Transduction of stx2a mediated by phage (Φ11-3088) from Escherichia coli O104:H4 in vitro and in situ during sprouting of mung beans.

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::ampr and demonstrated phage transduction during sprouting. The Φ11-3088∆stx transduced 5/45 strains, including generic E. coli, pap-positive E. coli O103:H2, ETEC, and S. sonnei. The expression level of Φ11-3088∆stx differed among lysogens upon induction. Of the 3 highly induced lysogens, the lytic cycle was induced in E. coli O104:H4∆stx2::gfp::ampr and O103:H2 but not in S. sonnei. E. coli DH5α was the only strain susceptible to lytic infection by Φ11-3088∆stx. To explore the effect of drying and rehydration during seed storage and sprouting on phage induction and transduction, mung beans inoculated with the phage donor E. coli O104:H4∆stx2::gfp::ampr (8 log CFU/g) were dried, rehydrated, and incubated with the phage recipient E. coli DH5α (7 log CFU/g) for 96 h. Sprouted seeds harbored about 3 log CFU/g of putative lysogens that acquired ampicillin resistance. At the end of sprouting, 71 % of putative lysogens encoded gfp, confirming phage transduction. Overall, stx transfer by phages may increase the cell counts of STEC during sprouting by converting generic E. coli to STEC.

The Use of Disinfectant in Barn Cleaning Alters Microbial Composition and Increases Carriage of Campylobacter jejuni in Broiler Chickens.

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.

Comparison of a Miniaturized Cassette PCR System with a Commercially Available Platform for Detecting Escherichia coli in Beef Carcass Swabs.

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.

Contribution of the Locus of Heat Resistance to Growth and Survival of Escherichia coli at Alkaline pH and at Alkaline pH in the Presence of Chlorine.

The locus of heat resistance (LHR) confers resistance to extreme heat, chlorine and oxidative stress in Escherichia coli. This study aimed to determine the function of the LHR in maintaining bacterial cell envelope homeostasis, the regulation of the genes comprising the LHR and the contribution of the LHR to alkaline pH response. The presence of the LHR did not affect the activity of the Cpx two-component regulatory system in E. coli, which was measured to quantify cell envelope stress. The LHR did not alter E. coli MG1655 growth rate in the range of pH 6.9 to 9.2. However, RT-qPCR results indicated that the expression of the LHR was elevated at pH 8.0 when CpxR was absent. The LHR did not improve survival of E. coli MG1655 at extreme alkaline pH (pH = 11.0 to 11.2) but improved survival at pH 11.0 in the presence of chlorine. Therefore, we conclude that the LHR confers resistance to extreme alkaline pH in the presence of oxidizing agents. Resistance to alkaline pH is regulated by an endogenous mechanism, including the Cpx envelope stress response, whereas the LHR confers resistance to extreme alkaline pH only in the presence of additional stress such as chlorine.

Effect of in-package atmospheric cold plasma discharge on microbial safety and quality of ready-to-eat ham in modified atmospheric packaging during storage.

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 < 0.05) influenced the inactivation of L. monocytogenes, irrespective of ham formulations. When MAP1 (20% O2 + 40% CO2 + 40% N2 ) was used, there was a significantly higher log reduction (>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.

Heat and Pressure Resistance in Escherichia coli Relates to Protein Folding and Aggregation.

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.

Lethality of high-pressure carbon dioxide on Shiga toxin-producing Escherichia coli, Salmonella and surrogate organisms on beef jerky.

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.

The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli.

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), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 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 BODIPY581/591, 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 H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::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.

Effect of drying on oxidation of membrane lipids and expression of genes encoded by the Shiga toxin prophage in Escherichia coli.

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.

Consumer Sensory Comparisons Among Beef, Horse, Elk, and Bison Using Preferred Attributes Elicitation and Check-All-That-Apply Methods.

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.

Tolerance to stress conditions associated with food safety in Campylobacter jejuni strains isolated from retail raw chicken.

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.

Detection of pathogenic Escherichia coli on potentially contaminated beef carcasses using cassette PCR and conventional PCR.

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.

Stretchable, tough, self-recoverable, and cytocompatible chitosan/cellulose nanocrystals/polyacrylamide hybrid hydrogels.

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.

Cold plasma treatment of ready-to-eat ham: Influence of process conditions and storage on inactivation of Listeria innocua.

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.

Application of lab-on-a-chip multiplex cassette PCR for the detection of enterohemorrhagic Escherichia 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.

Effect of chitosan, and bacteriocin - Producing Carnobacterium maltaromaticum on survival of Escherichia coli and Salmonella Typhimurium on beef.

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.

Comparative Genomics and Characterization of the Late Promoter pR' from Shiga Toxin Prophages in Escherichia coli.

Shiga-toxin producing Escherichia coli (STEC) causes human illness ranging from mild diarrhea to death. The bacteriophage encoded stx genes are located in the late transcription region, downstream of the antiterminator Q. The transcription of the stx genes is directly under the control of the late promoter pR', thus the sequence diversity of the region between Q and stx, here termed the pR' region, may affect Stx toxin production. Here, we compared the gene structure of the pR' region and the stx subtypes of nineteen STECs. The sequence alignment and phylogenetic analysis suggested that the pR' region tends to be more heterogeneous than the promoter itself, even if the prophages harbor the same stx subtype. Furthermore, we established and validated transcriptional fusions of the pR' region to the DsRed reporter gene using mitomycin C (MMC) induction. Finally, these constructs were transformed into native and non-native strains and examined with flow cytometry. The results showed that induction levels changed when pR' regions were placed under different regulatory systems. Moreover, not every stx gene could be induced in its native host bacteria. In addition to the functional genes, the diversity of the pR' region plays an important role in determining the level of toxin induction.

Effect of Pressure, Reconstituted RTE Meat Microbiota, and Antimicrobials on Survival and Post-pressure Growth of Listeria monocytogenes on Ham.

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 investigated the impact of pressure and RTE meat microbiota, with or without nisin and rosemary oil, on survival of Listeria after refrigerated storage. Ham was inoculated with a 5-strain cocktail of L. monocytogenes alone or with a cocktail of RTE meat microbiota consisting of Brochothrix thermosphacta, Carnobacterium maltaromaticum, Leuconostoc gelidum, and Lactobacillussakei. 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. After 4 weeks of storage, products were also analyzed by high throughput sequencing of 16S rRNA amplicons. Pressure treatment reduced counts of Listeria by 1 to 2 log (CFU/g); inactivation of RTE meat microbiota was comparable. Counts of Listeria increased by 1-3 log (CFU/g) during refrigerated storage. RTE meat microbiota did not influence pressure inactivation of Listeria but prevented growth of Listeria during refrigerated storage. Rosemary extract did not influence bacterial inactivation or growth. 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 alone does not eliminate Listeria or other microbiota on RTE ham; however, the presence of non-pathogenic microbiota prevents growth of Listeria on pressure treated ham and has a decisive influence on post-pressure survival and growth.

Monitoring food pathogens: Novel instrumentation for cassette PCR testing.

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.

Frequent Implication of Multistress-Tolerant Campylobacter jejuni in Human Infections.

Campylobacter jejuni, a major cause of bacterial foodborne illnesses, is considered highly susceptible to environmental stresses. In this study, we extensively investigated the stress tolerance of 121 clinical strains of C. jejuni against 5 stress conditions (aerobic stress, disinfectant exposure, freeze-thaw, heat treatment, and osmotic stress) that this pathogenic bacterium might encounter during foodborne transmission to humans. In contrast to our current perception about high stress sensitivity of C. jejuni, a number of clinical strains of C. jejuni were highly tolerant to multiple stresses. We performed population genetics analysis by using comparative genomic fingerprinting and showed that multistress-tolerant strains of C. jejuni constituted distinct clades. The comparative genomic fingerprinting subtypes belonging to multistress-tolerant clades were more frequently implicated in human infections than those in stress-sensitive clades. We identified unique stress-tolerant C. jejuni clones and showed the role of stress tolerance in human campylobacteriosis.