BackgroundIdentification of specific genes and gene expression patterns important for bacterial survival, transmission and pathogenesis is critically needed to enable development of more effective pathogen control strategies. The stationary phase stress response transcriptome, including many σB-dependent genes, was defined for the human bacterial pathogen Listeria monocytogenes using RNA sequencing (RNA-Seq) with the Illumina Genome Analyzer. Specifically, bacterial transcriptomes were compared between stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic ΔsigB mutant, which does not express the alternative σ factor σB, a major regulator of genes contributing to stress response, including stresses encountered upon entry into stationary phase.ResultsOverall, 83% of all L. monocytogenes genes were transcribed in stationary phase cells; 42% of currently annotated L. monocytogenes genes showed medium to high transcript levels under these conditions. A total of 96 genes had significantly higher transcript levels in 10403S than in ΔsigB, indicating σB-dependent transcription of these genes. RNA-Seq analyses indicate that a total of 67 noncoding RNA molecules (ncRNAs) are transcribed in stationary phase L. monocytogenes, including 7 previously unrecognized putative ncRNAs. Application of a dynamically trained Hidden Markov Model, in combination with RNA-Seq data, identified 65 putative σB promoters upstream of 82 of the 96 σB-dependent genes and upstream of the one σB-dependent ncRNA. The RNA-Seq data also enabled annotation of putative operons as well as visualization of 5'- and 3'-UTR regions.ConclusionsThe results from these studies provide powerful evidence that RNA-Seq data combined with appropriate bioinformatics tools allow quantitative characterization of prokaryotic transcriptomes, thus providing exciting new strategies for exploring transcriptional regulatory networks in bacteria.See minireivew http://jbiol.com/content/8/12/107.

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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. Listeria monocytogenes is an opportunistic food-borne pathogen responsible for approximately 250 deaths per year in the United States (1). The annual costs of listeriosis, including morbidity, mortality, and lost wages, are estimated at $2.8 billion (2). A 2003 risk assessment attributed 90% of listeriosis cases in the United States to consumption of contaminated ready-to-eat (RTE) deli meats (3), and most (Ͼ60%) of U.S. listeriosis cases linked to RTE deli meats were estimated to be due contamination during retail handling and slicing (4, 5). Consequently, the retail deli environment is a focal point for listeriosis reduction efforts.L. monocytogenes can persist in food-associated environments for months to years (6-8). Persistent strains have been linked to outbreaks of food-borne disease. For example, L. monocytogenes that was responsible for an outbreak linked to 29 cases and 4 deaths persisted in the source plant for at least 12 years (9). Therefore, the food processing industry has implemented the seek-anddestroy process to identify and eliminate point sources of persistence through enhanced environmental monitoring, sanitation, and equipment and process design (10).One challenge for persistent pathogen control is to differentiate true persistence from repeated reintroduction of a give...

Listeria monocytogenes HrcA and CtsR negatively regulate class I and III stress response genes, respectively, while B positively regulates the transcription of class II stress response genes. To define the HrcA regulon and identify interactions between HrcA, CtsR, and B , we characterized newly generated L. monocytogenes ⌬hrcA, ⌬ctsR ⌬hrcA, and ⌬hrcA ⌬sigB strains, along with previously described ⌬sigB, ⌬ctsR, and ⌬ctsR ⌬sigB 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 ⌬hrcA 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 ⌬hrcA 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 B (31 genes), or all three regulators (5 genes, e.g., gadCB). Combined with previous evidence that B appears to directly regulate hrcA transcription, our data suggest that HrcA and B , as well as CtsR, form a regulatory network that contributes to the transcription of a number of L. monocytogenes genes.Listeria monocytogenes is a gram-positive food-borne pathogen that can cause severe invasive disease in humans, as well as in a number of different animal species (31). The capacity of L. monocytogenes to survive and multiply under a wide range of environmental stress conditions appears to be critical for the food-borne transmission of this pathogen (10). A number of transcriptional regulators (e.g., PrfA, B , HrcA, and CtsR) that are important for the transcription of stress response and virulence genes have been identified in this organism (20,25,32,37). While clear evidence for interactions between PrfA and B has been reported (26,40,45), our understanding of interactions among other L. monocytogenes transcriptional regulators is limited. As no L. monocytogenes hrcA null mutant appears to have previously been reported, our understanding of the contributions of the negative regulator HrcA to stress response, transcriptional regulation, and regulatory networks is limited. In a number of gram-positive bacteria, including Bacillus subtilis, HrcA (heat regulation at CIRCE) has been found to repress the dnaK and groESL operons by binding to a region designated as the controlling inverted-repeat chaperone expression (CIRCE) element (38). Sequence analyses in L. monocytogenes also identified putative CIRCE ele...

BackgroundAntimicrobial disinfectants are used as primary treatment options against pathogens on surfaces in healthcare facilities to help prevent healthcare associated infections (HAIs). On many surfaces, pathogenic microorganisms exist as biofilms and form an extracellular matrix that protects them from the antimicrobial effects of disinfectants. Disinfectants are used as all-purpose antimicrobials though very few specifically make biofilm efficacy claims. The objective of this study was to evaluate the efficacy of eight registered disinfectants (six registered by the Environmental Protection Agency and two products registered in by the European Chemical Agency) with general bactericidal claims, but currently no biofilm efficacy claims, against Staphylococcus aureus ATTC-6538 and Pseudomonas aeruginosa ATCC-15442 biofilms. We hypothesized that hydrogen peroxide and sodium hypochlorite disinfectant products would be more effective than quaternary ammonium chlorides.MethodsThis study tested the bactericidal efficacy of eight registered disinfectant products against S. aureus ATCC-6538 and P. aeruginosa ATCC-15442 grown on glass coupons using a Center for Disease Control (CDC) biofilm reactor and EPA MLB SOP MB-19. Bactericidal efficacy was determined after treating coupons with disinfectants following standard EPA MLB SOP MB-20.ResultsOverall, sodium hypochlorite and hydrogen peroxide disinfectants had significantly higher bactericidal efficacies than quaternary ammonium chloride disinfectants. We also found that all tested disinfectants except for quaternary ammonium chloride disinfectants met and exceeded the EPA standard for bactericidal efficacy against biofilms.ConclusionIn general, bactericidal efficacy against biofilms differed by active ingredient. The efficacies of sodium hypochlorite and hydrogen peroxide disinfectants did not vary between strains, but there were significant differences between strains treated with quaternary ammonium chloride disinfectants.