Identification and taxonomic characterization of Bordetella pseudohinzii sp. nov. isolated from laboratory-raised mice.

Bordetella hinzii is known to cause respiratory disease in poultry and has been associated with a variety of infections in immunocompromised humans. In addition, there are several reports of B. hinzii infections in laboratory-raised mice. Here we sequenced and analysed the complete genome sequences of multiple B. hinzii-like isolates, obtained from vendor-supplied C57BL/6 mice in animal research facilities on different continents, and we determined their taxonomic relationship to other Bordetella species. The whole-genome based and 16S rRNA gene based phylogenies each identified two separate clades in B. hinzii, one was composed of strains isolated from poultry, humans and a rabbit whereas the other clade was restricted to isolates from mice. Distinctly different estimated DNA-DNA hybridization values, average nucleotide identity scores, gene content, metabolic profiles and host specificity all provide compelling evidence for delineation of the two species, B. hinzii - from poultry, humans and rabbit - and Bordetella pseudohinzii sp. nov. type strain 8-296-03T (=NRRL B-59942T=NCTC 13808T) that infect mice.

Vibrio cholerae leuO Transcription Is Positively Regulated by ToxR and Contributes to Bile Resistance.

UNLABELLED: Vibrio cholerae is an aquatic organism and facultative human pathogen that colonizes the small intestine. In the small intestine, V. cholerae is exposed to a variety of antimicrobial compounds, including bile. V. cholerae resistance to bile is multifactorial and includes alterations in the membrane permeability barrier that are mediated by ToxR, a membrane-associated transcription factor. ToxR has also been shown to be required for activation of the LysR family transcription factor leuO in response to cyclic dipeptides. LeuO has been implicated in the regulation of multiple V. cholerae phenotypes, including biofilm production and virulence. In this study, we investigated the effects of bile on leuO expression. We show that leuO transcription increased in response to bile and bile salts but not in response to other detergents. The bile-dependent increase in leuO expression was dependent on ToxR, which was found to bind directly to the leuO promoter. The periplasmic domain of ToxR was required for basal leuO expression and for the bile-dependent induction of both leuO and ompU transcription. V. cholerae mutants that did not express leuO exhibited increased bile susceptibility, suggesting that LeuO contributes to bile resistance. Our collective results demonstrate that ToxR activates leuO expression in response to bile and that LeuO is a component of the ToxR-dependent responses that contribute to bile resistance. IMPORTANCE: The success of Vibrio cholerae as a human pathogen is dependent upon its ability to rapidly adapt to changes in its growth environment. Growth in the human gastrointestinal tract requires the expression of genes that provide resistance to host antimicrobial compounds, including bile. In this work, we show for the first time that the LysR family regulator LeuO mediates responses in V. cholerae that contribute to bile resistance.

Reciprocal regulation of resistance-nodulation-division efflux systems and the Cpx two-component system in Vibrio cholerae.

The Cpx two-component regulatory system has been shown in Escherichia coli to alleviate stress caused by misfolded cell envelope proteins. The Vibrio cholerae Cpx system was previously found to respond to cues distinct from those in the E. coli system, suggesting that this system fulfills a different physiological role in the cholera pathogen. Here, we used microarrays to identify genes that were regulated by the V. cholerae Cpx system. Our observations suggest that the activation of the V. cholerae Cpx system does not induce expression of genes involved in the mitigation of stress generated by misfolded cell envelope proteins but promotes expression of genes involved in antimicrobial resistance. In particular, activation of the Cpx system induced expression of the genes encoding the VexAB and VexGH resistance-nodulation-division (RND) efflux systems and their cognate outer membrane pore protein TolC. The promoters for these loci contained putative CpxR consensus binding sites, and ectopic cpxR expression activated transcription from the promoters for the RND efflux systems. CpxR was not required for intrinsic antimicrobial resistance, but CpxR activation enhanced resistance to antimicrobial substrates of VexAB and VexGH. Mutations that inactivated VexAB or VexGH efflux activity resulted in the activation of the Cpx response, suggesting that vexAB and vexGH and the cpxP-cpxRA system are reciprocally regulated. We speculate that the reciprocal regulation of the V. cholerae RND efflux systems and the Cpx two-component system is mediated by the intracellular accumulation of an endogenously produced metabolic by-product that is normally extruded from the cell by the RND efflux systems.

The Impact of Water, Sanitation and Hygiene Interventions to Control Cholera: A Systematic Review.

BACKGROUND AND METHODS: Cholera remains a significant threat to global public health with an estimated 100,000 deaths per year. Water, sanitation and hygiene (WASH) interventions are frequently employed to control outbreaks though evidence regarding their effectiveness is often missing. This paper presents a systematic literature review investigating the function, use and impact of WASH interventions implemented to control cholera. RESULTS: The review yielded eighteen studies and of the five studies reporting on health impact, four reported outcomes associated with water treatment at the point of use, and one with the provision of improved water and sanitation infrastructure. Furthermore, whilst the reporting of function and use of interventions has become more common in recent publications, the quality of studies remains low. The majority of papers (>60%) described water quality interventions, with those at the water source focussing on ineffective chlorination of wells, and the remaining being applied at the point of use. Interventions such as filtration, solar disinfection and distribution of chlorine products were implemented but their limitations regarding the need for adherence and correct use were not fully considered. Hand washing and hygiene interventions address several transmission routes but only 22% of the studies attempted to evaluate them and mainly focussed on improving knowledge and uptake of messages but not necessarily translating this into safer practices. The use and maintenance of safe water storage containers was only evaluated once, under-estimating the considerable potential for contamination between collection and use. This problem was confirmed in another study evaluating methods of container disinfection. One study investigated uptake of household disinfection kits which were accepted by the target population. A single study in an endemic setting compared a combination of interventions to improve water and sanitation infrastructure, and the resulting reductions in cholera incidence. DISCUSSION AND RECOMMENDATIONS: This review highlights a focus on particular routes of transmission, and the limited number of interventions tested during outbreaks. There is a distinct gap in knowledge of which interventions are most appropriate for a given context and as such a clear need for more robust impact studies evaluating a wider array of WASH interventions, in order to ensure effective cholera control and the best use of limited resources.

Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels.

Type Six Secretion System of Bordetella bronchiseptica and Adaptive Immune Components Limit Intracellular Survival During Infection.

The Type Six Secretion System (T6SS) is required for Bordetella bronchiseptica cytotoxicity, cytokine modulation, infection, and persistence. However, one-third of recently sequenced Bordetella bronchiseptica strains of the predominantly human-associated Complex IV have lost their T6SS through gene deletion or degradation. Since most human B. bronchiseptica infections occur in immunocompromised patients, we determine here whether loss of Type Six Secretion is beneficial to B. bronchiseptica during infection of immunocompromised mice. Infection of mice lacking adaptive immunity (Rag1-/- mice) with a T6SS-deficient mutant results in a hypervirulent phenotype that is characterized by high numbers of intracellular bacteria in systemic organs. In contrast, wild-type B. bronchiseptica kill their eukaryotic cellular hosts via a T6SS-dependent mechanism that prevents survival in systemic organs. High numbers of intracellular bacteria recovered from immunodeficient mice but only low numbers from wild-type mice demonstrates that B. bronchiseptica survival in an intracellular niche is limited by B and T cell responses. Understanding the nature of intracellular survival during infection, and its effects on the generation and function of the host immune response, are important to contain and control the spread of Bordetella-caused disease.

Host Specificity of Ovine Bordetella parapertussis and the Role of Complement.

The classical bordetellae are comprised of three subspecies that differ from broad to very limited host specificity. Although several lineages appear to have specialized to particular host species, most retain the ability to colonize and grow in mice, providing a powerful common experimental model to study their differences. One of the subspecies, Bordetella parapertussis, is composed of two distinct clades that have specialized to different hosts: one to humans (Bpphu), and the other to sheep (Bppov). While Bpphu and the other classical bordetellae can efficiently colonize mice, Bppov strains are severely defective in their ability to colonize the murine respiratory tract. Bppov genomic analysis did not reveal the loss of adherence genes, but substantial mutations and deletions of multiple genes involved in the production of O-antigen, which is required to prevent complement deposition on B. bronchiseptica and Bpphu strains. Bppov lacks O-antigen and, like O-antigen mutants of other bordetellae, is highly sensitive to murine complement-mediated killing in vitro. Based on these results, we hypothesized that Bppov failed to colonize mice because of its sensitivity to murine complement. Consistent with this, the Bppov defect in the colonization of wild type mice was not observed in mice lacking the central complement component C3. Furthermore, Bppov strains were highly susceptible to killing by murine complement, but not by sheep complement. These data demonstrate that the failure of Bppov to colonize mice is due to sensitivity to murine, but not sheep, complement, providing a mechanistic example of how specialization that accompanies expansion in one host can limit host range.

Vibrio cholerae ToxR downregulates virulence factor production in response to cyclo(Phe-Pro).

UNLABELLED: Vibrio cholerae is an aquatic organism that causes the severe acute diarrheal disease cholera. The ability of V. cholerae to cause disease is dependent upon the production of two critical virulence determinants, cholera toxin (CT) and the toxin-coregulated pilus (TCP). The expression of the genes that encode for CT and TCP production is under the control of a hierarchical regulatory system called the ToxR regulon, which functions to activate virulence gene expression in response to in vivo stimuli. Cyclic dipeptides have been found to be produced by numerous bacteria, yet their biological function remains unknown. V. cholerae has been shown to produce cyclo(Phe-Pro). Previous studies in our laboratory demonstrated that cyclo(Phe-Pro) inhibited V. cholerae virulence factor production. For this study, we report on the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We have demonstrated that exogenous cyclo(Phe-Pro) activated the expression of leuO, a LysR-family regulator that had not been previously associated with V. cholerae virulence. Increased leuO expression repressed aphA transcription, which resulted in downregulation of the ToxR regulon and attenuated CT and TCP production. The cyclo(Phe-Pro)-dependent induction of leuO expression was found to be dependent upon the virulence regulator ToxR. Cyclo(Phe-Pro) did not affect toxR transcription or ToxR protein levels but appeared to enhance the ToxR-dependent transcription of leuO. These results have identified leuO as a new component of the ToxR regulon and demonstrate for the first time that ToxR is capable of downregulating virulence gene expression in response to an environmental cue. IMPORTANCE: The ToxR regulon has been a focus of cholera research for more than three decades. During this time, a model has emerged wherein ToxR functions to activate the expression of Vibrio cholerae virulence factors upon host entry. V. cholerae and other enteric bacteria produce cyclo(Phe-Pro), a cyclic dipeptide that we identified as an inhibitor of V. cholerae virulence factor production. This finding suggested that cyclo(Phe-Pro) was a negative effector of virulence factor production and represented a molecule that could potentially be exploited for therapeutic development. In this work, we investigated the mechanism by which cyclo(Phe-Pro) inhibited virulence factor production. We found that cyclo(Phe-Pro) signaled through ToxR to activate the expression of leuO, a new virulence regulator that functioned to repress virulence factor production. Our results have identified a new arm of the ToxR regulon and suggest that ToxR may play a broader role in pathogenesis than previously known.

Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus.

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.