Bacterial Populations in Complementary Foods and Drinking-water in Households with Children Aged 10-15 Months in Zanzibar, Tanzania

Bacteria were quantified in samples of drinking-water and in two porridges prepared for infant-feeding [fortified instant soy-rice porridge (SRP) and cooked porridge (Lishe bora, LB)] in 54 households. Bacterial numbers were measured again after the porridges had been held at room temperature for four hours (T4). Findings were benchmarked against bacterial numbers in traditional complementary foods sampled from 120 households. Total bacteria, coliform, and Enterobacteriaceae counts were enumerated using Petrifilm™. The mean log bacterial numbers were the lowest for LB at T0 (2.24±0.84 cfu/g aerobic counts) and the highest for SRP at T4 (4.63±0.56 cfu/g aerobic counts). The total bacteria, coliform and Enterobacteriaceae counts were higher at T4 than at T0 for LB (p≤0.001); however, only the coliform and Enterobacteriaceae counts were higher at T4 than at T0 for SRP (p<0.001). Drinking-water, SRP0, traditional foods, and SRP4 all had the mean aerobic counts higher than the acceptable cut-off but the total bacterial count in SRP0 was not significantly (p=0.543) different from drinking-water. However, coliform and Enterobacteriaceae counts in SRP0 were higher than in drinking-water (p<0.001). Also, although the aerobic counts of SRP4 were not significantly (p>0.999) different from traditional foods, the coliform and Enterobacteriaceae counts were significantly higher in SRP4 than in traditional foods (p<0.001). It is, therefore, recommended that food safety concerns be addressed when improving complementary foods

Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs

BACKGROUND: Identification 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 σ(B)-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 σ(B), a major regulator of genes contributing to stress response, including stresses encountered upon entry into stationary phase. RESULTS: Overall, 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 σ(B)-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 σ(B )promoters upstream of 82 of the 96 σ(B)-dependent genes and upstream of the one σ(B)-dependent ncRNA. The RNA-Seq data also enabled annotation of putative operons as well as visualization of 5'- and 3'-UTR regions. CONCLUSIONS: The 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

Bacterial Stress Responses: What Doesn't Kill Them Can Make Them Stronger

Identifying specific mechanisms that contribute to microbial survival under rapidly changing conditions could provide insight into stress response systems across life forms

A Small RNA Controls Expression of the Chitinase ChiA in Listeria monocytogenes

In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for efficient binding to its target. In the present study, we sought to gain more insight into the functional role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment, and we provide evidence that both LhrA and Hfq act to down-regulate the expression of lmo0302 and chiA. Furthermore, in vitro binding experiments show that Hfq stimulates the base pairing of LhrA to chiA mRNA. Finally, we demonstrate that LhrA has a negative effect on the chitinolytic activity of L. monocytogenes. In marked contrast to this, we found that Hfq has a stimulating effect on the chitinolytic activity, suggesting that Hfq plays multiple roles in the complex regulatory pathways controlling the chitinases of L. monocytogenes

Effects of Acid Stress on \u3cem\u3eVibrio parahaemolyticus\u3c/em\u3e Survival and Cytotoxicity

For several foodbome bacterial pathogens, an acid tolerance response appears to be an important strategy for counteracting acid stress imposed either during food processing or by the human host. The acid tolerance response enhances bacterial survival of lethal acid challenge following prior exposure to sublethal acidic conditions. Previous studies have revealed relationships between a foodbome pathogen\u27s ability to survive acid challenge and its infectious dose. Vibrio parahaemolyticus is capable of causing gastroenteritis when sufficient cells of pathogenic strains are consumed. This study was designed to characterize acid sensitivities and to compare the effect of sublethal acid exposure (adaptation) on survival capabilities and cytotoxicities of different V. parahaemolyticus strains. Survival of acid challenge by stationary-phase cells differed by up to 3 log CFU/ml among the 25 isolates tested. No differences in acid resistance were found between strains when they were grouped by source (clinical isolates versus those obtained from food). Survival at pH 3.6 for log-phase cells that had been previously exposed to sublethal acidic conditions (pH 5.5) was enhanced compared with that for cells not previously exposed to pH 5.5. However, for stationary-phase cells, exposure to pH 5.5 impaired both subsequent survival at pH 3.6 and cytotoxicity to human epithelial cells. Relative cytotoxicities of nonadapted starionary-phase cells were 1.2- to 4.8-fold higher than those of adapted cells. Sublethal acid exposure appears to impose measurable growth phase-dependent effects on subsequent lethal acid challenge survival and cytotoxicity of

Epidemiology, Pathogenesis, and Prevention of Foodbome \u3cem\u3eVibrio parahaemolyticus\u3c/em\u3e Infections

Since its discovery about 50 years ago, Vibrio parahaemolyticus has been implicated as a major cause of foodborne illness around the globe. V. parahaemolyticus is a natural inhabitant of marine waters. Human infections are most commonly associated with the consumption of raw, undercooked or contaminated shellfish. A few individual V. parahaemolyticus virulence factors, including the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), have been investigated in depth, yet a comprehensive understanding of this organism\u27s ability to cause disease remains unclear. Since 1996, serotype 03:K6 strains have been associated with an increased incidence of gastroenteritis in India and in Southeast Asia, and with large-scale foodborne outbreaks in the United States (US). In light of the emerging status of pathogenic V. parahaemolyticus, the US Food and Drug Administration conducted a microbial risk assessment to characterize the risk of contracting V. parahaemolyticus infections from consuming raw oysters. This review summarizes epidemiological findings, discusses recognized and putative V. parahaemolyticus virulence factors and pathogenicity mechanisms, and describes strategies for preventing V. parahaemolyticus infections

Evaluation of a Tissue Culture-Based Approach for Differentiating between Virulent and Avirulent \u3cem\u3eVibrio parahaemolyticus\u3c/em\u3e Strains Based on Cytotoxicity

The ability of only a subset of Vibrio parahaemolyticus strains to cause human infection underscores the need for an analytical method that can effectively differentiate between pathogenic strains and those that do not cause disease. We tested the feasibility of a tissue culture-based assay to determine whether clinical isolates could be differentiated from nonclinical isolates based on relative isolate cytopathogenicity. To screen for cytotoxic capability, we measured relative extracellular lactate dehydrogenase as an indicator of host cell damage in five different mammalian cell lines in the presence of V. parahaemolyticus. Isolates originating from clinical sources exhibited 15.5 to 59.3% relative cytotoxicity, whereas those originating from food source exhibited 4.4 to 54.9% relative cytotoxicity. In the presence of ~1.2 X 10n6 cells, cytotoxicity was 1.6- to 3.5-fold higher (P \u3c 0.05) for clinical isolates than for nonclinical isolates in L2, Henle 407, and Caco-2 cell lines. V. parahaemolyticus serotype 03:K6 clinical isolates had 1.6- to 2.l-fold higher cytotoxicity than did the non-03:K6 clinical isolates, with significantly higher cytotoxicity in HeLa, J774A.1, and Henle 407 cells than in L2 and Caco-2 ceUs. Because V. parahaemolyticus often is found in oysters, the effect of the presence of an oyster matrix on assay efficacy was also tested with L2 cells. The cytotoxicity elicited by a highly cytotoxic V. parahaemolyticus isolate was not affected by the presence of oyster tissue, suggesting that an oyster matrix will not interfere with assay sensitivity. In the present format, this assay can detect the presence of \u3e10n5 cells of a virulent V. parahaemolyticus strain in an oyster matrix

Listeria monocytogenes σ(B) Contributes to Invasion of Human Intestinal Epithelial Cells

The role of σ(B) in Listeria monocytogenes infection of human intestinal epithelial cells was investigated. Invasion defects associated with loss of σ(B) paralleled those of a ΔinlA strain independently of the σ(B)-dependent P2(prfA) promoter. Concomitantly, amounts of inlA transcript and InlA protein were significantly decreased in the ΔsigB strain