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Escherichia coli O104:H4 strain 11-3088 encoding Stx2a is epidemiologically related to the foodborne outbreak associated with sprouts in Germany, 2011. Sprouting provides suitable conditions for bacterial growth and may lead to transduction of non-pathogenic strains of E. coli with Stx phages. Although transduction of E. coli by Stx phages in food has been documented, data on the phages from E. coli O104:H4 is limited. This study determined the host range of the bacteriophage Φ11-3088 from E. coli O104:H4 using E. coli O104:H4 ∆stx2::gfp::amp

To maintain food safety and flock health in broiler chicken production, biosecurity approaches to keep chicken barns free of pathogens are important. Canadian broiler chicken producers must deep clean their barns with chemical disinfectants at least once annually (full disinfection [FD]) and may wash with water (water wash [WW]) throughout the year. However, many producers use FD after each flock, assuming a greater efficacy of more stringent cleaning protocols, although little information is known regarding how these two cleaning practices affect pathogen population and gut microbiota. In the present study, a crossover experiment over four production cycles was conducted in seven commercial chicken barns to compare WW and FD. We evaluated the effects of barn cleaning methods on commercial broiler performance, cecal microbiota composition, Campylobacter and Salmonella occurrence, and Campylobacter jejuni and Clostridium perfringens abundance, as well as on short-chain fatty acid (SCFA) concentrations in the month-old broiler gut. The 30-day body weight and mortality rate were not affected by the barn cleaning methods. The WW resulted in a modest but significant effect on the structure of broiler cecal microbiota (weighted-UniFrac; adonis P = 0.05, and unweighted-UniFrac; adonis P = 0.01), with notable reductions in C. jejuni occurrence and abundance. In addition, the WW group had increased cecal acetate, butyrate, and total SCFA concentrations, which were negatively correlated with C. jejuni abundance. Our results suggest that WW may result in enhanced activity of the gut microbiota and reduced zoonotic transmission of C. jejuni in broiler production relative to FD in the absence of a disease challenge. IMPORTANCE We compared the effects of barn FD and WW methods on gut microbial community structures and pathogen prevalence of broiler chickens in a nonchallenging commercial production setting. The results revealed that barn cleaning methods had little impact on the 30-day body weight and mortality rate of broiler chickens. In addition, the FD treatment had a subtle but significant effect on the broiler cecal microbiota with increased abundances of Campylobacter and decreased SCFA concentrations, which would support the adoption of WW as a standard practice. Thus, compared to FD, WW can be beneficial to broiler chicken production by inhibiting zoonotic pathogen colonization in the chicken gut with reduced cost and labor of cleaning.

Detection sensitivity of cassette PCR was compared with a commercial BAX® PCR system for detection of eae and stx genes in Escherichia coli from 806 beef carcass swabs. Cassette PCR detects multiple genetic markers on multiple samples using PCR and melt curve analysis. Conventional PCR served as a gold standard. Overall, for positive and negative concordance, cassette PCR was 98.6% concordant with conventional PCR, and BAX PCR was 65.4% concordant. Of 806 beef carcass swabs, 339 by cassette PCR and 84 by BAX PCR harbored eae + stx+E. coli. For BAX PCR reactions, 84% of eae+ swabs, 79% of stx+ swabs, and 86% of eae + stx+ swabs were also detected by cassette PCR. For cassette PCR reactions, 457 swabs were eae+ with only 117 scored as eae+ using BAX PCR for 26% positive concordance. For stx primers, cassette PCR scored 480 samples as stx+ but only 215 samples were stx+ by BAX PCR, giving 45% positive concordance. Importantly, cassette PCR scored 339 swabs as harboring eae + stx+ E. coli, but BAX PCR detected only 71 positives giving only 21% positive concordance, with many false negatives. Cassette PCR is a highly sensitive method for detection of STEC genes in E. coli found in carcass swabs.

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.

The aim of this study was to investigate the synergistic effect of chitosan and bacteriocins against Escherichia coli and Salmonella in media and in lean beef. The inhibitory effects of chitosan and bacteriocins against E. coli AW1.7 and S. enterica Typhimurium in media were determined by a critical dilution assay. The efficacy a bacteriocin-producing strain of Carnobacterium maltaromaticum and high molecular weight chitosan (HMWC) in inactivation of E. coli AW1.7 and S. Typhimurium was evaluated on beef. Current interventions applied in the beef industry, steaming coupled with lactic acid, were used as reference. HMWC demonstrated higher antibacterial activity than water soluble chitosan (WSC) or chitosan oligosaccharides (COS) in media, and the addition of partially purified bacteriocins from C. maltaromaticum UAL307 increased the activity of the chitosan in vitro. The hurdle combinations associated with HMWC inactivated E. coli AW1.7 and S. enterica Typhimurium more effectively on lean beef when compared to steam or steam coupled with lactic acid. When used on beef, addition of bacteriocins and chitosan did not increase the antibacterial efficacy. Cell counts of S. enterica were further reduced during storage in presence of C. maltaromaticum and chitosan; however, this decrease was not dependent on bacteriocin production. In conclusion, addition of chitosan alone or in combination with C. maltaromaticum UAL 307 as protective culture significantly reduces cell counts of E. coli and Salmonella on beef. Results will be useful to improve pathogen intervention treatments in beef processing.

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H(2)O(2) (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO(4), 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log(10) CFU/ml higher than that for LHR-positive strains of E. coli. The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY(581/591), respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H(2)O(2), NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx(2)::gfp::amp. Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx(2)::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island. IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Decontamination of low aw foods without impairing the food quality is challenging, particularly for sprouting seeds. This study aimed to explore the lethality of high pressure carbon dioxide (HPCD) to Salmonella enterica, E. coli AW1.7, A. niger, P. roquforti, and Fusarium on beans, cereal grains, and ground pepper. The optimal antimicrobial effect of HPCD was achieved by equilibration of the aw to 0.75, followed by soaking in water and treatments with water-saturated CO2. HPCD reduced the viable cell counts of E. coli and Salmonella by 3–10 log(CFU/g), and prevented fungal growth during 10 d of incubation. More than 90% of mung beans germinated but the germination of oats was impaired after fungicidal or bactericidal treatments with HPCD. Overall, HPCD can be a promising antimicrobial treatment but treatment parameters need to be optimized for each type of seed.

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.

Low water activity (aW) foods permit the survival of low-infectious dose pathogens including Escherichia coli and Salmonella. Desiccation of non-heat resistant E. coli and Salmonella enterica increases their heat resistance; therefore, alternative methods are necessary to ensure the safety of low aW foods. High-pressure carbon dioxide (HPCD) reduced microbial contaminants in high aW foods. This study aimed to identify HPCD conditions that reduce pathogenic E. coli and Salmonella in low aW conditions. Four strains of Shiga toxin-producing E. coli (STEC) and one strain of enteropathogenic E. coli were treated as a cocktail, and five strains of Salmonella were treated individually. The suitability of E. coli AW1.7, Pediococcus acidilactici FUA 3072, Enterococcus faecium NRRL B-2354 and Staphylococcus carnosus R6 FUA 2133 as surrogate organisms was evaluated. Treatments were validated in beef jerky. Samples were equilibrated to aW 0.75 and treated with heat, HPCD or pressurized N2. Treatment of desiccated E. coli AW1.7 and the STEC cocktail with dry gaseous CO2 (5.7 MPa and 65 °C) did not reduce cell counts; however, treatment with gaseous CO2 saturated with water reduced cell counts of all strains of E. coli. Treatment of beef jerky inoculated with E. coli and Salmonella with saturated gaseous CO2 resulted in >5-log reductions for all strains. E. faecium NRRL B-2354 and S. carnosus R6 were suitable surrogates for Salmonella on beef jerky treated with HPCD. Treatment of beef jerky with water-saturated gaseous CO2 was more effective than treatment with supercritical CO2 or treatments with N2 at the same temperature and pressure. Overall, the treatment of low aW foods with water-saturated gaseous HPCD can meet industry standards by achieving a >5-log reductions of E. coli and Salmonella. Additionally, surrogate organisms representing pathogenic E. coli and Salmonella have been validated.

Listeria monocytogenes is often responsible for postprocessing contamination of ready-to-eat (RTE) products including cooked ham. As an emerging technology, atmospheric cold plasma (ACP) has the potential to inactivate L. monocytogenes in packaged RTE meats. The objectives of this study were to evaluate the effect of treatment time, modified atmosphere gas compositions (MAP), ham formulation, and post-treatment storage (1 and 7 days at 4 °C) on the reduction of a five-strain cocktail of L. monocytogenes and quality changes in ham subjected to in-package ACP treatment. Initial average cells population on ham surfaces were 8 log CFU/cm2 . The ACP treatment time and gas composition significantly (P 2 log reduction) in L. monocytogenes on ham in comparison to MAP2 (50% CO2 + 50% N2 ) and MAP3 (100% CO2 ), irrespective of ham formulation. Addition of preservatives (that is, 0.1% sodium diacetate and 1.4% sodium lactate) or bacteriocins (that is, 0.05% of a partially purified culture ferment from Carnobacterium maltaromaticum UAL 307) did not significantly reduce cell counts of L. monocytogenes after ACP treatment. Regardless of type of ham, storage of 24 hr after ACP treatment significantly reduced cells counts of L. monocytogenes to approximately 4 log CFU/cm2 . Following 7 days of storage after ACP treatment, L. monocytogenes counts were below the detection limit (>6 log reduction) when samples were stored in MAP1. However, there were significant changes in lipid oxidation and color after post-treatment storage. In conclusion, the antimicrobial efficacy of ACP is strongly influenced by gas composition inside the package and post-treatment storage. PRACTICAL APPLICATION: Surface contamination of RTE ham with L. monocytogenes may occur during processing steps such as slicing and packaging. In-package ACP is an emerging nonthermal technology, which can be used as a postpackaging decontamination step in industrial settings. This study demonstrated the influence of in-package gas composition, treatment time, post-treatment storage, and ham formulation on L. monocytogenes inactivation efficacy of ACP. Results of present study will be helpful to optimize in-package ACP treatment and storage conditions to reduce L. monocytogenes, while maintaining the quality of ham.

Campylobacter jejuni is a microaerophilic foodborne pathogen that is sensitive to stress conditions. However, it is not yet understood how this stress-sensitive pathogen may cause a significant number of cases of human gastroenteritis worldwide. In this study, we examined stress tolerance in 70 C. jejuni strains isolated from retail chicken under several stress conditions related to food safety. Compared to oxygen-sensitive (OS) strains of C. jejuni, C. jejuni strains with increased aerotolerance, such as hyper-aerotolerant (HAT) and aerotolerant (AT) strains, were more tolerant to peracetic acid, refrigeration and freeze-thaw stresses. However, the levels of thermotolerance and hyper-osmotolerance were not associated with the aerotolerance level of C. jejuni. The HAT and AT strains of C. jejuni exhibited significantly increased activities of catalase and superoxide dismutase (SOD), compared to the OS strains. Consistently, the HAT and AT strains were highly tolerant to oxidants, such as hydrogen peroxide, cumene hydroperoxide and menadione, compared to the OS strains. The AT and HAT strains that were tolerant to stresses, particularly peracetic acid and refrigeration, predominantly belonged to multilocus sequence typing (MLST) clonal complex (CC)-21. This study shows that oxidative stress resistance plays a role in determining the differential level of aerotolerance in C. jejuni and that AT and HAT strains of C. jejuni are more tolerant to oxidants and low temperatures than OS strains.

Background Over a one year period, swabs of 820 beef carcasses were tested for the presence of Shiga toxin-producing Escherichia coli by performing Polymerase Chain Reaction (PCR) in a novel technology termed “cassette PCR”, in comparison to conventional liquid PCR. Cassette PCR is inexpensive and ready-to-use. The operator need only add the sample and press “go”. Cassette PCR can simultaneously test multiple samples for multiple targets. Carcass swab samples were first tested for the presence of STEC genes (O157, eae, stx1 and stx2). Samples were considered to be pathogenic if positive for eae plus stx1 and/or stx2. For samples scored as pathogenic, further testing screened for 6 additional high frequency O-antigens (O26, O45, O103, O111, O121, and O145). Results Of the 820 samples, 41% were pathogenic and 30% were O157 positive. Of these, 19% of samples were positive for O157 and carried potentially pathogenic E. coli (eae plus stx1 and/or stx2). Of all samples identified as carrying pathogenic E. coli, 18.9, 38.8, 41.4, 0, 36.1, and 4.1% respectively were positive for O26, O45, O103, O111, O121, and O145. To validate cassette PCR testing, conventional PCR using STEC primers was performed on each of the 820 samples. Only 148 of 3280 cassette PCR tests were discordant with conventional PCR results. However, further fractional testing showed that 110 of these 148 PCRs reflected low numbers of E. coli in the enrichment broth and could be explained as due to Poisson limiting dilution of the template, affecting both cassette PCR and conventional PCR. Of the remaining 38 discordant tests, 27 initial capillary PCRs and 10 initial conventional tests were nominally discordant between cassette and conventional PCR, perhaps reflecting human/technical error on both sides of the comparison. Conclusions Contaminated beef carcass swabs were often complex, likely harboring more than one strain of pathogenic E. coli. Cassette PCR had 98.8% concordance with parallel conventional PCR for detection of STEC genes. This indicates that cassette PCR is highly reliable for detecting multiple pathogens in beef carcass swabs from processing plants.

The locus of heat resistance (LHR) confers extreme heat resistance in Escherichia coli. This study explored the role of the LHR in heat and pressure resistance of E. coli, as well as its relationship with protein folding and aggregation in vivo. The role of LHR was investigated in E. coli MG1655 and the pressure resistant E. coli LMM1010 expressing an ibpA-yfp fusion protein to visualize inclusion bodies by fluorescence microscopy. The expression of proteins by the LHR was determined by proteomic analysis; inclusion bodies of untreated and treated cells were also analyzed by proteomics, and by fluorescent microscopy. In total, 11 proteins of LHR were expressed: sHSP20, ClpKGI, sHSP, YdfX1 and YdfX2, HdeD, KefB, Trx, PsiE, DegP, and a hypothetical protein. The proteomic analysis of inclusion bodies revealed a differential abundance of proteins related to oxidative stress in strains carrying the LHR. The LHR reduced the presence of inclusion bodies after heat or pressure treatment, indicating that proteins expressed by the LHR prevent protein aggregation, or disaggregate proteins. This phenotype of the LHR was also conferred by expression of a fragment containing only sHSP20, ClpKGI, and sHSP. The LHR and the fragment encoding only sHSP20, ClpKGI, and sHSP also enhanced pressure resistance in E. coli MG1655 but had no effect on pressure resistance of E. coli LMM1010. In conclusion, the LHR confers pressure resistance to some strains of E. coli, and reduces protein aggregation. Pressure and heat resistance are also dependent on additional LHR-encoded functions.

Drying processes do not eliminate pathogenic Escherichia coli in foods but induce sublethal injury, which may also induce the Shiga toxin (Stx) prophage. This study investigated the effect of drying on membrane lipid oxidation and stx expression in E. coli. Lipid peroxidation was probed with C11-BODIPY581/591; and stx expression was assayed by quantification of GFP in E. coli O104:H4 Δstx2a:gfp:ampr. Treatment of E. coli with H2O2 oxidized the probe; probe oxidation was also observed after drying and rehydration. Lipid oxidation and the lethality of drying were reduced when cells were dried with trehalose under anaerobic condition; in addition, viability and probe oxidation differed between E. coli AW1.7 and E. coli AW1.7Δcfa. Desiccation tolerance thus relates to membrane lipid oxidation. Drying also resulted in expression of GFP in 5% of the population. Overexpression of gfp and recA after drying and rehydration suggested that the expression of Stx prophage was regulated by the SOS response. Overall, C11-BODIPY581/591 allowed investigation of lipid peroxidation in bacteria. Drying causes lipid oxidation, DNA damage and induction of genes encoded by the Stx prophage in E. coli.

Background Fast molecular detection methods benefit from ready-to-run lab-on-a-chip molecular assays with minimum preparation time. Detection efficiency of such methods can improve if multiple targets are detected simultaneously per given reaction. Detection of food pathogens, i.e. Escherichia coli (E. coli), is generally performed in two stages with the detection of multiple targets in each stage.With simultaneous testing, screening for pathogens is fast and efficient. Results In this study, we show the application of multiplex PCR performed on a ready-made cassette to detect 10 targets each for eight samples known to harbor E. coli. In cassette PCR, the aluminum cassette (38.6 mm × 31.4 mm) contains 10 trenches having a total of 50 capillaries with microliter volumes of desiccated acrylamide gels holding all reagents required for the PCR including internal positive and negative controls. The gel contains LCGreen dye to detect double stranded DNA. Fluorescence monitoring allows the detection of the amplified products by melt curve analysis. In this application, each of the five capillaries in a given trench contains two of the primer sets for the detection of 10 targets in pathogenic E. coli, namely, O157, Eae, Stx1, Stx2 and six O-antigen genes. Primer specificity was confirmed. Each trench tests one sample. Eight minimally processed enriched beef carcass swab samples were analyzed for parallel detection of 10 targets within 1 h and 15 min. Samples were delivered to the capillaries by capillary forces thereby hydrating the gels. Multiplex cassette PCR results were confirmed with conventional multiplex PCRs performed in a commercial real-time PCR system. Conclusions Cassette PCR technology is ideally suited to multi-target detection of pathogens in food products. The cassette performs multiple PCR reactions in parallel, with multiplex detection of targets within each reaction unit. Cassette PCR/ melt curve analysis results for the simultaneous detection of 10 targets of pathogenic E.coli in beef carcass swab samples were confirmed with a conventional real-time PCR/ melt curve analysis as well as with agarose gel electrophoresis. Although designed for the detection of E. coli, this multiplex cassette PCR technique can be applied to any other assay where the fast detection of multiple targets is required.

Despite their nutritional benefits, consumption of red meat from alternative sources such as bison, elk, and horse is low when compared to beef. Sensory attributes and drivers of liking were identified for these meats using the Preferred Attributes Elicitation (PAE) and Check-All-That-Apply (CATA) methods. For the PAE study, 25 panelists evaluated beef, horse, bison, and elk meats in three different group sessions (n = 7, 7, and 11), whereas 63 panelists participated in the CATA study. Consumers in both PAE and CATA studies associated horse meat with dry and fibrous appearance, whereas beef was associated with meaty/beefy flavor and aroma: bison with metallic and livery aroma and intense aftertaste and elk meat with livery, fishy, metallic flavor, musky aroma, and bloody aftertaste. Penalty analysis on the CATA data identified similar drivers of meat liking as the PAE groups. The attributes were juiciness, meaty/beefy aroma, tender texture, meaty/beefy flavor, and mild flavor and aroma. Attributes with significantly negative mean impact on liking were dryness, tough texture, livery flavor, and aftertaste. Association of these attributes with horse and elk meats has implication on drivers of dislike for these meat types. Cluster analysis identified a small group of consumers with preference for horse and elk meats, and this may present niche market opportunities for these meat types. Results showed that the PAE method was comparable to CATA for the evaluation of meat from different species and for identification of drivers of liking and that both methods are effective for meat sensory characterization. PRACTICAL APPLICATION: Lean red meat from unconventional sources such as elk, bison, and horse has unique sensory attributes that may influence acceptance. This study characterized the sensory attributes of these meats and their impact on liking using two rapid consumer descriptive profiling methods-PAE and CATA. Undesirable flavor and aftertaste attributes were identified as the major drivers of disliking for these unconventional meats. Both methods gave similar description of the samples, thus confirming the suitability of PAE for descriptive meat profiling by consumer panels.

The refrigerated storage life of selected meat sandwiches packed in elevated carbon dioxide atmospheres with air or nitrogen was studied by acceptability scores of untrained panelists and by microbiological analysis. Commercially produced sandwiches, including processed meats, roast beef and hamburgers, were packed in gas impermeable film with 50% CO2 and 50% air, stored at 4°C, and compared with a frozen reference sample. Processed meat products were still acceptable up to 35 d storage, roast beef for 28 to 35 d storage, and hamburger for only 14 d. The effect of gas atmospheres with 30, 50, or 70% CO2 and either air or nitrogen, on acceptability of laboratory-packaged bologna, ham, roast beef, and hamburger, was also studied. The most significant effect was observed with hamburger samples, in which exclusion of oxygen extended storage life from 14 to 35 d. Initial product quality was a more important factor determining the length of storage life than the range of gas atmospheres between 30 and 70% CO2, except for hamburger packaged in atmospheres containing air. A universal storage life for these products cannot be recommended. However acceptance of the products by an untrained taste panel generally extends beyond achievement of maximum microbial load.

As medical practitioners’ interest in hydrogels continues to grow, their new expectations in terms of mechanical properties, biocompatibility and durability are changed. Here, we demonstrated a new strategy to improve both mechanical properties and self-recovery of double network (DN) hydrogels by introducing a self-healing network, consisting of carboxymethyl chitosan (CMC) and dialdehyde cellulose nanocrystals (DACNC). Notably, the hydrogel could be repeatedly stretched to 4 times its initial length and has tensile strength of 244 kPa, and completely recovered its shape when compressed by 90% and had the compressive strength up to 8 MPa. In addition, the deformed hydrogel recovered 81.3% of its dissipated energy at room temperature without any external stimuli. The hydrogel also exhibited good biocompatibility. We have developed a new method to fabricate stretchable and tough hydrogels that could spontaneously self-repair following mechanical deformation. They are promising for controlled drug release and dye adsorption.

Cattle are a reservoir for enterohemorrhagic Escherichia coli (EHEC), and ground beef is a major vehicle for human infection with EHEC. Heat resistance of E. coli, including EHEC, is impacted by NaCl and other additives. This study aimed to evaluate the effect of NaCl and other additives on the heat resistance of E. coli in beef patties. E. coli AW1.7ΔpHR1(pLHR) with the locus of heat resistance (LHR), E. coli AW1.7ΔpHR1(pRK767) without LHR, or a 5-strain cocktail of EHEC were inoculated (107–108 CFU/g) into ground beef (15% fat) with NaCl (0–3%), marinade, carvacrol (0.1%), potassium lactate (3%) or chitosan (0.1%) following different protocols. Patties were grilled immediately, or stored in sterile bags for two days at 4 °C prior to grilling to a core temperature of 71 °C. Cell counts of LHR-positive E. coli AW1.7ΔpHR1(pLHR) were higher than that of the isogenic LHR-negative E. coli AW1.7ΔpHR1(pRK767) by >3 log10 (CFU/g) after cooking. Addition of 3% NaCl increased survival of E. coli AW1.7ΔpHR1(pRK767) and the EHEC cocktail while cell counts of the heat resistant strains were not changed. A protective effect of NaCl was not observed with E. coli AW1.7ΔpHR1(pRK767) or EHEC if cells of E. coli were cooled to 4 °C prior to mixing with cold meat and NaCl, indicating that the response of E. coli to osmotic shock contributes to this effect. Chitosan enhanced the thermal destruction of LHR-positive E. coli AW1.7ΔpHR1(pLHR) in ground beef stored at 4 °C for 2 days, while marinade, carvacrol, or potassium lactate had no such effect, indicating that chitosan can be characterized as an effective hurdle concept to reduce the potential risk of LHR-positive pathogen to meat safety.

Ready-to-eat (RTE) deli meat has been linked to several Listeria monocytogenes associated recalls. Recent studies demonstrated the potential antimicrobial effects of atmospheric cold plasma treatment on various food surfaces including RTE meat products. However, the influence of intrinsic and extrinsic factors, determining the efficacy of cold plasma to reduce Listeria has not been reported. This study investigated the influence of rosemary extract, salt (% NaCl), and treatment temperature on the efficacy of plasma to reduce numbers of L. innocua on RTE ham. The effect of post-treatment storage on L. innocua inactivation was also investigated. When the cold plasma treatment temperature was 4 °C, we observed a significant reduction in L. innocua of 1.75 and 1.51 log CFU/cm2 on 1% and 3% NaCl ham surface without rosemary extract respectively, after 180 s treatment. At a treatment temperature of 23 °C, the L. innocua cells were reduced by 1.78 and 1.43 log CFU/cm2, respectively on these surfaces after 180 s. No significant effects of salt concentration and treatment temperature were observed on L. innocua inactivation during cold plasma treatment of ham. The post treatment storage at 4 °C for 6 h after 180 s of plasma treatment enhanced further reduction of L. innocua on 1% NaCl ham without rosemary. We also observed the increased concentration of malondialdehyde (MDA) equivalent lipid oxidation of plasma treated samples and was significantly higher (1.53 MDA mg/ kg ham) compared to untreated samples (0.92 MDA mg/kg ham). However, no significant differences in surface color parameters, L* and b* values were observed after plasma treatment, except a significant increase in a* values. The water content of plasma exposed samples decreased significantly for all treatment conditions whereas the water activity values were not changed significantly. In conclusion, the atmospheric cold plasma could be applied as a means for surface decontamination of RTE ham. However, the drying and oxidation of ham should be controlled in an open atmospheric plasma treatment condition.

The pressure resistance of Shiga-toxin producing Escherichia coli (STEC) depends on food matrix. This study compared the resistance of two five-strain E. coli cocktails, as well as the pressure resistant strain E. coli AW1.7, to hydrostatic pressure application in bruschetta, tzatziki, yoghurt and ground beef at 600 MPa, 20 °C for 3 min and during post-pressure survival at 4 °C. Pressure reduced STEC in plant and dairy products by more than 5 logs (cfu/ml) but not in ground beef. The pH affected the resistance of STEC to pressure as well as the post-pressure survival. E. coli with food constituents including calcium, magnesium, glutamate, caffeic acid and acetic acid were treated at 600 MPa, 20 °C. All compounds exhibited a protective effect on E. coli. The antimicrobial compounds ethanol and phenylethanol enhanced the inactivation by pressure. Calcium and magnesium also performed protective effects on E. coli during storage. Glutamate, glutamine or glutathione did not significantly influence the post-pressure survival over 12 days. Preliminary investigation on cell membrane was further performed through the use of fluorescence probe 1-N-phenylnaphthylamine. Pressure effectively permeabilised cell membrane, whereas calcium showed no effects on membrane permeabilisation.

This study investigated the effect of growth temperature (8–32 °C), process temperature (−17 to 32 °C), and sodium chloride concentration (0–3 %) on the lethality of pressure to Listeria monocytogenes. Pressure treatments were performed using a 5-strain cocktail of L. monocytogenes. Cultures grown at 8 °C were more resistant to pressure than cultures grown at 20 or 32 °C. Pressure treatments of the Listeria cocktail indicated that Listeria were most resistant to pressure at −5 or +5 °C. The effect of pressure was further evaluated at 500 MPa and +5 °C in buffer containing 1 or 3 % NaCl. Cultures treated in the presence of 3 % NaCl were more resistant than cultures treated in the presence of 1 % NaCl. Results obtained in buffer were compared to treatment of cooked ham containing 1 or 3 % NaCl. L. monocytogenes was more resistant in ham with 3 % NaCl when compared to ham with 1 % NaCl. L. monocytogenes grown at 32 °C were slightly more resistant to pressure when compared to cultures grown at 8 °C. Refrigerated storage of treated samples for 4 weeks demonstrated that L. monocytogenes recovered from all treatments with a pressure-holding time of 8 min or less. In conclusion, the effect of high-pressure processing strongly depends on growth temperature, process temperature, and the food matrix. To generally achieve a 5-log reduction of L. monocytogenes on ready-to-eat meats, combinations of pressure with elevated temperature or other antimicrobial hurdles are necessary.

Pressure treatment of ready-to-eat (RTE) meats extends the shelf life and reduces risks associated with Listeria monocytogenes. However, pressure reduces numbers of Listeria on ham by less than 5 log (CFU/g) and pressure effects on other meat microbiota are poorly documented. This study aimed to investigate the impact of pressure and meat microbiota, with or without antimicrobials, on survival of Listeria after refrigerated storage. Ham was inoculated with a 5-strain cocktail of L. monocytogenes alone or together with a cocktail of competitive meat microbiota consisting of Brochothrix thermosphacta, Carnobacterium maltaromaticum, Leuconostoc gelidum, and Lactobacillus sakei. Products were treated at 500 MPa at 5°C for 1 or 3 min, with or without rosemary extract or nisin. Surviving cells were differentially enumerated after pressure treatment and after 4 weeks of refrigerated storage; meat microbiota after 4 weeks of storage were also analysed by high throughput sequencing of 16S rRNA amplicons. Pressure treatment of Listeria on ham for 1 or 3 min reduced counts by 1 and 2 log (CFU/g), respectively; inactivation of other meat microbiota was comparable. Counts of Listeria increased by 3 and 1 log (CFU/g) during refrigerated storage after 1 or 3 min of treatment, respectively. The presence of meat microbiota did not influence pressure inactivation of Listeria but prevented growth of Listeria during refrigerated storage. The addition of rosemary extract did not influence inactivation of Listeria or the meat microbiota, or growth of microorganisms during storage. The combination of nisin with pressure treatment for 3 min reduced counts of Listeria and meat microbiota by >5 log (CFU/g); after 4 weeks of storage, counts were below the detection limit. In conclusion, pressure application alone does not eliminate Listeria or meat microbiota on RTE ham; however, the presence of meat microbiota prevents growth of Listeria on pressure treated ham and has a decisive influence on post pressure survival and growth.

Horse meat is a good source of iron with low cholesterol but consumers consider tenderness most important when purchasing meat. Myofibers and intramuscular connective tissue, the main structural components of muscle, play major roles in meat toughness. The effects of myofibers and perimysium characteristics on Warner-Bratzler Shear Force (WBSF) values of horse semimembranosus were investigated on muscles previously assessed as tough (mean WBSF 68.87 ± 3.21 N, n = 8) or tender (mean WBSF 46.21 ± 2.27 N, n = 8). Mean Type IIA myofiber diameter was significantly greater in tender than in tough muscles. Secondary thick perimysium was thicker and collagen fibers more loosely arranged in tender muscles than in tough. A negative correlation was observed between WBSF values and perimysium thickness and a trend between WBSF and intramuscular fat. Adipocytes in perimysium of tender muscles may have contributed to thickening of perimysium and loosened collagen fibers thus contributing to tenderness of the meat.

In this manuscript, we report the design and development of a fast, reliable instrument to run gel-based cassette polymerase chain reactions (PCR). Here termed the GelCycler Mark II, our instrument is a miniaturized molecular testing system that is fast, low cost and sensitive. Cassette PCR utilizes capillary reaction units that carry all reagents needed for PCR, including primers and Taq polymerase, except the sample, which is loaded at the time of testing. Cassette PCR carries out real time quantitative PCR followed by melt curve analysis (MCA) to verify amplicon identity at the expected melt temperature (Tm). The cassette PCR technology is well developed, particularly for detecting pathogens, and has been rigorously validated for detecting pathogenic Escherichia coli in meat samples. However, the work has been hindered by the lack of a robust and stable instrument to carry out the PCR, which requires fast and accurate temperature regulation, improved light delivery and fluorescent recording, and faster PCR reactions that maintain a high sensitivity of detection. Here, we report design and testing of a new instrument to address these shortcomings and to enable standardized testing by cassette PCR and commercial manufacture of a robust and accurate instrument that can be mass produced to deliver consistent performance. As a corollary to our new instrument development, we also report the use of an improved design approach using a machined aluminum cassette to meet the new instrument standards, prevent any light bleed across different trenches in each cassette, and allow testing of a larger number of samples for more targets in a single run. The GelCycler Mark II can detect and report E. coli contamination in 41 minutes. Sample positives are defined in as having a melt curve comparable to the internal positive control, with peak height exceeding that of the internal negative control. In a fractional analysis, as little as 1 bacterium per capillary reaction unit is directly detectable, with no enrichment step, in 35 cycles of PCR/MCA, in a total time of 53 minutes, making this instrument and technology among the very best for speed and sensitivity in screening food for pathogenic contamination.

Many strains of verotoxigenic Escherichia coli (VTEC) are highly resistant to pressure. To facilitate future studies to improve the elimination of VTEC by pressure processing of food, this study developed and validated a cocktail of non-pathogenic strains of E. coli with equal or higher resistance to pressure when compared to pressure resistant strains of VTEC. Strains of E. coli obtained from a beef processing plant were screened for their resistance to heat and pressure. Treatments were carried out in LB broth. Cell counts of 3 out of 16 strains were reduced by 5–6 log (cfu/mL) after 30 min at 60 °C, and cell counts of 10 out of 16 strains were reduced by 5–6 log (cfu/mL) after 30 min at 40 °C and 400 MPa. All highly heat resistant strains were also pressure resistant but not all pressure resistant strains were also heat resistant. Pressure resistant and -sensitive strains of E. coli were treated in presence of 0 or 2% NaCl and at 3, 20, or 40 °C. The effect of these parameters on the lethality of pressure treatments was comparable for all strains. The addition of 2% NaCl did not increase pressure resistance. The bactericidal effect of treatments at 3 and 20 °C and 600 MPa was comparable but inactivation of E. coli was faster at 40 °C and 600 MPa. The resistance to treatment with 600 MPa at 20 °C of a cocktail of 5 non-pathogenic strains of E. coli was compared to a 5 strain cocktail of pressure resistant VTEC. Treatments were performed in ground beef containing 15% fat. Survival and sublethal injury of the two cocktails was comparable; cell counts of beef inoculated with either cocktail were reduced by about 4 log (cfu/mL) after 30 min of treatment. In conclusion, this study validated a cocktail of non-pathogenic strains of E. coli for use as surrogate organisms in studies on the elimination of E. coli by pressure.

Daikon radish (Raphanus sativus) fermented with lactic acid bacteria, especially Leuconostoc or Lactobacillus spp., can be used to make kimchi, a traditional Korean fermented vegetable. Commercial Leuconostoc/radish root ferment filtrates are claimed to have broad spectrum antimicrobial activity. Leuconostoc kimchii fermentation products are patented as preservatives for cosmetics, and certain strains of this organism are reported to produce antimicrobial peptides (bacteriocins). We examined the antimicrobial agents in commercial Leuconostoc/radish root ferment filtrates. Both activity-guided fractionation with Amberlite XAD-16 and direct extraction with ethyl acetate gave salicylic acid as the primary agent with activity against Gram-negative bacteria. Further analysis of the ethyl acetate extract revealed that a didecyldimethylammonium salt was responsible for the Gram-positive activity. The structures of these compounds were confirmed by a combination of (1)H- and (13)C NMR, high-performance liquid chromatography, high-resolution mass spectrometry, and tandem mass spectrometry analyses. Radiocarbon dating indicates that neither compound is a fermentation product. No antimicrobial peptides were detected.

This study evaluated the heat and pressure resistance of 112 strains of Escherichia coli, including 102 strains of verotoxigenic E. coli (VTEC) representing 23 serotypes and four phylogenetic groups. In an initial screening, the heat and pressure resistance of 100 strains, including 94 VTEC strains, were tested in phosphate-buffered saline (PBS). Treatment at 60°C for 5 min reduced cell counts by 2.0 to 5.5 log CFU/ml; treatment at 600 MPa for 3 min at 25°C reduced the cell counts by 1.1 to 5.5 log CFU/ml. Heat or pressure resistance did not correlate to the phylogenetic group or the serotype. A smaller group of E. coli strains was evaluated for heat and pressure resistance in Luria-Bertani (LB) broth. Generally, the levels of heat resistance of E. coli strains in LB and PBS were similar; however, the levels of pressure resistance observed for treatments in LB broth or PBS were variable. The cell counts of pressure-resistant strains of VTEC were reduced by less than 1.5 log CFU/ml after treatment at 600 MPa for 3 min. E. coli strains were also treated with 600 MPa for 3 min in ground beef or inoculated into beef patties and grilled to 63 or 71°C. The cell counts of the VTEC E. coli O26:H11 strain 05-6544 were reduced by 2 log CFU/g by pressure treatment in ground beef. The cell counts of the heat-resistant E. coli strain AW1.7 were reduced by 1.4 and 3.4 log CFU/g in beef patties grilled to internal temperatures of 63 and 71°C, respectively. The cell counts of E. coli 05-6544 were reduced by less than 3 and 6 log CFU/g in beef patties grilled to internal temperatures of 63 and 71°C, respectively. To study whether the composition of the beef patties influenced heat resistance, E. coli strains AW1.7, AW1.7 Δ pHR1, MG1655, and LMM1030 were mixed into beef patties containing 15 or 35% fat and 0 or 2% NaCl, and the patties were grilled to an internal temperature of 63°C. The highest heat resistance of E. coli was observed in patties containing 15% fat and 2% NaCl.

The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae , including pathogenic strains of Salmonella enterica and Escherichia coli . The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1 GI , yfdX2 , hdeD GI , orf11 , trx GI , kefB , and psiE GI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript “GI” [genomic island] if an ortholog of the same gene is present in genomes of E. coli .) LHR-encoded heat shock proteins sHSP20, ClpK GI , and sHSP GI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trx GI , kefB , and psiE GI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA . In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food.

Enterococcus canintestini 49, isolated from dog feces, is active against Clostridium perfringens, vancomycin-resistant enterococci, and Listeria monocytogenes. Its draft genome sequence reported herein contains a gene cluster encoding multiple bacteriocins and indicates the absence of genes for virulence factors. These characteristics signify the strain’s potential for use as a probiotic.

Enterococcus canintestini 49, isolated from dog feces, is active against Clostridium perfringens , vancomycin-resistant enterococci, and Listeria monocytogenes . Its draft genome sequence reported herein contains a gene cluster encoding multiple bacteriocins and indicates the absence of genes for virulence factors. These characteristics signify the strain’s potential for use as a probiotic.

Bacterial spoilage and safety are major concerns in the marketing of raw and processed meats. When meat is packaged under modified atmosphere with elevated levels of carbon dioxide (including vacuum packaging), the prevailing microflora of meat is changed from aerobic, putrefactive bacteria to lactic acid bacteria. Some "new generation" convenience foods rely almost entirely on refrigeration for assurance of safety against growth of pathogenic bacteria. With the emergence of cold-tolerant foodborne pathogens it is desirable to increase the "hurdles" to pathogen growth. Lactic acid bacteria preserve meats by competitive exclusion of other microorganisms but they also produce inhibitory substances, including lactic and acetic acids and bacteriocins. Bacteriocins are naturally produced peptides that are antagonistic to other closely related bacteria. Although bacteriocins are expected to have a narrow range of antibacterial activity, nisin is a bacteriocin that is active against a relatively broad spectrum of gram-positive bacteria, including inhibition of the outgrowth of Clostridium botulinum spores. Nisin is not effective in meat systems; as a result, research on the lactic acid bacteria of meat is focused on the selection of lactic acid bacteria that do not cause meat spoilage and that enhance product safety.

Egg production in Canada is highly controlled under a supply management system, and all registered egg producers must conduct regular testing of the production facilities for Salmonella . In most provinces, the testing is conducted under the guidelines of the “Start Clean-Stay Clean” on-farm food safety programs. This Hazard Analysis Critical Control Point-based program incorporates on-farm activities that help to reduce the risk of Salmonella Enteritidis contamination of eggs, thus reducing the risk of human illness. Although human illnesses associated with Salmonella enterica serovar Enteritidis have increased in Canada, there is little evidence to link the increase to consumption of eggs produced through the supply management system, as many other risk factors for Salmonella Enteritidis infections have been identified. However, safe food handling practices throughout the food chain need to be improved to mitigate infections from handling or consumption of eggs.

The aim of this study was to examine the filament formation and differential gene expression of Listeria monocytogenes 08-5923 grown on refrigerated vacuum-packaged ham products with various NaCl concentrations. Filament formation of L. monocytogenes was observed on ham products with 1.35% and 2.35% NaCl, which was monitored using flow cytometry by measuring forward light scatter. Quantitative real-time PCR was used to study the differential expression of genes in filamented cells of L. monocytogenes grown on hams following 2 or 3 months of storage at 4 °C. The genes involved in cell division (ftsX/lmo2506), cell wall synthesis (murZ/lmo2552), and NADPH production (gnd/lmo1376) were significantly downregulated in filamented cells of L. monocytogenes grown on ham with 2.35% NaCl stored at 4 °C. To our knowledge, this study reports the first evidence of filament formation of Listeria grown on meat products, which could impact the food safety risk and tolerance levels of L. monocytogenes set by regulatory agencies.

Carnocyclin A (CCLA) is an antimicrobial peptide produced by Carnobacterium maltaromaticum ATCC PTA-5313, which can be used to control the growth of Listeria monocytogenes in ready-to-eat meat products. The aim of this research was to elucidate the cellular responses of L. monocytogenes 08-5923 exposed to a sublethal dose of CCLA. Microarray, quantitative reverse transcription-PCR, tandem mass spectrometry, and electron microscopy were used to investigate the alteration in gene expression, protein production, and morphological changes in cells of Listeria following treatment with CCLA. The genes involved in metabolism ( baiE , trn , and pykA ), cell wall synthesis ( murZ and dacB2 ), and cell division ( clpE and divIVA ) were upregulated following a 15-min exposure to CCLA as a result of stress responses. Genes involved in cell division, cell wall synthesis, flagellar synthesis, and metabolism were downregulated after 4 h as a result of adaptation. Analysis of total soluble proteins confirmed the downregulation of pykA and gnd after 4 h of exposure to CCLA. The absence of flagella was observed in L. monocytogenes following 30 h of exposure to CCLA. A sublethal dose of CCLA induced adaptation in L. monocytogenes 08-5923 by inhibition of expression of genes and proteins critical for synthesis of cell wall structures and maintaining metabolic functions. Both the mannose- and cellobiose-specific phosphotransferase systems could be targets for CCLA.