Summary Whole genome sequencing is increasing used in epidemiology, e.g. for tracing outbreaks of food‐borne diseases. This requires in‐depth understanding of pathogen emergence, persistence and genomic diversity along the food production chain including in food processing plants. We sequenced the genomes of 80 isolates of Listeria monocytogenes sampled from Danish food processing plants over a time‐period of 20 years, and analysed the sequences together with 10 public available reference genomes to advance our understanding of interplant and intraplant genomic diversity of L. monocytogenes. Except for three persisting sequence types (ST) based on Multi Locus Sequence Typing being ST7, ST8 and ST121, long‐term persistence of clonal groups was limited, and new clones were introduced continuously, potentially from raw materials. No particular gene could be linked to the persistence phenotype. Using time‐based phylogenetic analyses of the persistent STs, we estimate the L. monocytogenes evolutionary rate to be 0.18–0.35 single nucleotide polymorphisms/year, suggesting that the persistent STs emerged approximately 100 years ago, which correlates with the onset of industrialization and globalization of the food market.

The degU (lmo2515) gene encodes a putative response regulator in the food-borne pathogen Listeria monocytogenes. It has 63% amino acid identity to the DegU response regulator of Bacillus subtilis. We have characterized the degU gene product in L. monocytogenes EGD by generation of a deletion mutant. The DeltadegU mutant was found to be non-motile in motility plate assay and no flagellin was detected. The mutant was attenuated in challenge of mice. Northern blot analysis suggested that the degU gene product is a transcriptional activator of the flagellin gene, flaA, at 25 degrees C. However, the degU gene product had no influence on the transcription of prfA encoding the major virulence regulator, PrfA. The results indicate that the putative DegU response regulator is a pleiotropic regulator involved in expression of both motility at low temperature and in vivo virulence in mice.

BackgroundIn comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice.Methodology/Principal FindingsWe constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence.Conclusions/SignificanceOur results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type [1]. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.

Listeria monocytogenes can cause the serious infection listeriosis, which despite antibiotic treatment has a high mortality. Understanding the response of L. monocytogenes to antibiotic exposure is therefore important to ensure treatment success. Some bacteria survive antibiotic treatment by formation of persisters, which are a dormant antibiotic-tolerant subpopulation. The purpose of this study was to determine whether L. monocytogenes can form persisters and how bacterial physiology affects the number of persisters in the population. A stationary-phase culture of L. monocytogenes was adjusted to 10 8 CFU ml ؊1, and 10 3 to 10 4 CFU ml ؊1 survived 72-h treatment with 100 g of norfloxacin ml ؊1 , indicating a persister subpopulation. This survival was not caused by antibiotic resistance as regrown persisters were as sensitive to norfloxacin as the parental strain. Higher numbers of persisters (10 5 to 10 6 ) were surviving when older stationary phase or surface-associated cells were treated with 100 g of norfloxacin ml ؊1. The number of persisters was similar when a ⌬sigB mutant and the wild type were treated with norfloxacin, but the killing rate was higher in the ⌬sigB mutant. Dormant norfloxacin persisters could be activated by the addition of fermentable carbohydrates and subsequently killed by gentamicin; however, a stable surviving subpopulation of 10 3 CFU ml ؊1 remained. Nitrofurantoin that has a growth-independent mode of action was effective against both growing and dormant cells, suggesting that eradication of persisters is possible. Our study adds L. monocytogenes to the list of bacterial species capable of surviving bactericidal antibiotics in a dormant stage, and this persister phenomenon should be borne in mind when developing treatment regimens.

Salmonella enterica serovar Typhimurium requires the type III secretion system encoded by Salmonella pathogenicity island 1 (SPI1) and controlled by the master regulator, HilA, to penetrate the intestinal epithelium. Numerous regulators affect virulence through influence on this system, including the proteolytic component ClpP, the stationary phase regulator RpoS and the carbon-storage regulator CsrA. However, the mechanism behind the ClpP regulation is not fully understood. To elucidate this we examined differentially expressed genes in a DclpP mutant compared with WT using global transcriptomic analysis. SPI1 and SPI4 virulence genes were significantly downregulated in the DclpP mutant, whereas several RpoS-dependent genes and the fliC gene encoding flagellin were upregulated. While the DclpP mutant was attenuated in cell invasion, this attenuation was not present in a DclpP/rpoS : : amp double mutant, suggesting the repression of invasion was directed through RpoS. The expression of the csrA virulence regulator was increased in the DclpP mutant and decreased in the rpoS : : amp and DclpP/rpoS : : amp mutants, indicating that ClpP affects the csrA expression level as well. Thus, this study suggests that ClpP affects SPI1 expression and thereby virulence indirectly through its regulation of both RpoS and CsrA.