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Recent outbreaks traced to contaminated flour have created a need in the milling industry for a process that reduces pathogens in wheat while maintaining its functional properties. Vacuum steam treatment is a promising technology for treatment of low-moisture foods. Traditional thermal treatment methods can compromise wheat functionality due to high temperatures; thus, maintaining the functional quality of the wheat protein was critical for this research. The objective of this study was to evaluate the effect of vacuum steam treatment of hard red spring (HRS) wheat kernels on final flour quality and the overall efficacy of vacuum stream treatment for reducing pathogens on HRS wheat kernels. HRS wheat samples were treated with steam under vacuum at 65, 70, 75, and 85°C for 4 and 8 min. Significant changes in dough and baked product functionality were observed for treatments at ≥70°C. Treatment time had no significant effect on the qualities evaluated. After determining that vacuum steam treatment at 65°C best preserved product quality, HRS wheat was inoculated with Escherichia coli O121 and Salmonella Enteritidis PT 30 and processed at 65°C for 0, 2, 4, 6, or 8 min. The treatments achieved a maximum average reduction of 3.57 ± 0.33 log CFU/g for E. coli O121 and 3.21 ± 0.27 log CFU/g for Salmonella. Vacuum steam treatment could be an effective pathogen inactivation method for the flour milling industry. HIGHLIGHTS

Purpose. Listeria monocytogenes is a foodborne pathogen that causes central nervous system (CNS) and maternal-neonatal (MN) infections, bacteremia (BAC), and gastroenteritis in humans and ruminants. Specific clonal complexes (CC) have been associated with severe listeriosis cases, however, less is known about differences among subgroup virulence patterns. This study aimed to assess variation in virulence across different CC and clinical outcomes. Methodology. Galleria mellonella larvae were used to compare virulence phenotypes of 34 L . monocytogenes strains representing isolates from CC1, CC6 (from lineage I), and CC7, CC9, CC14, CC37 and CC204 (from lineage II) classified by clinical outcome: BAC, CNS and MN infection. Larvae survival, LD50, cytotoxicity, health index scores and bacterial concentrations post-infection were evaluated as quantifiable indicators of virulence. Results. Isolates belonging to CC14 and MN-associated infections are hypervirulent in G. mellonella as they led to lower G. mellonella survival rates and health index scores, as well as reduced cytotoxic effects when compared to other CC and clinical outcomes included here. CC14 isolates also showed increased bacterial concentrations at 8 and 24 h post-infection, indicating ability to survive the initial immune response and proliferate within G. mellonella larvae. Conclusion. Subgroups of L. monocytogenes possess different virulence phenotypes that may be associated with niche-specificity. While hypervirulent clones have been identified so far in lineage I, our data demonstrate that hypervirulent clones are not restricted to lineage I, as CC14 belongs to lineage II. Identification of subgroups with a higher ability to cause disease may facilitate surveillance and management of listeriosis.

Manure runoff can transfer pathogens to farmlands or to water sources, leading to subsequent contamination of produce. Untreated biological soil amendments, like manure, can be contaminated with foodborne pathogens, such as Salmonella Newport, which may lead to transfer of the pathogen to fruits or vegetables. Studies have reported the occurrence and survival of Salmonella in manure or manure slurries. However, data on the survival and growth of Salmonella Newport is lacking in matrices simulating runoff. We quantified the survival and growth of wild-type (WT) Salmonella Newport and rpoS-deficient (Δ rpoS) strains in sterile and nonsterile soil extracts prepared with (amended) or without (unamended) heat-treated poultry pellets at 25°C. Salmonella Newport WT and Δ rpoS populations reached a maximum cell density of 6 to 8 log CFU/mL in 24 to 30 h in amended and unamended soil extracts and remained in stationary phase for up to 4 days. Salmonella Newport in amended soil extracts exhibited a decreased lag phase (λ , 2.87 ± 1.01 h) and greater maximum cell densities ( Nmax, 6.84 ± 1.25 CFU/mL) compared with λ (20.10 ± 9.53 h) and Nmax (5.22 ± 0.82 CFU/mL) in unamended soil extracts. In amended soil extract, the Δ rpoS strain had no measurable λ , similar growth rates (μmax) compared with WT, and a lower Nmax compared with the WT strain. Unamended, nonsterile soil extracts did not support the growth of Salmonella Newport WT and led to a decline in populations for the Δ rpoS strain. Salmonella Newport had lower cell densities in nonsterile soil extracts (5.94 ± 0.95 CFU/mL) than it did in sterile soil extracts (6.66 ± 1.50 CFU/mL), potentially indicating competition for nutrients between indigenous microbes and Salmonella Newport. The most favorable growth conditions were provided by amended sterile and nonsterile soil extracts, followed by sterile, unamended soil extracts for both Salmonella Newport strains. Salmonella Newport may grow to greater densities in amended extracts, providing a route for increased Salmonella levels in the growing environments of produce.

Wheat flour has been connected to outbreaks of foodborne illnesses with increased frequency in recent years, specifically, outbreaks involving Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC). However, there is little information regarding the survival of these pathogens on wheat grain during long-term storage in a low-moisture environment. This study aims to evaluate the long-term survival of these enteric pathogens on wheat grain over the course of a year. Hard red spring wheat was inoculated with strains of four serovars of Salmonella (Enteritidis, Agona, Tennessee, and Montevideo) and six serotypes of EHEC (O157:H7, O26:H11, O121:H19, O45:NM, O111:H8, and O103:H2) in triplicate, sealed in Mylar bags to maintain the water activity, and stored at room temperature (22 ± 1°C). The survival of each pathogen was evaluated by plating onto differential media. Viable counts of strains from all four serovars of Salmonella (Enteritidis, Agona, Tennessee, and Montevideo) were detected on wheat grain stored at room temperature (22 ± 1°C) for the duration of the study (52 weeks). Viable counts of strains from EHEC serotypes O45:NM, O111:H8, and O26:H11 were only detected for 44 weeks, and strains from serotypes O157:H7, O121:H19, and O103:H2 were only detected for 40 weeks until they passed below the limit of detection (2.0 log CFU/g). The D-values were found to be significantly different between Salmonella and EHEC (adjusted P ≤ 0.05) with Salmonella D-values ranging from 22.9 ± 2.2 weeks to 25.2 ± 1.0 weeks and EHEC D-values ranging from 11.4 ± 0.6 weeks to 13.1 ± 1.8 weeks. There were no significant differences among the four Salmonella strains or among the six EHEC strains (adjusted P > 0.05). These observations highlight the wide range of survival capabilities of enteric pathogens in a low-moisture environment and confirm these pathogens are a food safety concern when considering the long shelf life of wheat grain and its products. HIGHLIGHTS

Flaxseed is an oilseed with many health benefits. Flaxseed may be consumed raw or in processed form. In the raw form, there is a potential for microbial contamination. Several pasteurization methods have been used to reduce microbial contamination. However, such treatments may affect chemical properties of foods. In this study, vacuum steam‐pasteurization was conducted on whole flaxseed and milled flaxseed using 4 different conditions (3 min at 75 °C, 3 min at 90 °C, 9 min at 90 °C, and 3 min at 105 °C). Microbial and chemical shelf‐life was monitored for 28 wk (36 wk for aerobic plate counts). Significant reduction (P < 0.05) in microbial counts (total aerobic plate counts, and yeast and mold counts) occurred after pasteurization and during storage of both whole flaxseed and milled flaxseed. Although both the moisture content and aw increased after pasteurization, they were similar to the unpasteurized samples during storage. Peroxide value, free fatty acid, headspace volatiles, fatty acid profiles, oil content, and secoisolariciresinol diglucoside (SDG) content were chemical indices measured. Only small changes were observed in the chemical indices after vacuum steam‐pasteurization for both pasteurized whole flaxseed and milled flaxseed as compared to the unpasteurized flaxseed at most instances. Vacuum steam‐pasteurization can be used as a safe alternative for the microbial reduction of low‐moisture products, such as flaxseed, without significantly affecting chemical stability. PRACTICAL APPLICATION: Vacuum steam‐pasteurization can be effectively used for the treatment of whole flaxseed and milled flaxseed to reduce spoilage microorganisms, such as total aerobes and yeasts and molds. In addition, this pasteurization method had minimal effects on several chemical shelf‐life parameters with positive impact on SDG of the processed flaxseed.

Immunomagnetic separation used with culture based methods has been a useful technique in the detection of pathogens. However, previous studies have not answered many of the necessary questions for real world applications. The objective of this study was to assess the efficacy of different immunomagnetic separation (IMS) bead types in recovery of the correct serogroup from a mixture of big six non-O157 Shiga toxin-producing Escherichia coli strains. To determine the impact of different matrices on recovery, samples of sterile phosphate buffered saline (PBS), sterile and non-sterile cattle faeces, ground beef and lettuce were inoculated with 10 CFU per ml mixture of isolates representing the six serogroups. After a 6 h incubation at 37°C, samples were mixed with IMS beads from three different commercial sources and plated on eosin methylene blue agar (EMB). Three suspect E. coli colonies were selected from each EMB plate and multiplex polymerase chain reaction was used to determine the serogroup. The rate of correct identification varied with the serogroup, IMS bead manufacturer and matrix. Overall, recovery of the correct serogroup became less likely with increase in matrix complexity, with enrichments containing lettuce having the greatest number of bead types with significantly lower likelihood of correct recovery compared to recovery in PBS. Significance and Impact of the Study The need to accurately and efficiently detect Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121 and O145, which have caused outbreaks on numerous occasions, is a major public health and food safety concern in the United States. Detecting these STEC serogroups can be challenging because methods to detect non-O157 serogroups have not been refined as compared to those for O157. Immunomagnetic separation (IMS) has the potential to isolate STEC from a mixture in complex matrices. Our results highlight the need for optimization of IMS-based detection of STEC to effectively recover the targeted serogroup from a variety of sample matrices.

Low moisture foods such as nuts, spices, and seeds have been implicated in several outbreaks due to Salmonella or E. coli O157:H7 contamination. Such foods may be consumed raw, and can be used as ingredients in other food products. While numerous thermal inactivation studies have been conducted for Salmonella on nuts, studies on other seeds and grains are minimal. Product water activity can influence the thermal resistance of pathogens, where thermal resistance increases as water activity decreases, leading to a requirement for higher temperatures and longer exposure times to achieve significant reduction of pathogen numbers. Vacuum steam pasteurization uses steam under vacuum, which can be operated at temperatures above and below 100°C. The objective of this study was to determine the efficacy of vacuum steam pasteurization for inactivation of pathogens on whole flaxseed, quinoa, sunflower kernels, milled flaxseed and whole black peppercorns. The use of E. faecium as a potential surrogate for Salmonella and E. coli O157:H7 in vacuum steam pasteurization was also evaluated. Pasteurization for 1min at 75°C yielded average log reductions of 5.48±1.22, 5.71±0.40 and 5.23±0.61 on flaxseed, 4.29±0.92, 5.89±0.26 and 2.39±0.83 on quinoa, and 4.01±0.74, 5.40±0.83 and 2.99±0.92 on sunflower kernels for Salmonella PT 30, E. coli O157:H7 and E. faecium, respectively. Similarly, on milled flaxseed and black peppercorns average log reductions of 3.02±0.79 and 6.10±0.64CFU/g were observed for Salmonella PT 30 after 1min of treatment at 75°C but, on average, >6.0 log reductions were observed after pasteurization at 85°C. Our data demonstrate that vacuum steam pasteurization can be effectively used to reduce pathogens on these low moisture foods at temperature as low as 75 and 85°C, and that E. faecium may be used as a potential surrogate for Salmonella PT 30 and E. coli O157:H7.

AIMS Exposure of Listeria monocytogenes to osmotic stress can induce increased resistance to subsequent lethal exposure to cell envelope stressors, such as nisin and bile salts. We wanted to determine if similar cross-protection phenotypes could occur when L. monocytogenes strains were treated with osmotic stress and exposed to sublethal levels of the cell envelope stressor, bile. METHOD AND RESULTS Growth phenotypes were measured for six L. monocytogenes strains exposed to 6% NaCl, 0·3 and 1% bile in BHI. To evaluate cross-protection, cells were pre-exposed to 6% NaCl, followed by exposure to BHI+1% bile for 26 h and vice versa. Significant increases in λ (lag phase) and doubling time were observed under salt and bile stresses compared with BHI alone. Average λ and Nmax (maximum cell density) in 0·3 and 1% bile for all strains were significantly lower than that in 6% NaCl. Pre-exposure to 6% NaCl followed by exposure to 1% bile significantly increased λ (P

Untreated biological soil amendments of animal origin (BSAAO) are commonly used as biological fertilizers but can harbor foodborne pathogens like Salmonella enterica, leading to potential transfer from soils to fruits and vegetables intended for human consumption. Heat-treated poultry pellets (HTPP) can provide produce growers with a slow-release fertilizer with a minimized risk of pathogen contamination. Little is known about the impact of HTPP-amended soil on the survival of Salmonella enterica. The contributions of RpoS and formation of viable but nonculturable cells to Salmonella survival in soils are also inadequately understood. We quantified the survival of Salmonella enterica subsp. enterica serovar Newport wild-type (WT) and rpoS-deficient (ΔrpoS mutant) strains in HTPP-amended and unamended soil with or without spinach plants over 91 days using culture and quantitative PCR methods with propidium monoazide (PMA-qPCR). Simulated “splash” transfer of S. Newport from soil to spinach was evaluated at 35 and 63 days postinoculation (dpi). The S. Newport WT and ΔrpoS mutant reached the limit of detection, 1.0 log CFU/g (dry weight), in unamended soil after 35 days, whereas 2 to 4 log CFU/g (dry weight) was observed for both WT and ΔrpoS mutant strains at 91 dpi in HTPP-amended soil. S. Newport levels in soils determined by PMA-qPCR and plate count methods were similar (P > 0.05). HTPP-amended soils supported higher levels of S. Newport transfer to and survival on spinach leaves for longer periods of time than did unamended soils (P

Sublethal heating, which can occur during slow cooking of meat products, is known to induce increased thermal resistance in Salmonella. However, very few studies have addressed the kinetics of this response. Although several recent studies have reported improved thermal inactivation models that include the effect of prior sublethal history on subsequent thermal resistance, none of these models were based on cellular-level responses to sublethal thermal stress. The goal of this study was to determine whether a nonlinear model could accurately portray the response of Salmonella to heat stress induced by prolonged exposure to sublethal temperatures. To accomplish this, stationary-phase Salmonella Montevideo cultures were subjected to various heating profiles (held at either 40 or 45°C for 0, 5, 10, 15, 30, 60, 90, 180, or 240 min) using a PCR thermal cycler. Differential plating on selective and nonselective media was used to confirm the presence of cellular injury. Reverse transcription quantitative PCR was used to screen the transcript levels of six heat stress-related genes to find candidate genes for nonlinear modeling. Injury was detected in populations of Salmonella held at 45°C for 30, 60, and 90 min and at 40°C for 0, 5, and 90 min (P0.05), whereas no significant injury was found at 180 and 240 min (P0.05). The transcript levels of ibpA, which codes for a small heat shock protein associated with the ClpB and DnaK-DnaJ-GrpE chaperone systems, showed the greatest increase relative to the transcript levels at 0 min, which was significant at 5, 10, 15, 30, 60, 90, and 180 min at 45°C and at 5, 10, 15, 30, 60, and 90 min at 40°C (P0.05). Using ibpA transcript levels as an indicator of adaptation to thermal stress, a nonlinear model for sublethal injury is proposed. The use of variables indicating the physiological state of the pathogen during stress has the potential to increase the accuracy of thermal inactivation models that must account for prolonged exposure to sublethal temperatures.

For the last few decades, the application of biopolymers such as poly(lactic acid, PLA) as an alternative to synthetic polymers in the packaging industry is in great demand. However, to rival the traditional polymers, the barrier properties and antimicrobial activities of PLA need to be adapted to the food packaging requirements. The purpose of this research was to compare the impact of thin-film fabrication techniques in combination with different contents of cellulose nanocrystals (CNCs) (1–5 wt%) on the barrier and antimicrobial properties of PLA films. The nanocomposite thin films were manufactured via solvent casting and spin-coating techniques. The spin-coating method has been employed for the first time in this work to prepare nanocomposite thin films with a thickness of ˂ 0.2 mm. The nanocomposite films were tested for surface morphology, degree of crystallinity, water vapor permeability, contact angle, and antimicrobial properties. Scanning electron microscopy (SEM) micrographs revealed the strong tendency of CNCs to establish microscale aggregates, however, a high drying rate in the spin-coating method decreases the self-assembly in CNCs. Thus, a significantly higher degree of crystallinity and glass transition temperature were observed in spin-coated films due to a more uniform dispersion of CNCs in PLA. Higher contact angle and lower water vapor transfer rate in spin-coated samples confirmed the lower hydrophilicity character of spin-coated thin films. Two separate assays were employed for evaluating the anti-microbial activity of PLA on the foodborne pathogens. The results showed that the film manufacturing technique and CNC concentration have a strong influence on microbial reduction.

Thermal resistance among Salmonella serovars has been shown to vary, however, such data are minimal for Salmonella inoculated onto low moisture foods. We evaluated survival and subsequent thermal resistance for 32 strains of Salmonella from four serovars (Agona, Enteritidis, Montevideo, and Tennessee) on flaxseed over 24 weeks. After inoculation, flaxseeds were adjusted to aw = 0.5 and stored at 22 °C. After 24 weeks at 22 °C, strains of serovar Agona had a significantly slower rate of reduction compared to those of Enteritidis and Montevideo (adj. p

One strategy bacteria use to acclimate to changing environmental conditions is modulation of gene expression. Alterations in gene expression are indicative of activation or repression of certain physiological responses. In order to understand which genetic responses are utilized to cope with various environmental conditions by analyzing transcriptomes, obtaining RNA of high quality, yield, and integrity is paramount. Here, we describe an acid phenol-chloroform method employed to extract RNA from laboratory grown cell cultures, as well as cultures inoculated onto complex matrices such as lettuce and cold-smoked salmon. The method results in high-quality RNA, which can be used for various downstream processes such as cDNA library construction, RNA sequencing, real-time quantitative PCR, and northern analysis. Extraction of RNA from bacterial foodborne pathogens in conjunction with transcriptome sequencing is a useful technique to elucidate pathogens' transcriptional responses.

One strategy bacteria use to acclimate to changing environmental conditions is modulation of gene expression. Alterations in gene expression are indicative of activation or repression of certain physiological responses. In order to understand which genetic responses are utilized to cope with various environmental conditions by analyzing transcriptomes, obtaining RNA of high quality, yield, and integrity is paramount. Here, we describe an acid phenol-chloroform method employed to extract RNA from laboratory grown cell cultures, as well as cultures inoculated onto complex matrices such as lettuce and cold-smoked salmon. The method results in high-quality RNA, which can be used for various downstream processes such as cDNA library construction, RNA sequencing, real-time quantitative PCR, and northern analysis. Extraction of RNA from bacterial foodborne pathogens in conjunction with transcriptome sequencing is a useful technique to elucidate pathogens' transcriptional responses.

Aims Listeria monocytogenes nisin resistance increases when first exposed to NaCl and other stresses, such as low pH. In addition to environmental stressors, specific genomic elements can confer nisin resistance, such as the stress survival islet (SSI-1). As SSI-1 is variably present among L. monocytogenes strains, we wanted to determine if SSI-1 was associated with salt-induced nisin resistance. Methods and Results The presence of SSI-1 was determined using PCR for 48 strains of L. monocytogenes. When combined with multi-locus sequence typing data, we found that the distribution of SSI-1 is clonal, where strains from clonal complexes (CC) 2, 6 and 11 do not have SSI-1, while strains from CCs 3, 5, 7 and 9 contain SSI-1. The impact of SSI-1 on salt-induced nisin resistance was dependent on CC. The average log decrease after 24 h exposure to nisin at 7°C under salt-inducing conditions was 2.6 ± 1.1 for CC 9 strains and 2.3 ± 0.7 for CC 11 strains, which was significantly lower survival compared to the other CCs, such as 1.3 ± 0.3 for CC 6. Deletion of SSI-1 from a CC 7 strain demonstrated the role SSI-1 plays in salt-induced nisin resistance, as the deletion mutant had lower resistance compared to the parent strain. Conclusions These data suggest that inducible nisin resistance in L. monocytogenes can be influenced by environmental conditions as well as the genetic composition of the strain, which should be considered when selecting control measures for ready-to-eat foods. Significance and impact of the study The foodborne pathogen L. monocytogenes can grow in suboptimal conditions, including low temperature and high osmolarity, which makes it a safety concern for ready-to-eat foods. When using antimicrobial peptide inhibitors such as nisin, it is important to understand how food components can impact antimicrobial resistance across the genetic diversity of L. monocytogenes. This article is protected by copyright. All rights reserved.

Contamination of cold-smoked salmon by Listeria monocytogenes is a major concern for the seafood industry. The objectives of this study were to (i) determine the most effective bactericidal treatment for L. monocytogenes on salmon and (ii) optimize bactericidal and bacteriostatic treatment combinations to identify cost-effective treatments against L. monocytogenes on salmon. L. monocytogenes challenge trials were conducted in brain heart infusion (BHI) and on salmon disks that were supplemented with bactericidal compounds nisin (NIS), lauric arginate (LAE), e-polylysine (EPL), and chitosan (CHIT). Subsequently, the most effective bactericidal compound was further tested by concurrent application of a blend of organic acid salts containing potassium lactate and sodium diacetate (PLSDA). L. monocytogenes populations were measured at 7 °C over 60 days, and initial cell density ( N 0 ), maximum initial log reduction ( N r ), lag phase (λ), maximum growth rate (μ max ), and maximum cell density ( N max ) over 60 days storage were estimated. Time to recover to initial cell density ( T initial ) was also compared for combinations of bactericidal and bacteriostatic treatments. Varying degrees of antimicrobial effects were observed with bactericidal compounds in BHI. However, when tested on salmon, only NIS significantly decreased initial L. monocytogenes populations by approximately 2 log CFU/g, and reduced N max by approximately 1.5 log CFU/g compared to untreated control (CTRL). N r achieved by the combined treatment of NIS and PLSDA was approximately 2 log CFU/g regardless of the presence of PLSDA, and a dose-dependent increase in N r was observed with increasing NIS concentrations. PLSDA alone or in combination with 20 ppm NIS was most effective at delaying growth of L. monocytogenes . The greatest reduction in N max was observed with the combination of 20 ppm NIS and PLSDA; N max was 3.1 log CFU/g lower compared to CTRL. Comparison of T initial indicated that PLSDA with NIS can effectively retard growth of L. monocytogenes to its initial level (following initial reduction) and offers a cost benefit over using high concentrations of NIS alone. In summary, the combined application of NIS (for a bactericidal effect) and PLSDA (for a bacteriostatic effect) proved to be an effective treatment option to reduce initial levels as well as minimize subsequent growth of L. monocytogenes throughout the expected shelf-life of cold-smoked salmon.

Genomics, transcriptomics, and proteomics are rapidly transforming our approaches to the detection, prevention, and treatment of foodborne pathogens. Microbial genome sequencing in particular has evolved from a research tool into an approach that can be used to characterize foodborne pathogen isolates as part of routine surveillance systems. Genome sequencing efforts will not only improve outbreak detection and source tracking, but will also create large amounts of foodborne pathogen genome sequence data, which will be available for data-mining efforts that could facilitate better source attribution and provide new insights into foodborne pathogen biology and transmission. Although practical uses and application of metagenomics, transcriptomics, and proteomics data and associated tools are less prominent, these tools are also starting to yield practical food safety solutions.

In the field, foodborne pathogens such as enterohemorrhagic Escherichia coli (EHEC) are capable of surviving on produce over time, yet little is known about how these pathogens adapt to this environment. To assess the impact of pre-harvest environmental conditions on EHEC survival, we quantified survival on romaine lettuce under two relative humidity (75% and 45%) and seasonal conditions (March and June). Greenhouse-grown lettuce was spray-inoculated with EHEC and placed in a growth chamber, mimicking conditions typical for June and March in Salinas Valley, California. Bacteria were enumerated on days 0, 1, 3, and 5 post-inoculation. Overall, we found that the effect of relative humidity on EHEC survival depended on the seasonal conditions. Under June seasonal conditions, higher relative humidity led to lower survival, and lower relative humidity led to greater survival, five days post-inoculation. Under March seasonal conditions, the impact of relative humidity on EHEC survival was minimal over the five days. The bacteria were also tested for their ability to survive a chlorine decontamination wash. Inoculated lettuce was incubated under the June 75% relative humidity conditions and then washed with a 50 ppm sodium hypochlorite solution (40 ppm free chlorine). When incubated under June seasonal conditions for three to five days, EHEC strains showed increased tolerance to chlorine (adj. p &lt, 0.05) compared to chlorine tolerance upon inoculation onto lettuce. This indicated that longer incubation on lettuce led to greater EHEC survival upon exposure to chlorine. Subsequent transcriptome analysis identified the upregulation of osmotic and oxidative stress response genes by EHEC after three and five days of incubation on pre-harvest lettuce. Assessing the physiological changes in EHEC that occur during association with pre-harvest lettuce is important for understanding how changing tolerance to post-harvest control measures may occur.

Growth of Listeria monocytogenes on refrigerated, ready-to-eat food is a significant food safety concern. Natural antimicrobials, such as nisin, can be used to control this pathogen on food, but little is known about how other food-related stresses may impact how the pathogen responds to these compounds. Prior work demonstrated that exposure of L. monocytogenes to salt stress at 7°C led to increased expression of genes involved in nisin resistance, including the response regulator liaR . We hypothesized that exposure to salt stress would increase subsequent resistance to nisin and that LiaR would contribute to increased nisin resistance. Isogenic deletion mutations in liaR were constructed in 7 strains of L. monocytogenes , and strains were exposed to 6% NaCl in brain heart infusion broth and then tested for resistance to nisin (2 mg/ml Nisaplin) at 7°C. For the wild-type strains, exposure to salt significantly increased subsequent nisin resistance ( P < 0.0001) over innate levels of resistance. Compared to the salt-induced nisin resistance of wild-type strains, Δ liaR strains were significantly more sensitive to nisin ( P < 0.001), indicating that induction of LiaFSR led to cross-protection of L. monocytogenes against subsequent inactivation by nisin. Transcript levels of LiaR-regulated genes were induced by salt stress, and lmo1746 and telA were found to contribute to LiaR-mediated salt-induced nisin resistance. These data suggest that environmental stresses similar to those on foods can influence the resistance of L. monocytogenes to antimicrobials such as nisin, and potential cross-protective effects should be considered when selecting and applying control measures for this pathogen on ready-to-eat foods.

We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes serotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or “epidemic clones” that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread in L. monocytogenes and is not limited to the recognized epidemic clones. Pairwise comparisons between epidemiologically related isolates within clonal complexes showed that genome-level variation differed by 2 orders of magnitude between different comparisons, and the distribution of point mutations (core versus accessory genome) also varied. In addition, genetic divergence between one closely related pair of isolates from a single outbreak was driven primarily by changes in phage regions. The evolutionary analysis showed that the changes could be attributed to horizontal gene transfer; members of the diverse bacterial community found in the production facility could have served as the source of novel genetic material at some point in the production chain. The results raise the question of how to best utilize information contained within the accessory genome in outbreak investigations. The full magnitude and complexity of genetic changes revealed by genome sequencing could not be discerned from traditional subtyping methods, and the results demonstrate the challenges of interpreting genetic variation among isolates recovered from a single outbreak. Epidemiological information remains critical for proper interpretation of nucleotide and structural diversity among isolates recovered during outbreaks and will remain so until we understand more about how various population histories influence genetic variation.

A set of seven Listeria monocytogenes 10403S mutant strains, each bearing an in-frame null mutation in a gene encoding a key regulatory protein, was used to characterize transcriptional networks in L. monocytogenes ; the seven regulatory proteins addressed include all four L. monocytogenes alternative sigma factors (σ B , σ C , σ H , and σ L ), the virulence gene regulator PrfA, and the heat shock-related negative regulators CtsR and HrcA. Whole-genome microarray analyses, used to identify regulons for each of these 7 transcriptional regulators, showed considerable overlap among regulons. Among 188 genes controlled by more than one regulator, 176 were coregulated by σ B , including 92 genes regulated by both σ B and σ H (with 18 of these genes coregulated by σ B , σ H , and at least one additional regulator) and 31 genes regulated by both σ B and σ L (with 10 of these genes coregulated by σ B , σ L , and at least one additional regulator). Comparative phenotypic characterization measuring acid resistance, heat resistance, intracellular growth in J774 cells, invasion into Caco-2 epithelial cells, and virulence in the guinea pig model indicated contributions of (i) σ B to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, σ B , and CtsR to virulence-associated characteristics. Loss of the remaining transcriptional regulators (i.e., sigH , sigL , or sigC ) resulted in limited phenotypic consequences associated with stress survival and virulence. Identification of overlaps among the regulons provides strong evidence supporting the existence of complex regulatory networks that appear to provide the cell with regulatory redundancies, along with the ability to fine-tune gene expression in response to rapidly changing environmental conditions.

Escherichia coli O157:H7 has caused serious outbreaks of food-borne illness via transmission in a variety of food vehicles, including unpasteurized apple juice, dried salami, and spinach. To understand how this pathogen responds to the multiple stresses of the food environment, we compared global transcription patterns before and after exposure to model apple juice. Transcriptomes of mid-exponential- and stationary-phase cells were evaluated after 10 min in model apple juice (pH 3.5) using microarrays probing 4,886 open reading frames. A total of 331 genes were significantly induced upon exposure of cells to model apple juice, including genes involved in the acid, osmotic, and oxidative stress responses as well as the envelope stress response. Acid and osmotic stress response genes, including asr, osmC, osmB , and osmY , were significantly induced in response to model apple juice. Multiple envelope stress responses were activated as evidenced by increased expression of CpxR and Rcs phosphorelay-controlled genes. Genes controlled by CpxR ( cpxP, degP , and htpX ) were significantly induced 2- to 15-fold upon exposure to apple juice. Inactivation of CpxRA resulted in a significant decrease in survival of O157:H7 in model apple juice compared to the isogenic parent strain. Of the 331 genes induced in model apple juice, 104 are O157-specific genes, including those encoding type three secretion effectors ( espJ, espB, espM2, espL3 , and espZ ). Elucidating the response of O157:H7 to acidic foods provides insight into how this pathogen is able to survive in food matrices and how exposure to foods influences subsequent transmission and virulence.

Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of the glutamate decarboxylase (GAD) system, the most efficient acid resistance (AR) mechanism in E. coli . The full contribution of GadE to the AR and virulence of E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles of the mutant with that of the wild type in the exponential and stationary phases of growth. Inactivation of gadE significantly altered the expression of 60 genes independently of the growth phase and of 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly downregulated the expression of gadA, gadB , and gadC and of many acid fitness island genes. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler , showed a significant increase in expression upon gadE inactivation. Inactivation of ler in the Δ gadE strain reversed the effect of gadE deletion on LEE expression, indicating that Ler is necessary for LEE repression by GadE. GadE is also involved in downregulation of LEE expression under conditions of moderately acidic pH. Characterization of AR of the Δ gadE strain revealed that GadE is indispensable for a functional GAD system and for survival of E. coli O157:H7 in a simulated gastric environment. Altogether, these data indicate that GadE is critical for the AR of E. coli O157:H7 and that it plays an important role in virulence by downregulating expression of LEE.

The foodborne pathogen Listeria monocytogenes is able to survive and grow in ready-to-eat foods, in which it is likely to experience a number of environmental stresses due to refrigerated storage and the physicochemical properties of the food. Little is known about the specific molecular mechanisms underlying survival and growth of L. monocytogenes under different complex conditions on/in specific food matrices. Transcriptome sequencing (RNA-seq) was used to understand the transcriptional landscape of L. monocytogenes strain H7858 grown on cold smoked salmon (CSS; water phase salt, 4.65%; pH 6.1) relative to that in modified brain heart infusion broth (MBHIB; water phase salt, 4.65%; pH 6.1) at 7°C. Significant differential transcription of 149 genes was observed (false-discovery rate [FDR], L. monocytogenes were not significantly different between CSS and MBHIB, indicating that the transcriptomic differences reflect how L. monocytogenes is able to facilitate growth under these different conditions. Differential expression analysis and Gene Ontology enrichment analysis indicated that genes encoding proteins involved in cobalamin biosynthesis as well as ethanolamine and 1,2-propanediol utilization have significantly higher transcript levels in H7858 grown on CSS than in that grown in MBHIB. Our data identify specific transcriptional profiles of L. monocytogenes growing on vacuum-packaged CSS, which may provide targets for the development of novel and improved strategies to control L. monocytogenes growth on this ready-to-eat food.

The objective of this study was to assess the genetic diversity of non-O157Shiga toxin-producing Escherichia coli (STEC) isolates from cattle. Multi-locussequence typing (MLST) was used to identify and compare the sequence types(STs) of 43 non-O157 STEC cattle isolates using the EcMLST database curatedby the STEC Center at Michigan State University. For the 43 isolates, 19 STswere identified and 10 of those STs were novel compared to those in EcMLST.For the 43 isolates, 19 different serotypes were identified. STEC O22:H8, O174:H28 and O8:H19 were most common, and STEC O8 isolates were the mostdiverse, with seven different STs for isolates with that O group. STEC strainswith O types identified in this study have been isolated from cattle by otherresearchers, as well as from cases of human gastroenteritis. Of the 10 novel STsidentified, six were found to be closely related to previously identified STs,indicating that populations of non-O157 STEC in cattle are similar to thosefrom other sources, including human clinical cases.IntroductionShiga toxin-producing Escherichia coli (STEC), alsoknown as Vero toxin-producing E. coli (VTEC), arehuman pathogens of zoonotic origin which have beenlinked to serious disease such as haemorrhagic colitis andhaemolytic uremic syndrome (Smith et al. 2014). Over400 serotypes of STEC/VTEC are identified to be patho-genic due to the presence of one or more genes encodingShiga toxin (Blanco et al. 2004a,b; Brooks et al. 2005;Hussein 2007; Tozzoli and Scheutz 2014). The most stud-ied serotype of STEC to date has been O157:H7, due tolarge outbreaks in the 1980s and 1990s associated withstrains of this serotype (Goldwater and Bettelheim 1998).The importance of non-O157 STEC serotypes has beenincreasingly recognized, and the most recent estimatesfrom the US Centers for Disease Control and Preventionindicate that non-O157 STEC are responsible for themajority of human illness caused by STEC (Scallan et al.2011). Surveillance data of foodborne outbreaks in theUnited States from 1998 to 2008 report that 308 out-breaks during this period were linked to STEC, and ofthose outbreaks that could be attributed to a specific foodsource, 78 were linked to beef (Gould et al. 2013).Cattle are considered to be the main reservoir of STEC,and either directly or indirectly contribute to the presenceof STEC in the food supply. The prevalence of STECO157 in cattle can range from 0 3to27 3%, and dependson a number of factors such as feed type and season(Hussein 2007). The prevalence of non-O157 STEC in

This chapter focuses on the application of molecular subtyping methods for identifying and tracing sources of microbial pathogens in food and food-borne human diseases. It should be noted, however, that in addition to the food-borne pathogens, harmless or nonpathogenic microorganisms can also contaminate the food supply and contribute to spoilage. These microbes are generally part of the natural microbiota of raw foods and food products, which serves as an ideal growth medium for many organisms with nearly neutral pH, high water activity, and richness in nutrients. If left unchecked, growth of these harmless microorganisms on foods results in off odors and flavors and, ultimately, in an inedible food product. Many of the methods developed to inhibit the growth of nonpathogenic microbes in foods also restrict the growth of food-borne pathogens. The major advantage of pulsed-field gel electrophoresis (PFGE) profiling is that the technique can be widely applied to different bacterial species and is relatively simple and straightforward to perform in the laboratory. Outbreak investigation and epidemiological tracing have been the primary incentives encouraging the development of the PulseNet and FoodNet national systems for food-borne disease surveillance. In the future, the authors expect to see the application of new molecular genotyping systems with rapid throughput, such as those based on single-nucleotide polymorphisms, to generate high-quality genotypic data for microbial source tracking.

Tom Whittam's pioneering research into the structure of Escherichia coli populations not only advanced the field of microbial evolution and genetics, but it also provided a contextual framework for investigating variation in the epidemiology and virulence of bacterial populations. This chapter highlights the work of his laboratory involving the use of various population genetic strategies for characterizing E. coli pathotypes, particularly enterohemorrhagic E. coli (EHEC) O157:H7, while focusing on contributions to epidemiology and public health. To determine whether specific genotypes are associated with human enteric disease, multilocus enzyme electrophoresis (MLEE) was used to examine 1,300 E. coli strains representing 16 serotypes, including EHEC O157:H7 from patients with hemorrhagic colitis and hemolytic uremic syndrome (HUS). Similar to MLEE and multilocus sequence typing (MLST) data, systematic analysis of single nucleotide polymorphisms (SNPs) is amenable to both population genetic and phylogenetic analyses. SNPs data can also be used to examine epidemiological associations between bacterial genotypes and clinical disease. By using the SNP genotyping phylogeny as a framework, it is possible to investigate virulence gene diversity, allelic variation, and gene expression differences among genotypes to identify bacterial genomic determinants of colonization, pathogenesis, and transmissibility. The chapter describes differences in stress resistance properties among clades, nonrandom distribution of stx variants, variation in adherence to epithelial cells, and differential expression among shared genes.

Listeria monocytogenes is well known to survive and grow under several stress conditions, including salt stress, which is important for growth in certain foods as well as for host infection. To characterize the contributions, to salt stress response, of transcriptional regulators important for stress response and virulence (i.e., σ(B) and PrfA), we analyzed three L. monocytogenes parent strains and isogenic mutants (ΔsigB, ΔprfA, and ΔsigBΔprfA), representing different serotypes and lineages, for their ability to grow, at 25 °C, in BHI with 1.9 M NaCl. With regard to growth rate, only the lineage IV strain presented a significant difference between the parent strain and both of its respective mutants lacking prfA (ΔprfA and ΔsigBΔprfA). Conversely, the lineage I and II parent strains showed significantly shorter lag phase in comparison to their respective ΔsigB mutant strains. Intestinal epithelial cell invasion assay and hemolytic activity assays showed a significant role for σ(B) in the former and for PrfA in the latter. To explore the mechanism that may contribute to the extended lag phase in the ΔsigB mutant strain and survival and growth of the parent strain upon salt shock, whole genome transcription profiling was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic ΔsigB mutant after 30 min of lag phase growth at 25 °C in the presence of 1.9 M NaCl (salt shock) without aeration. Microarray data showed significantly higher transcript levels for 173 genes in the parent strain as compared to the ΔsigB strain. Overall, 102 of the 173 σ(B) up-regulated genes had been identified in previous studies, indicating that 71 genes were newly identified as being up-regulated by σ(B) in this study. We hypothesize that, among these genes newly identified as σ(B) up-regulated, four genes (lmo2174, lmo0530, lmo0527 and lmo0529) may play a major role in response to salt stress. Lmo2174 contains domains that facilitate sensing and producing a transduction signal in the form of cyclic di-GMP, which may activate the enzymes Lmo0527, Lmo0529 and Lmo0530, which encode proteins similar to those responsible for synthesis of exopolysaccharides that may protect the cell by changing the cell wall structure during salt stress. Overall, our data showed that σ(B), but not PrfA, contributes to growth under salt stress. Moreover, we show that the σ(B) regulon of a L. monocytogenes lineage I strain challenged with salt shock includes salt stress-specific as well as previously unidentified σ(B) up-regulated genes.

Listeria monocytogenes is of particular concern in cold-smoked fish products as it can survive curing and cold-smoking, and can subsequently grow from low numbers to potentially hazardous levels during refrigerated storage. The purpose of this study was to (i) quantify the effects of organic acids, nisin, and their combinations on controlling L. monocytogenes growth on cold-smoked salmon at refrigeration temperatures, (ii) identify synergistic interactions of binary combinations of these antimicrobials, and (iii) determine if results from laboratory growth media can predict antimicrobial efficacy on cold-smoked salmon. Strains representing the genetic diversity of L. monocytogenes lineages I and II were grown in brain heart infusion (BHI) broth as well as on the surface of commercially produced wet-cured, cold-smoked salmon slices at 7 °C. BHI broth and cold-smoked salmon were supplemented with sodium diacetate (SDA, 0.14% water phase (w.p.)), potassium lactate (PL, 2% w.p.), nisin (NI, 50 ppm), and binary combinations of inhibitors at the same levels. Cell densities of L. monocytogenes were measured over time and used to calculate growth parameters, including initial cell density ( N 0 ), lag phase (λ), maximum growth rate (μ max ), and maximum cell density ( N max ) for each antimicrobial treatment. N 0 was significantly lowered by addition of NI with a similar average reduction on salmon (2.02 ± 0.99 log(CFU/g)) and in BHI (1.51 ± 0.83 log(CFU/ml)). Among all antimicrobial treatments, the combination of PL and SDA led to the greatest increase in λ both on salmon (7.1 ± 3.6 days) and in BHI (9.7 ± 3.8 days) when compared to the controls. The combination of PL and SDA had synergistic effects on increasing λ and lowering N max both in BHI and on salmon. Among all the treatments tested, the combination of NI and PL led to the greatest reductions in N max on salmon. We observed positive correlations between the growth parameters obtained from BHI broth and cold-smoked salmon, indicating that growth of L. monocytogenes in broth, to some extent, qualitatively reflected characteristics of growth on cold-smoked salmon under antimicrobial stresses. Results from BHI could quantitatively predict the variability of growth parameters obtained from salmon for lineage II strains, but not for lineage I strains. Although results from laboratory growth medium may not provide exact predictions of antimicrobial efficacy on cold-smoked salmon, they could be used to rapidly identify effective combinations for further examination on cold-smoked salmon.

The presence of the foodborne pathogen Listeria monocytogenes on cold-smoked salmon is a major concern for the seafood industry. Understanding processing and postprocessing handling factors that affect the ability of this pathogen to grow on cold-smoked salmon is critical for developing effective control strategies. In this study, we investigated the effect of curing method and freeze-thawing of cold-smoked salmon on (i) physicochemical properties and (ii) subsequent growth of genetically diverse strains of L. monocytogenes (inoculated after freeze-thawing) and endogenous lactic acid bacteria. The majority of the measured physicochemical properties were unaffected by freezing and thawing. Overall, wet-cured cold-smoked salmon had higher pH, water activity, and moisture, as well as lower fat, water-phase salt, and phenolic content compared with dry-cured cold-smoked salmon. The curing method and freeze-thawing did not affect growth of endogenous lactic acid bacteria. Freeze-thawing cold-smoked salmon prior to inoculation led to pronounced growth of L. monocytogenes at 7°C. The increase in cell density between days 0 and 30 was significantly (P = 0.0078) greater for cold-smoked salmon that was frozen and thawed prior to inoculation compared with nonfrozen cold-smoked salmon. On dry-cured, freeze-thawed cold-smoked salmon, L. monocytogenes had a lag phase ranging from 3.7 ± 0.1 to 11.2 ± 1.4 days compared with salmon that was wet cured and freeze-thawed, on which L. monocytogenes began to grow within 24 h. Variation in growth among L. monocytogenes strains was also observed, indicating the significance of assessing multiple strains. Further efforts to understand the impact of processing and postprocessing handling steps of cold-smoked salmon on the growth of genetically diverse L. monocytogenes will contribute to improved challenge study designs and data. This, in turn, will likely lead to more reliable and unbiased risk assessments and control measures.

The food-borne pathogen Listeria monocytogenes experiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. During transmission, L. monocytogenes is likely to experience osmotic stress at different temperatures and may adapt to osmotic stress in a temperature-dependent manner. To understand the impact of temperature on the responses this pathogen uses to adapt to osmotic stress, we assessed genome-wide changes in the L. monocytogenes H7858 transcriptome during short-term and long-term adaptation to salt stress at 7°C and 37°C. At both temperatures, the short-term response to salt stress included increased transcript levels of sigB and SigB-regulated genes, as well as mrpABCDEFG , encoding a sodium/proton antiporter. This antiporter was found to play a role in adaptation to salt stress at both temperatures; Δ mrpABCDEFG had a significantly longer lag phase than the parent strain in BHI plus 6% NaCl at 7°C and 37°C. The short-term adaptation to salt stress at 7°C included increased transcript levels of two genes encoding carboxypeptidases that modify peptidoglycan. These carboxypeptidases play a role in the short-term adaptation to salt stress only at 7°C, where the deletion mutants had significantly different lag phases than the parent strain. Changes in the transcriptome at both temperatures suggested that exposure to salt stress could provide cross-protection to other stresses, including peroxide stress. Short-term exposure to salt stress significantly increased H 2 O 2 resistance at both temperatures. These results provide information for the development of knowledge-based intervention methods against this pathogen, as well as provide insight into potential mechanisms of cross-protection.

The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic ability to inhibit the growth of Listeria monocytogenes . Full-genome microarrays were used to investigate the synergistic transcriptomic responses of two L. monocytogenes strains, H7858 (serotype 4b) and F6854 (serotype 1/2a), to these two organic acids under conditions representing osmotic and cold stress encountered in foods. Strains were exposed to brain heart infusion (BHI) broth at 7°C with 4.65% water-phase (w.p.) NaCl at pH 6.1 with (i) 2% w.p. potassium lactate, (ii) 0.14% w.p. sodium diacetate, (iii) the combination of both at the same levels, or (iv) no organic acids as a control. RNA was extracted 8 h after exposure, during lag phase, to capture gene transcription changes during adaptation to the organic acid stress. Significant differential transcription of 1,041 genes in H7858 and 640 genes in F6854 was observed in at least one pair of the 4 different treatments. The effects of combined treatment with lactate and diacetate included (i) synergistic transcription differences for 474 and 209 genes in H7858 and F6854, respectively, (ii) differential transcription of genes encoding cation transporters and ABC transporters of metals, and (iii) altered metabolism, including induction of a nutrient-limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional treatments that interfere with cellular energy generation processes could more efficiently inhibit the growth of L. monocytogenes .

In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for efficient binding to its target. In the present study, we sought to gain more insight into the functional role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment, and we provide evidence that both LhrA and Hfq act to down-regulate the expression of lmo0302 and chiA. Furthermore, in vitro binding experiments show that Hfq stimulates the base pairing of LhrA to chiA mRNA. Finally, we demonstrate that LhrA has a negative effect on the chitinolytic activity of L. monocytogenes. In marked contrast to this, we found that Hfq has a stimulating effect on the chitinolytic activity, suggesting that Hfq plays multiple roles in the complex regulatory pathways controlling the chitinases of L. monocytogenes.

A set of seven Listeria monocytogenes 10403S mutant strains, each bearing an in-frame null mutation in a gene encoding a key regulatory protein, was used to characterize transcriptional networks in L. monocytogenes; the seven regulatory proteins addressed include all four L. monocytogenes alternative sigma factors (σ(B), σ(C), σ(H), and σ(L)), the virulence gene regulator PrfA, and the heat shock-related negative regulators CtsR and HrcA. Whole-genome microarray analyses, used to identify regulons for each of these 7 transcriptional regulators, showed considerable overlap among regulons. Among 188 genes controlled by more than one regulator, 176 were coregulated by σ(B), including 92 genes regulated by both σ(B) and σ(H) (with 18 of these genes coregulated by σ(B), σ(H), and at least one additional regulator) and 31 genes regulated by both σ(B) and σ(L) (with 10 of these genes coregulated by σ(B), σ(L), and at least one additional regulator). Comparative phenotypic characterization measuring acid resistance, heat resistance, intracellular growth in J774 cells, invasion into Caco-2 epithelial cells, and virulence in the guinea pig model indicated contributions of (i) σ(B) to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, σ(B), and CtsR to virulence-associated characteristics. Loss of the remaining transcriptional regulators (i.e., sigH, sigL, or sigC) resulted in limited phenotypic consequences associated with stress survival and virulence. Identification of overlaps among the regulons provides strong evidence supporting the existence of complex regulatory networks that appear to provide the cell with regulatory redundancies, along with the ability to fine-tune gene expression in response to rapidly changing environmental conditions.

Listeria monocytogenes can survive and grow under wide-ranging environmental stress conditions encountered both in foods and in the host. The ability of certain L. monocytogenes subtypes to thrive under stress conditions present in specific niches was hypothesized to reflect genetic characteristics and phenotypic capabilities conserved among strains within a subtype. To quantify variations in salt stress phenotypes among 40 strains selected to represent the diversity of the three major L. monocytogenes genetic lineages and to determine if salt stress phenotypes were associated with genetic relatedness, we measured growth under salt stress at both 7°C and 37°C. At 7°C, in brain–heart infusion with 6% NaCl, average growth rates among the lineages were similar. A comparison of doubling times after exposure to salt stress at 7°C or 37°C indicated that growth at 7°C provided crossprotection to subsequent salt stress for strains in lineages I and II. At 37°C, in brain–heart infusion with 6% NaCl, lineage I and III strains grew significantly faster (p

Listeria monocytogenesσBand positive regulatory factor A (PrfA) are pleiotropic transcriptional regulators that coregulate a subset of virulence genes. A positive regulatory role for σBinprfAtranscription has been well established; therefore, observations of increased virulence gene expression and hemolytic activity in a ΔsigBstrain initially appeared paradoxical. To test the hypothesis thatL. monocytogenesσBcontributes to a regulatory network critical for appropriate repression as well as induction of virulence gene expression, genome-wide transcript profiling and follow-up quantitative reverse transcriptase PCR (qRT-PCR), reporter fusion, and phenotypic experiments were conducted usingL. monocytogenes prfA*,prfA* ΔsigB, ΔprfA, and ΔprfAΔsigBstrains. Genome-wide transcript profiling and qRT-PCR showed that in the presence of active PrfA (PrfA*), σBis responsible for reduced expression of the PrfA regulon. σB-dependent modulation of PrfA regulon expression reduced the cytotoxic effects of a PrfA* strain in HepG2 cells, highlighting the functional importance of regulatory interactions between PrfA and σB. The emerging model of the role of σBin regulating overall PrfA activity includes a switch from transcriptional activation at the P2prfApromoter (e.g., in extracellular bacteria when PrfA activity is low) to posttranscriptional downregulation of PrfA regulon expression (e.g., in intracellular bacteria when PrfA activity is high).

Background: Global patterns of gene expression of Escherichia coli K-12 during growth transitions have been deeply investigated, however, comparable studies of E. coli O157:H7 have not been explored, particularly with respect to factors regulating virulence genes and genomic islands specific to this pathogen. To examine the impact of growth phase on the dynamics of the transcriptome, O157:H7 Sakai strain was cultured in MOPS minimal media (0.1% glucose), RNA harvested at 10 time points from early exponential to full stationary phase, and relative gene expression was measured by co-hybridization on high-density DNA microarrays. Expression levels of 14 genes, including those encoding Shiga toxins and other virulence factors associated with the locus of enterocyte effacement (LEE), were confirmed by Q-PCR. Results: Analysis of variance (R/MAANOVA, Fs test) identified 442 (36%) of 1239 O157-specific ORFs and 2110 (59%) of 3647 backbone ORFs that changed in expression significantly over time. QT cluster analysis placed 2468 of the 2552 significant ORFs into 12 groups; each group representing a distinct expression pattern. ORFs from the largest cluster (n = 1078) decreased in expression from late exponential to early stationary phase: most of these ORFs are involved in functions associated with steady state growth. Also represented in this cluster are ORFs of the TAI island, encoding tellurite resistance and urease activity, which decreased ~4-fold. Most ORFs of the LEE pathogenicity island also decreased ~2-fold by early stationary phase. The ORFs encoding proteins secreted via the LEE encoded type III secretion system, such as tccP and espJ, also decreased in expression from exponential to stationary phase. Three of the clusters (n = 154) comprised genes that are transiently upregulated at the transition into stationary phase and included genes involved in nutrient scavenging. Upregulated genes with an increase in mRNA levels from late exponential to early stationary phase belonged to one cluster (n = 923) which includes genes involved in stress responses (e.g. gadAB, osmBC, and dps). These transcript levels remained relatively high for > 3 h in stationary phase. The Shiga toxin genes (stx 1AB and stx 2B) were significantly induced after transition into stationary phase. Conclusion: Expression of more than 300 O157-specific ORFs, many implicated in virulence of the O157 pathogen, was modulated in a growth dependent manner. These results provide a baseline transcriptional profile that can be compared to patterns of gene expression of this important foodborne pathogen under adverse environmental conditions.