these, 141 (55.51%) were STEC and 113 (44.48%) were generic E. coli. The biofilm-forming capabilities of these isolates were assessed, and their desiccation tolerance was investigated to understand the relationships between growth temperature, relative humidity (RH), and bacterial survival. Only 28% of the STEC isolates had the ability to form biofilms, compared to 60% of the generic E. coli. Stainless steel surfaces were exposed to different combinations of temperature (0 °C or 35 °C) and relative humidity (75% or 100%), and the bacterial attachment and survival rates were measured over 72 h and compared to controls. The results revealed that all the strains exposed to 75% relative humidity (RH) at any temperature had reduced growth (p < 0.001). In contrast, 35 °C and 100% RH supported bacterial proliferation, except for isolates forming the strongest biofilms. The ability of E. coli to form a biofilm did not impact growth reduction at 75% RH. Therefore, desiccation treatment at 75% RH at temperatures of 0 °C or 35 °C holds promise as a novel antimicrobial hurdle for the removal of biofilm-forming E. coli from challenging-to-clean surfaces and equipment within food processing facilities.

Evaluating disinfectant efficacy on mixed biofilms comprising Shiga toxigenic Escherichia coli, lactic acid bacteria, and spoilage microorganisms.

This study aimed to investigate the impact of temperature and the presence of other microorganisms on the susceptibility of STEC to biocides. Mature biofilms were formed at both 10°C and 25°C. An inoculum of planktonic bacteria comprising 106 CFU/mL of spoilage bacteria and 103 CFU/mL of a single E. coli strain (O157, O111, O103, and O12) was used to form mixed biofilms. The following bacterial combinations were tested: T1: Carnobacterium piscicola + Lactobacillus bulgaricus + STEC, T2: Comamonas koreensis + Raoultella terrigena + STEC, and T3: Pseudomonas aeruginosa + C. koreensis + STEC. Tested biocides included quaternary ammonium compounds (Quats), sodium hypochlorite (Shypo), sodium hydroxide (SHyd), hydrogen peroxide (HyP), and BioDestroy®-organic peroxyacetic acid (PAA). Biocides were applied to 6-day-old biofilms. Minimum Bactericidal Concentrations (MBC) and Biofilm Eradication Concentrations (BEC) were determined. Planktonic cells and single-species biofilms exhibited greater susceptibility to sanitizers (p < 0.0001). Lactobacillus and Carnobacterium were more susceptible than the rest of the tested bacteria (p < 0.0001). Single species biofilms formed by E. coli O111, O121, O157, and O45 showed resistance (100%) to Shypo sanitizer (200 ppm) at 25°C. From the most effective to the least effective, sanitizer performance on single-species biofilms was PAA > Quats > HyP > SHyd > Shypo. In multi-species biofilms, spoilage bacteria within T1, T2, and T3 biofilms showed elevated resistance to SHyd (30%), followed by quats (23.25%), HyP (15.41%), SHypo (9.70%), and BioDestroy® (3.42%; p < 0.0001). Within T1, T2, and T3, the combined STEC strains exhibited superior survival to Quats (23.91%), followed by HyP (19.57%), SHypo (18.12%), SHyd (16.67%), and BioDestroy® (4.35%; p < 0.0001). O157:H7-R508 strains were less tolerant to Quats and Shypo when combined with T2 and T3 (p < 0.0001). O157:H7 and O103:H2 strains in mixed biofilms T1, T2, and T3 exhibited higher biocide resistance than the weak biofilm former, O145:H2 (p < 0.0001). The study shows that STEC within multi-species biofilms' are more tolerant to disinfectants.