Fate of salmonella and shiga-toxin producing Escherichia coli on wheat grain during tempering.

Outbreaks of Salmonella and Shiga-toxin producing Escherichia coli (STEC) linked to wheat flour led to increased interest in characterizing the fate of Salmonella and STEC on wheat during processing. Tempering is the stage of wheat processing where water is added to toughen the bran prior to milling, which has the potential to influence pathogen behavior on the kernels. This study aimed to quantify changes in the numbers of STEC and Salmonella inoculated onto soft red winter wheat, and to observe potential changes in the distribution of the pathogens on the surface of kernels during tempering. Lab-scale tempering experiments were conducted to quantify the water activity of and bacterial populations on wheat grain at various time points during 16 h of tempering. The highest water activity observed throughout 16 h of tempering was 0.88. There was no significant change (p > 0.05) in numbers of Salmonella, STEC, or native mesophiles. Using confocal microscopy, observation of Salmonella and STEC cells expressing mCherry on wheat kernels showed an even distribution of inoculated cells, though the localization of cells on kernels did not change significantly after tempering. Even though the environment was not favorable for pathogen replication on grain, the population remained stable, suggesting that disinfection of the kernels prior to milling could reduce food safety concerns in flour.

Survival and thermal resistance among four Salmonella serovars inoculated onto flaxseeds.

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 < 0.05). Inoculated flaxseeds were processed at 71 °C with vacuum steam pasteurization at 4 time points during storage. Average initial D71°C values ranging from 1.0 to 1.5 min were similar across serovars. Over 24 weeks, D71°C varied in a serovar-dependent manner. D71°C at 8, 16, and 24 weeks did not change significantly for Enteritidis and Montevideo but did for Tennessee and Agona. While significant, the differences in D71°C over time were less than 1 min, indicating that storage time prior to heat treatment would have a minimal effect on the processing time required to inactivate Salmonella on flaxseed.

Salmonella enterica in Soils Amended with Heat-Treated Poultry Pellets Survived Longer than Bacteria in Unamended Soils and More Readily Transferred to and Persisted on Spinach.

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 < 0.05). Salmonella Newport introduced to HTPP-amended soils survived for longer periods and was more likely to transfer to and persist on spinach plants than was S. Newport introduced to unamended soils.IMPORTANCE Heat-treated poultry pellets (HTPP) often are used by fruit and vegetable growers as a slow-release fertilizer. However, contamination of soil on farms may occur through contaminated irrigation water or scat from wild animals. Here, we show that the presence of HTPP in soil led to increased S. Newport survival in soil and to greater likelihood of its transfer to and survival on spinach plants. There were no significant differences in survival durations of WT and ΔrpoS mutant isolates of S. Newport. The statistically similar populations recovered by plate count and estimated by PMA-qPCR for both strains in the amended and unamended soils in this study indicate that all viable populations of S. Newport in soils were culturable.

Genomic and phenotypic diversity of Listeria monocytogenes clonal complexes associated with human listeriosis.

Listeria monocytogenes is a pathogen of significant concern in many ready to eat foods due to its ability to survive and multiply even under significant environmental stresses. Listeriosis in humans is a concern, especially to high-risk populations such as those who are immunocompromised or pregnant, due to the high rates of morbidity and mortality. Whole genome sequencing has become a routine part of assessing L. monocytogenes isolated from patients, and the frequency of different genetic subtypes associated with listeriosis is now being reported. The recent abundance of genome sequences for L. monocytogenes has provided a wealth of information regarding the variation in core and accessory genomic elements. Newly described accessory genomic regions have been linked to greater virulence capabilities as well as greater resistance to environmental stressors such as sanitizers commonly used in food processing facilities. This review will provide a summary of our current understanding of stress response and virulence phenotypes of L. monocytogenes, within the context of the genetic diversity of the pathogen.

Determination of Evolutionary Relationships of Outbreak-Associated Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Whole-Genome Sequencing.

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.

VirR-Mediated Resistance of Listeria monocytogenes against Food Antimicrobials and Cross-Protection Induced by Exposure to Organic Acid Salts.

Formulations of ready-to-eat (RTE) foods with antimicrobial compounds constitute an important safety measure against foodborne pathogens such as Listeria monocytogenes. While the efficacy of many commercially available antimicrobial compounds has been demonstrated in a variety of foods, the current understanding of the resistance mechanisms employed by L. monocytogenes to counteract these stresses is limited. In this study, we screened in-frame deletion mutants of two-component system response regulators associated with the cell envelope stress response for increased sensitivity to commercially available antimicrobial compounds (nisin, lauric arginate, ε-polylysine, and chitosan). A virR deletion mutant showed increased sensitivity to all antimicrobials and significantly greater loss of membrane integrity when exposed to nisin, lauric arginate, or ε-polylysine (P < 0.05). The VirR-regulated operon, dltABCD, was shown to be the key contributor to resistance against these antimicrobial compounds, whereas another VirR-regulated gene, mprF, displayed an antimicrobial-specific contribution to resistance. An experiment with a ß-glucuronidase (GUS) reporter fusion with the dlt promoter indicated that nisin does not specifically induce VirR-dependent upregulation of dltABCD. Lastly, prior exposure of L. monocytogenes parent strain H7858 and the ΔvirR mutant to 2% potassium lactate enhanced subsequent resistance against nisin and ε-polylysine (P < 0.05). These data demonstrate that VirRS-mediated regulation of dltABCD is the major resistance mechanism used by L. monocytogenes against cell envelope-damaging food antimicrobials. Further, the potential for cross-protection induced by other food-related stresses (e.g., organic acids) needs to be considered when applying these novel food antimicrobials as a hurdle strategy for RTE foods.

Transcriptomic Analysis of the Adaptation of Listeria monocytogenes to Growth on Vacuum-Packed Cold Smoked Salmon.

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], <0.05; fold change, ≥2.5), and 88 and 61 genes were up- and downregulated, respectively, in H7858 grown on CSS relative to the genes in H7858 grown in MBHIB. In spite of these differences in transcriptomes under these two conditions, growth parameters for 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.

Clonal Clustering Using 10-Gene Multilocus Sequence Typing Reveals an Association Between Genotype and Listeria monocytogenes Maximum Growth Rate in Defined Medium.

We used a 10-gene (10G) multilocus sequence typing scheme to investigate the diversity and phylogenetic distribution of 124 Listeria monocytogenes strains across major lineages, major serotypes, and seven epidemic clones that have been previously associated with outbreaks. The 124 isolates proved to be diverse, with a total of 81 sequence types (10G-STs) belonging to 13 clonal complexes (CCs), where all STs of the same CC differ from one another in up to 3 of the 10 alleles (named as 10G-triple-locus-variant-clonal-complexes [10G-TLV-CCs]). Phenotypic characterization for 105 of the 124 strains showed that L. monocytogenes had variable maximum growth rate (µ(max)) in a defined medium at 16°C, and classification by lineage or serotype was not able to reflect the genetic basis for the difference of this phenotype. Among the six major 10G-TLV-CCs, 10G-TLV-CC4 that included lineage I strains had significantly lower µ(max) (Tukey honestly significant difference adjusted [adj.] p < 0.05) compared to 10G-TLV-CC1 and 10G-TLV-CC3 that both comprised lineage II strains, indicating a distinct difference in growth of these L. monocytogenes isolates under nutrient-limited conditions among some of the CCs. However, the other three (10G-TLV-CC2, 6, and 10) of the six major 10G-TLV-CCs containing either lineage I or lineage II strains did not show significantly different µ(max) compared to the others (adj. p < 0.05). Our findings highlighted the importance of using molecular typing methods that can be used in evolutionary analyses as a framework for further understanding the phenotypic characteristics of subgroups of L. monocytogenes.

Antimicrobial Solid Starch-Iodine Complex via Reactive Extrusion and Its Application in PLA-PBAT Blown Films.

In this study, a solid masterbatch of starch-iodine complex with 6.7 wt.% iodine was prepared in pellet form using a ZSK-30 twin-screw extruder. Thermogravimetric (TGA) and isothermal TGA analysis of the pellets revealed that there was no significant loss of iodine due to sublimation during reactive extrusion. These solid pellets demonstrated antifungal properties when applied to strawberries via dip coating in an aqueous solution, extending their shelf life from two days to eight days, thereby reducing fungal growth and visual decay. Furthermore, the solid pellets displayed antibacterial activity against E. coli, as evidenced by the clear zone of inhibition observed in the Kirby-Bauer test. To enhance practical application, these pellets were further blended with PLA-PBAT film formulations at 10 and 18% by wt. to make blown films with effective iodine loadings of 0.7 and 1.3% by wt. These films showed superior antibacterial activity against E. coli compared with PLA control films and the commercial silver antimicrobial-containing films during direct inoculation tests as per ISO 22196. Tensile strength and elongation at break in machine direction (MD) for the starch-iodine-containing blown films were comparable to the control films in MD, but tensile strength was reduced to 37-40% in the transverse direction (TD). This was due to a non-uniform dispersion of the starch-iodine complex in the films, as confirmed by the visual and SEM analyses. Thus, this study illustrates the practical utility of the solid starch-iodine complex as a safe and efficient means of introducing iodine into an environment, mitigating the typical hazards associated with handling solid iodine.

Impact of Biotic and Abiotic Factors on Listeria monocytogenes, Salmonella enterica, and Enterohemorrhagic Escherichia coli in Agricultural Soil Extracts.

Outbreaks of Enterohemorrhagic Escherichia coli (EHEC), Salmonella enterica, and Listeria monocytogenes linked to fresh produce consumption pose significant food safety concerns. These pathogens can contaminate pre-harvest produce through various routes, including contaminated water. Soil physicochemical properties and flooding can influence pathogen survival in soils. We investigated survival of EHEC, S. enterica, and L. monocytogenes in soil extracts designed to represent soils with stagnant water. We hypothesized pathogen survival would be influenced by soil extract nutrient levels and the presence of native microbes. A chemical analysis revealed higher levels of total nitrogen, phosphorus, and carbon in high-nutrient soil extracts compared to low-nutrient extracts. Pathogen survival was enhanced in high-nutrient, sterile soil extracts, while the presence of native microbes reduced pathogen numbers. A microbiome analysis showed greater diversity in low-nutrient soil extracts, with distinct microbial compositions between extract types. Our findings highlight the importance of soil nutrient composition and microbial dynamics in influencing pathogen behavior. Given key soil parameters, a long short-term memory model (LSTM) effectively predicted pathogen survival. Integrating these factors can aid in developing predictive models for pathogen persistence in agricultural systems. Overall, our study contributes to understanding the complex interplay in agricultural ecosystems, facilitating informed decision-making for crop production and food safety enhancement.

Nisin resistance of Listeria monocytogenes is increased by exposure to salt stress and is mediated via LiaR.

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, ΔliaR 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.

Correction: Wiedmann, M., et al. Exploration of the Role of the Non-Coding RNA SbrE in L. monocytogenes Stress Response. Int. J. Mol. Sci. 2013, 14, 378-393.

The original version of the paper in Section 3.8 reports that "The peptide mass tolerance and fragment mass tolerance values were 10 ppm and 30 mDa, respectively" [1] (p. 387). To help others who may want to use the same methods in the future, the authors would like to correct the wording to: "The peptide mass tolerance and fragment mass tolerance values were 30 ppm and 0.15 Da, respectively. In order to decrease the probability of false peptide identification, only peptides with significance scores above the identity threshold (at the 95% confidence interval), defined by Mascot probability analysis (www.matrixscience.com/help/scoring_help.html#PBM), were considered to be confidently identified and used for protein identification.  Furthermore, only proteins identified in all four iTRAQ samples through at least two peptides with a p-value of <0.05 (expectation value) were further analyzed". The authors would like to apologize for any inconvenience this may have caused to the readers of this journal.

Inactivation of Salmonella and Shiga-toxin producing Escherichia coli on soft wheat kernels using vacuum steam pasteurization.

Wheat, the raw material for flour milling, can be contaminated with enteric pathogens, leading to outbreaks linked to flour. In previous lab-scale studies, vacuum steam treatment was able to reduce Salmonella Enteritidis PT30 and Shiga-toxin producing E. coli (STEC) O121 levels on soft wheat kernels while maintaining flour quality and gluten functionality. This study used a newly designed lab-scale vacuum steam pasteurizer (VSP) to evaluate its efficacy to inactivate multiple strains of Salmonella and STEC on soft wheat by modeling the non-isothermal time-temperature history during treatment and reduction of the microbial populations. The results demonstrated that vacuum steam treatment could effectively disinfect wheat grains inoculated with enteric pathogens. In this study, Salmonella strains were less thermally resistant than STEC strains. The D75°C of Salmonella strains were 2.8 and 3.2 min, and the D75°C of STEC ranged from 3.1 to 4.6 min. E. faecium had a D75°C of 3.3 min, which indicates that it could be used as surrogate for larger scale evaluation of vacuum steam pasteurization in the future but was not conservative compared to some of the STEC strains.

Listeria monocytogenes shows temperature-dependent and -independent responses to salt stress, including responses that induce cross-protection against other stresses.

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; ΔmrpABCDEFG 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.

Exploration of the role of the non-coding RNA SbrE in L. monocytogenes stress response.

SbrE is a ncRNA in Listeria monocytogenes, reported to be up-regulated by the alternative sigma factor σB. Initial quantitative RT-PCR (qRT-PCR) experiments on parent strains and isogenic ΔsigB strains demonstrated σB-dependent expression of SbrE across the four L. monocytogenes lineages and in L. innocua. Microarray and proteomics (MDLC/MS/MS with iTRAQ labeling) experiments with the L. monocytogenes parent strain and an isogenic ΔsbrE strain identified a single gene (lmo0636) and two proteins (Lmo0637 and Lmo2094) that showed lower expression levels in the ΔsbrE strain. qRT-PCR demonstrated an increase in SbrE transcript levels in stationary phase L. monocytogenes and in bacteria exposed to oxidative stress (mean log2 transcript levels 7.68 ± 0.57 and 1.70 ± 0.71 greater than in mid-log phase cells, respectively). However, no significant differences in growth or survival between the parent strain and ΔsbrE strain were confirmed under a variety of environmental stress conditions tested. Our data suggest that σB-dependent transcription of SbrE represents a conserved mechanism that contributes, across Listeria species, to fine-tuning of gene expression under specific environmental conditions that remain to be defined.

Impact of Chlorinated Water on Pathogen Inactivation during Wheat Tempering and Resulting Flour Quality.

ABSTRACT: Outbreaks of enteric pathogens linked to wheat flour have led the wheat milling industry to seek solutions addressing this food safety concern. Chlorinated water at 400 to 700 ppm has been used in the flour milling industry as a tempering aid to control growth of yeast and mold in tempering bins. However, the effectiveness of chlorinated water for inactivating enteric pathogens on wheat kernels was unknown. Five strains of Shiga toxin-producing Escherichia coli and two strains of Salmonella were inoculated onto hard red spring wheat at 7 log CFU/g and stored at room temperature for 1 month. Inoculated wheat was tempered with four concentrations (0, 400, 800, and 1,200 ppm) of chlorinated water (pH 6.5). The reduction due to chlorine was determined by calculating change in microbial loads at each chlorine level by using the response at 0 ppm as a reference. Uninoculated wheat tempered with chlorinated water was used to measure flour quality parameters. Changes in pathogen population over 18 h ranged from -2.35 to -0.30 log CFU/g with 800 ppm of chlorinated water and were not significantly different from changes at 400 and 1,200 ppm. Significant (P < 0.05) differences in the extent of reduction were observed among strains. However, the effect of chlorinated water at reducing native microbes on wheat kernels was minimal, with an average reduction of 0.39 log CFU/g for all concentrations. No significant (P > 0.05) changes occurred in flour quality and gluten functionality or during bread making for grains tempered at 400 and 800 ppm of chlorinated water. There were small but significant (P < 0.05) changes in flour protein content, final viscosity, and water absorption when tempered with 1,200 ppm of chlorinated water. The data showed that the level of chlorinated water currently used in industry for tempering could reduce enteric pathogen numbers by 1.22 log CFU/g for Shiga toxin-producing Escherichia coli and 2.29 log CFU/g for Salmonella, with no significant effects on flour quality and gluten functionality.

Genome-Wide Association Study of Listeria monocytogenes Isolates Causing Three Different Clinical Outcomes.

Heterogeneity in virulence potential of L. monocytogenes subgroups have been associated with genetic elements that could provide advantages in certain environments to invade, multiply, and survive within a host. The presence of gene mutations has been found to be related to attenuated phenotypes, while the presence of groups of genes, such as pathogenicity islands (PI), has been associated with hypervirulent or stress-resistant clones. We evaluated 232 whole genome sequences from invasive listeriosis cases in human and ruminants from the US and Europe to identify genomic elements associated with strains causing three clinical outcomes: central nervous system (CNS) infections, maternal-neonatal (MN) infections, and systemic infections (SI). Phylogenetic relationships and virulence-associated genes were evaluated, and a gene-based and single nucleotide polymorphism (SNP)-based genome-wide association study (GWAS) were conducted in order to identify loci associated with the different clinical outcomes. The orthologous results indicated that genes of phage phiX174, transfer RNAs, and type I restriction-modification (RM) system genes along with SNPs in loci involved in environmental adaptation such as rpoB and a phosphotransferase system (PTS) were associated with one or more clinical outcomes. Detection of phenotype-specific candidate loci represents an approach that could narrow the group of genetic elements to be evaluated in future studies.

Comparative Genomics of Listeria monocytogenes Isolates from Ruminant Listeriosis Cases in the Midwest United States.

Ruminants are a well-known reservoir for Listeria monocytogenes. In addition to asymptomatic carriage of the pathogen, ruminants can also acquire listeriosis and develop clinical manifestations in the form of neurologic or fetal infections, similar to those occurring in humans. Genomic characterization of ruminant listeriosis cases in Europe have identified lineage 1 and 2 strains associated with infection, as well as clonal complexes (CCs) that are commonly isolated from human cases of listeriosis; however, there is little information on the diversity of L. monocytogenes from ruminant listeriosis in the United States. In this study, we characterized and compared 73 L. monocytogenes isolates from ruminant listeriosis cases from the Midwest and the Upper Great Plains collected from 2015 to 2020. Using whole-genome sequence data, we classified the isolates and identified key virulence factors, stress-associated genes, and mobile genetic elements within our data set. Our isolates belonged to three different lineages: 31% to lineage 1, 53% to lineage 2, and 15% to lineage 3. Lineage 1 and 3 isolates were associated with neurologic infections, while lineage 2 showed a greater frequency of fetal infections. Additionally, the presence of mobile elements, virulence-associated genes, and stress and antimicrobial resistance genes was evaluated. These genetic elements are responsible for most of the subgroup-specific features and may play a key role in the spread of hypervirulent clones, including the spread of hypervirulent CC1 clone commonly associated with disease in humans, and may explain the increased frequency of certain clones in the area. IMPORTANCE Listeria monocytogenes affects humans and animals, causing encephalitis, septicemia, and abortions, among other clinical outcomes. Ruminants such as cattle, goats, and sheep are the main carriers contributing to the maintenance and dispersal of this pathogen in the farm environment. Contamination of food products from farms is of concern not only because many L. monocytogenes genotypes found there are associated with human listeriosis but also as a cause of significant economic losses when livestock and food products are affected. Ruminant listeriosis has been characterized extensively in Europe; however, there is limited information about the genetic diversity of these cases in the United States. Identification of subgroups with a greater ability to spread may facilitate surveillance and management of listeriosis and contribute to a better understanding of the genome diversity of this pathogen, providing insights into the molecular epidemiology of ruminant listeriosis in the region.

The transcriptional response of Listeria monocytogenes during adaptation to growth on lactate and diacetate includes synergistic changes that increase fermentative acetoin production.

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.

Transcriptomic and phenotypic analyses identify coregulated, overlapping regulons among PrfA, CtsR, HrcA, and the alternative sigma factors sigmaB, sigmaC, sigmaH, and sigmaL in Listeria 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.