Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection, B. cereus G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the B. anthracis virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental B. cereus isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from B. cereus G9241. PCR analysis demonstrated that both clinical isolates contained B. anthracis pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victim's worksite contained the cap A, B, and C genes required for capsule biosynthesis in B. anthracis. Both clinical isolates expressed a capsule; however, neither was composed of poly-D-glutamic acid. Although most B. cereus isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some B. cereus strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.
Listeria monocytogenes must overcome a variety of stress conditions in the host digestive tract to cause foodborne infections. The alternative sigma factor s B , encoded by sigB, is responsible for regulating transcription of several L. monocytogenes virulence and stress-response genes, including genes that contribute to establishment of gastrointestinal infections. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the virulence genes inlA and bsh, the stress-response genes opuCA and lmo0669 (encoding a carnitine transporter and an oxidoreductase, respectively) and the housekeeping gene rpoB. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting osmotic (0?3 M NaCl) or acid (pH 4?5) conditions typical for the human intestinal lumen. In exponential-phase cells, as well as under osmotic and acid stress, inlA, opuCA and bsh showed significantly lower absolute expression levels in a L. monocytogenes DsigB null mutant compared to wild-type. A statistical model that normalized target gene expression relative to rpoB showed that accumulation of inlA, opuCA and bsh transcripts was significantly increased in the wild-type strain within 5 min of acid and osmotic stress exposure; lmo0669 transcript accumulation increased significantly only after acid exposure. It was concluded that s B is essential for rapid induction of the tested stress-response and virulence genes under conditions typically encountered during gastrointestinal passage. As inlA, bsh and opuCA are critical for gastrointestinal infections in animal models, the data also suggest that s B contributes to the ability of L. monocytogenes to cause foodborne infections.
Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.
The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, B , which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ⌬sigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ⌬sigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ⌬sigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is B independent. B-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functionalB reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms.B does not appear to contribute to pH i homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH i buffering by the GAD system under the conditions examined in this study. In summary, a functional B protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.
Listeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity. The alternative sigma factor σ B contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific σ B-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative σ B-dependent genes based on the presence of a predicted σ B-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm σ B-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes ΔsigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes ΔsigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0·5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of σ B activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA–gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0·5 M NaCl or 0·5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific σ B-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.
This study was designed to evaluate the hypothesis that some of the Listeria monocytogenes subtypes associated with foods, specifically smoked fish, may have an attenuated ability to cause human disease. We tested this hypothesis by using two different approaches: (i) comparison of molecular subtypes found among 117 isolates from smoked fish, raw materials, fish in process, and processing environments with subtypes found among a collection of 275 human clinical isolates and (ii) the evaluation of the cytopathogenicity of industrial isolates. Ribotyping and PCR-restriction fragment length polymorphism typing of the hlyA and actA genes differentiated 23 subtypes among the industrial isolates and allowed classification of the isolates into three genetic lineages. A significantly higher proportion of human isolates (69.1%) than industrial isolates (36.8%) were classified as lineage I, which contains human sporadic isolates and all epidemic isolates. All other industrial isolates (63.2%) were classified as lineage II, which contains only human sporadic isolates. Lineage I ribotypes DUP-1038B and DUP-1042B represented a significantly higher proportion of the human isolates than industrial isolates (5.1%). Lineage II ribotypes DUP-1039C, DUP-1042C, and DUP-1045, shown previously to persist in the smoked fish processing environment, represented nearly 50% of the industrial isolates, compared to 7.6% of the human isolates. Representatives of each subtype were evaluated with a tissue culture plaque assay. Lineage I isolates formed plaques that were significantly larger than those formed by lineage II isolates. Isolates from the smoked fish industry representing three ribotypes formed no plaques or small plaques, indicating that they had an impaired ability to infect mammalian cells. While L. monocytogenes clonal groups linked to human listeriosis cases and outbreaks were isolated, our data also suggest that at least some L. monocytogenes subtypes present in ready-to-eat foods may have limited human-pathogenic potential.Listeria monocytogenes is responsible for nearly one-fourth of all estimated food-borne-disease-related deaths caused by known pathogens in the United States each year, which highlights its significance as a public health concern (25). The majority of human listeriosis cases occur in pregnant women, neonates, immunosupressed individuals, and the elderly (12). As a growing segment of our population falls into high-risk groups, improved methods for reducing the levels of L. monocytogenes in foods are essential. A better understanding of the ecology, transmission, and pathogenicity of this organism should facilitate development of effective strategies.While L. monocytogenes causes relatively few human disease cases, particularly compared to many other food-borne pathogens (25), it appears to be commonly present in raw and readyto-eat foods. U.S. Department of Agriculture data, for example, indicated a 2.5% prevalence of L. monocytogenes in 3,547 samples of ready-to-eat products surveyed in 1998 and a 4.6 and 2...
The regulation of the compatible solute transport systems in Listeria monocytogenes by the stress-inducible sigma factor B was investigated. Using wild-type strain 10403S and an otherwise isogenic strain carrying an in-frame deletion in sigB, we have examined the role of B in regulating the ability of cells to utilize betaine and carnitine during growth under conditions of hyperosmotic stress. Cells lacking B were defective for the utilization of carnitine but retained the ability to utilize betaine as an osmoprotectant. When compatible solute transport studies were performed, the initial rates of uptake of both betaine and carnitine were found to be reduced in the sigB mutant; carnitine transport was almost abolished, whereas betaine transport was reduced to approximately 50% of that of the parent strain. Analysis of the cytoplasmic pools of compatible solutes during balanced growth revealed that both carnitine and betaine steady-state pools were reduced in the sigB mutant. Transcriptional reporter fusions to the opuC (which encodes an ABC carnitine transporter) and betL (which encodes an a secondary betaine transporter) operons were generated by using a promoterless copy of the gus gene from Escherichia coli. Measurement of -glucuronidase activities directed by opuC-gus and betL-gus revealed that transcription of opuC is largely B dependent, consistent with the existence of a potential B consensus promoter motif upstream from opuCA. The transcription of betL was found to be sigB independent. Reverse transcriptase PCR experiments confirmed these data and indicated that the transcription of all three known compatible solute uptake systems (opuC, betL, and gbu), as well as a gene that is predicted to encode a compatible solute transporter subunit (lmo1421) is induced in response to elevated osmolarity. The osmotic induction of opuCA and lmo1421 was found to be strongly B dependent. Together these observations suggest that B plays a major role in the regulation of carnitine utilization by L. monocytogenes but is not essential for betaine utilization by this pathogen.
Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and -lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A -lactamase and was previously implicated in resistance to -lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei. Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.
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