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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

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.

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.

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 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.

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.