Haley F. Oliver

Listeria monocytogenes σB Has a Small Core Regulon and a Conserved Role in Virulence but Makes Differential Contributions to Stress Tolerance across a Diverse Collection of Strains

ABSTRACT Listeria monocytogenes strains are classified in at least three distinct phylogenetic lineages. There are correlations between lineage classification and source of bacterial isolation; e.g., human clinical and food isolates usually are classified in either lineage I or II. However, human clinical isolates are overrepresented in lineage I, while food isolates are overrepresented in lineage II. σB, a transcriptional regulator previously demonstrated to contribute to environmental stress responses and virulence in L. monocytogenes lineage II strains, was hypothesized to provide differential abilities for L. monocytogenes survival in various niches (e.g., food and human clinical niches). To determine if the contributions of σB to stress response and virulence differ across diverse L. monocytogenes strains, ΔsigB mutations were created in strains belonging to lineages I, II, IIIA, and IIIB. Paired parent and ΔsigB mutant strains were tested for survival under acid and oxidative stress conditions, Caco-2 cell invasion efficiency, and virulence using the guinea pig listeriosis infection model. Parent and ΔsigB mutant strain transcriptomes were compared using whole-genome expression microarrays. σB contributed to virulence in each strain. However, while σB contributed significantly to survival under acid and oxidative stress conditions and Caco-2 cell invasion in lineage I, II, and IIIB strains, the contributions of σB were not significant for these phenotypes in the lineage IIIA strain. A core set of 63 genes was positively regulated by σB in all four strains; different total numbers of genes were positively regulated by σB in the strains. Our results suggest that σB universally contributes to L. monocytogenes virulence but specific σB-regulated stress response phenotypes vary among strains.

Whole genome sequencing allows for improved identification of persistent Listeria monocytogenes in food associated environments.

ABSTRACT While the food-borne pathogen Listeria monocytogenes can persist in food associated environments, there are no whole-genome sequence (WGS) based methods to differentiate persistent from sporadic strains. Whole-genome sequencing of 188 isolates from a longitudinal study of L. monocytogenes in retail delis was used to (i) apply single-nucleotide polymorphism (SNP)-based phylogenetics for subtyping of L. monocytogenes, (ii) use SNP counts to differentiate persistent from repeatedly reintroduced strains, and (iii) identify genetic determinants of L. monocytogenes persistence. WGS analysis revealed three prophage regions that explained differences between three pairs of phylogenetically similar populations with pulsed-field gel electrophoresis types that differed by ≤3 bands. WGS-SNP-based phylogenetics found that putatively persistent L. monocytogenes represent SNP patterns (i) unique to a single retail deli, supporting persistence within the deli (11 clades), (ii) unique to a single state, supporting clonal spread within a state (7 clades), or (iii) spanning multiple states (5 clades). Isolates that formed one of 11 deli-specific clades differed by a median of 10 SNPs or fewer. Isolates from 12 putative persistence events had significantly fewer SNPs (median, 2 to 22 SNPs) than between isolates of the same subtype from other delis (median up to 77 SNPs), supporting persistence of the strain. In 13 events, nearly indistinguishable isolates (0 to 1 SNP) were found across multiple delis. No individual genes were enriched among persistent isolates compared to sporadic isolates. Our data show that WGS analysis improves food-borne pathogen subtyping and identification of persistent bacterial pathogens in food associated environments.

Transcriptomic and Phenotypic Analyses Suggest a Network between the Transcriptional Regulators HrcA and σB in Listeria monocytogenes

ABSTRACT Listeria monocytogenes HrcA and CtsR negatively regulate class I and III stress response genes, respectively, while σB positively regulates the transcription of class II stress response genes. To define the HrcA regulon and identify interactions between HrcA, CtsR, and σB, we characterized newly generated L. monocytogenes ΔhrcA, ΔctsR ΔhrcA, and ΔhrcA ΔsigB strains, along with previously described ΔsigB, ΔctsR, and ΔctsR ΔsigB strains, using phenotypic assays (i.e., heat resistance, acid resistance, and invasion of human intestinal epithelial cells) and performed whole-genome transcriptome analysis of the ΔhrcA strain. The hrcA and sigB deletions had significant effects on heat resistance. While the hrcA deletion had no significant effect on acid resistance or invasion efficiency in Caco-2 cells, a linear regression model revealed a significant (P = 0.0493) effect of interactions between the hrcA deletion and the ctsR deletion on invasiveness. Microarray-based transcriptome analyses and promoter searches identified (i) 25 HrcA-repressed genes, including two operons (the groESL and dnaK operons, both confirmed as HrcA regulated by quantitative real-time PCR) and one gene directly repressed by HrcA, and (ii) 36 genes that showed lower transcript levels in the ΔhrcA strain and thus appear to be indirectly upregulated by HrcA. A number of genes were found to be coregulated by either HrcA and CtsR (2 genes), HrcA and σB (31 genes), or all three regulators (5 genes, e.g., gadCB). Combined with previous evidence that σB appears to directly regulate hrcA transcription, our data suggest that HrcA and σB, as well as CtsR, form a regulatory network that contributes to the transcription of a number of L. monocytogenes genes.

Prevalence, distribution, and diversity of Listeria monocytogenes in retail environments, focusing on small establishments and establishments with a history of failed inspections.

Despite growing concerns about cross-contamination of ready-to-eat foods with Listeria monocytogenes, our knowledge about the ecology and transmission of L. monocytogenes in retail establishments has remained limited. We conducted a cross-sectional study to characterize the prevalence, distribution, and subtype diversity of L. monocytogenes in 120 New York State retail deli establishments that were hypothesized to present an increased risk for environmental L. monocytogenes contamination (i.e., small establishments and establishments with a history of failed New York State Agriculture and Markets inspections). Analysis of these data along with previously reported data for 121 predominantly larger retail establishments in New York State identified establishment size, geographic location, and inspection history as significant predictors of L. monocytogenes presence and prevalence. The odds of an establishment being L. monocytogenes positive were approximately twice as high for large establishments, establishments located in New York City, or establishments with poor inspection history (as compared with establishments without these attributes), even though correlation between location and inspection history complicated interpretation of results. Within an establishment, L. monocytogenes was significantly more prevalent on nonfood contact surfaces than on food contact surfaces; prevalence was particularly high for floors and in floor drains, sinks, the dairy case, and milk crates. L. monocytogenes subtype diversity differed between sites, with lineage I isolates significantly associated with nonfood contact surfaces and lineage II isolates significantly associated with food contact surfaces. Isolates belonging to the same ribotype were often found dispersed across multiple sites within an operation.

Listeria monocytogenes and Listeria spp. contamination patterns in retail delicatessen establishments in three U.S. states.

Postprocessing contamination in processing plants has historically been a significant source of Listeria monocytogenes in ready-to-eat delicatessen meats, and therefore a major cause of human listeriosis cases and outbreaks. Recent risk assessments suggest that a majority of human listeriosis cases linked to consumption of contaminated deli meats may be due to L. monocytogenes contamination that occurs at the retail level. To better understand the ecology and transmission of Listeria spp. in retail delicatessens, food and nonfood contact surfaces were tested for L. monocytogenes and other Listeria spp. in a longitudinal study conducted in 30 retail delis in three U.S. states. In phase I of the study, seven sponge samples were collected monthly for 3 months in 15 delis (5 delis per state) prior to start of daily operation; in phase II, 28 food contact and nonfood contact sites were sampled in each of 30 delis during daily operation for 6 months. Among the 314 samples collected during phase I, 6.8% were positive for L. monocytogenes. Among 4,503 samples collected during phase II, 9.5% were positive for L. monocytogenes; 9 of 30 delis showed low L. monocytogenes prevalence (<1%) for all surfaces. A total of 245 Listeria spp. isolates, including 184 Listeria innocua, 48 Listeria seeligeri, and 13 Listeria welshimeri were characterized. Pulsed-field gel electrophoresis (PFGE) was used to characterize 446 L. monocytogenes isolates. PFGE showed that for 12 of 30 delis, one or more PFGE types were isolated on at least three separate occasions, providing evidence for persistence of a given L. monocytogenes subtype in the delis. For some delis, PFGE patterns for isolates from nonfood contact surfaces were distinct from patterns for occasional food contact surface isolates, suggesting limited cross-contamination between these sites in some delis. This study provides longitudinal data on L. monocytogenes contamination patterns in retail delis, which should facilitate further development of control strategies in retail delis.

Nano/Micro and Spectroscopic Approaches to Food Pathogen Detection

Despite continuing research efforts, timely and simple pathogen detection with a high degree of sensitivity and specificity remains an elusive goal. Given the recent explosion of sensor technologies, significant strides have been made in addressing the various nuances of this important global challenge that affects not only the food industry but also human health. In this review, we provide a summary of the various ongoing efforts in pathogen detection and sample preparation in areas related to Fourier transform infrared and Raman spectroscopy, light scattering, phage display, micro/nanodevices, and nanoparticle biosensors. We also discuss the advantages and potential limitations of the detection methods and suggest next steps for further consideration.

Identification of Core Competencies for an Undergraduate Food Safety Curriculum Using a Modified Delphi Approach.

Identification of core competencies for undergraduates in food safety is critical to assure courses and curricula are appropriate in maintaining a well-qualified food safety workforce. The purpose of this study was to identify and refine core competencies relevant to postsecondary food safety education using a modified Delphi method. Twenty-nine experts representing food safety professionals in academia, government, and industry were given 2 rounds of questionnaires that specified initial food safety competencies, core domains, and subdomains. Competencies were defined as a set of skills, knowledge, and abilities that correlate to success of a trainee. The framework for which competencies were classified consisted of (1) core domains, defined as broad food safety subjects; and (2) subdomains, or more specific food safety subjects. The expert panel used a 5-point Likert scale with an acceptance criterion, or consensus, of 75%, with a rating of “4” or greater. After 2 rounds of questionnaires and revisions from the expert panel, 5 core domains were established: (1) Food Production, Manufacturing, Retail, and Consumer; (2) Foodborne Hazards; (3) Public Health; (4) Legislation and Policy; and (5) Communication and Education. Specific responses from the experts highlighted areas in which further curriculum revision would be beneficial. This study provides a framework for the development of a vetted, standardized undergraduate food safety curriculum. The Delphi method, with its inclusion of professionals representing various sectors of food safety, provided relevant perspectives for curriculum design, and also allowed participants the opportunity to contribute to the education of future food safety professionals.

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.

Transcriptional and Phenotypic Responses of Listeria monocytogenes to Chlorine Dioxide

ABSTRACT Significant food-borne disease outbreaks have occurred from consumption of ready-to-eat foods, including produce, contaminated with Listeria monocytogenes. Challenging food matrices (e.g., cantaloupe, sprouts) with limited processing steps postharvest to reduce pathogen loads have underscored a need for new mitigation strategies. Chlorine dioxide (ClO2) is increasingly being used in produce and other food systems to reduce food-borne pathogen levels. The goal of this study was to characterize the transcriptional response and survival of L. monocytogenes 10403S exposed to ClO2. The transcriptional profile of log-phase cells exposed to 300 mg/liter ClO2 for 15 min was defined by whole-genome microarray. A total of 340 genes were significantly differentially expressed. Among the differentially expressed genes, 223 were upregulated (fold change ≥ 1.5; adjusted P value < 0.05) in role categories responsible for protein fate, cellular processes, and energy metabolism. There were 113 and 16 genes differentially expressed belonging to regulatory networks of σB and CtsR, respectively. We assessed L. monocytogenes 10403S survival after exposure to 100, 300, and 500 mg/liter aqueous ClO2 in brain heart infusion (BHI) broth; there was a significant difference between cells exposed to 500 mg/liter ClO2 and those exposed to all other conditions over time (P value < 0.05). Isogenic ΔsigB and ΔctsR mutants exposed to 300 mg/liter ClO2 were more sensitive to ClO2 than the wild type under the same conditions. These results provide an initial insight into the mechanisms that L. monocytogenes employs to survive sublethal ClO2 and further our understanding of the inactivation mechanisms of this increasingly used sanitizer.

Listeria monocytogenes Internalizes in Romaine Lettuce Grown in Greenhouse Conditions

Listeria monocytogenes has been implicated in a number of outbreaks involving fresh produce, including an outbreak in 2016 resulting from contaminated packaged salads. The persistence and internalization potential of L. monocytogenes in romaine lettuce was evaluated, and the persistence of two L. monocytogenes strains was assessed on three romaine lettuce cultivars. Seeds were germinated, and plants grown in three soil types (i.e., standard potting mix, autoclaved potting mix, and top soil) and sterile soft-top agar for up to 21 days. Average CFU per gram of L. monocytogenes on seeds and plants was calculated from five replicates per harvest day. Up to 8.2 log CFU/g L. monocytogenes persisted on romaine lettuce plants (Braveheart cultivar) grown in soft-top agar, while those grown in commercial potting mix (initial soil aerobic plate count of 4.0 × 104 CFU/g) had a final concentration of 5.4 log CFU/g, and autoclaved commercial potting mix had a final concentration of 3.8 ± 0.2 log CFU/g after a 21-day period. Pathogen levels dropped below the limit of detection (2 log CFU/g) by day 18 in 75% topsoil (initial soil aerobic plate count of 4.0 × 101 CFU/g); this did not occur in sterile media. Although L. monocytogenes strain differences and presence of a clay coating on seeds did not affect persistence, differences were observed in L. monocytogenes growth and survival among cultivars. To assess internalization, seeds were inoculated with L. monocytogenes expressing green fluorescent protein. Three plants were fixed, paraffin embedded, and sectioned; localization was studied by using standard immunohistochemistry techniques. A total of 539 internalized L. monocytogenes cells were visualized among three 20-day seedlings. L. monocytogenes cells were located in all major tissue types (pith followed by cortex, xylem, phloem, and epidermis). The presence of L. monocytogenes in the plant vasculature suggests potential for transport throughout the plant into edible tissue.