Increased iron utilization and oxidative stress tolerance in a Vibrio cholerae flrA mutant confers resistance to amoeba predation.

IMPORTANCE: Persistence of V. cholerae in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face predation pressure by heterotrophic protists such as the free-living amoeba A. castellanii. This study explores how a mutant of V. cholerae adapts to acquire essential nutrients and survive predation. Here, we observed that up-regulation of iron acquisition genes and genes regulating resistance to oxidative stress enhances pathogen fitness. Our data show that V. cholerae can defend predation to overcome nutrient limitation and oxidative stress, resulting in an enhanced survival inside the protozoan hosts.

A commensal-encoded genotoxin drives restriction of Vibrio cholerae colonization and host gut microbiome remodeling.

SignificanceIn a polymicrobial battlefield where different species compete for nutrients and colonization niches, antimicrobial compounds are the sword and shield of commensal microbes in competition with invading pathogens and each other. The identification of an Escherichia coli-produced genotoxin, colibactin, and its specific targeted killing of enteric pathogens and commensals, including Vibrio cholerae and Bacteroides fragilis, sheds light on our understanding of intermicrobial interactions in the mammalian gut. Our findings elucidate the mechanisms through which genotoxins shape microbial communities and provide a platform for probing the larger role of enteric multibacterial interactions regarding infection and disease outcomes.

Crystal structure of oligoribonuclease from Vibrio cholerae O1 El Tor with bound peptide.

Oligoribonuclease (Orn), a member of the DEDDh superfamily, can hydrolyse 2-5 nt nanoRNAs to mononucleotides. It is involved in maintaining the intracellular levels of RNA, c-di-GMP signalling and transcription initiation in many bacterial species. Here, the crystal structure of Orn from Vibrio cholerae O1 El Tor (VcOrn) is reported at a resolution of 1.7 Å. VcOrn, which consists of nine α-helices and six ß-strands, crystallizes with a single monomer in the asymmetric unit but forms a homodimer via crystallographic twofold symmetry. Electron density is observed in the active pocket that corresponds to an intersubunit N-terminal expression tag with sequence GPLGSHHH. The positively charged N-terminal tag binds in the negatively charged nucleotide-binding pocket with a buried surface area of ∼500 Å2. The N-terminal tag interacts with VcOrn via π-π stacking with two conserved residues involved in nucleotide binding, as well as via salt bridges and hydrogen bonds. The structure reported here reveals that the active pocket can accommodate polypeptides in addition to nucleotides, thus providing an important starting point for investigation into substrate modification and inhibitor design targeting VcOrn.

Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens.

Pathogenic bacteria can rapidly respond to stresses such as reactive oxygen species (ROS) using reversible redox-sensitive oxidation of cysteine thiol (-SH) groups in regulators. Here, we use proteomics to profile reversible ROS-induced thiol oxidation in Vibrio cholerae, the etiologic agent of cholera, and identify two modified cysteines in ArcA, a regulator of global carbon oxidation that is phosphorylated and activated under low oxygen. ROS abolishes ArcA phosphorylation but induces the formation of an intramolecular disulfide bond that promotes ArcA-ArcA interactions and sustains activity. ArcA cysteines are oxidized in cholera patient stools, and ArcA thiol oxidation drives in vitro ROS resistance, colonization of ROS-rich guts, and environmental survival. In other pathogens, such as Salmonella enterica, oxidation of conserved cysteines of ArcA orthologs also promotes ROS resistance, suggesting a common role for ROS-induced ArcA thiol oxidation in modulating ArcA activity, allowing for a balance of expression of stress- and pathogenesis-related genetic programs.

A Phase 2a randomized, single-center, double-blind, placebo-controlled study to evaluate the safety and preliminary efficacy of oral iOWH032 against cholera diarrhea in a controlled human infection model.

Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V. cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study. Trial registration: This study is registered with ClinicalTrials.gov as NCT04150250.

Immunomodulatory Effects of Adipose-derived Mesenchymal Stem Cells on Epithelial Cells Function in Response to Vibrio cholera in a Co-culture Model.

Inflammation-induced by the interaction of the Vibrio cholerae with the epithelial cells is considered as a main cause of bacteria spreading through the gastrointestinal tract and its consequences. Because of the immunomodulatory and antibacterial properties of adipose-derived mesenchymal stem cells (AD-MSCs), this study aimed to investigate the effect of AD-MSCs on the interaction of the bacterial-epithelial cell. Caco-2 differentiated to intestinal epithelial cells co-cultured with AD-MSCs in a 1:1 ratio of the surface area of six-well plates, for 48 hours. After exposure to Vibrio cholerae, bacterial attachment and internalization were evaluated. Secretions of interleukin (IL) -6, prostaglandin E2 (PGE2), and nitric oxide (NO) were also measured using ELISA, and Griess assay, respectively. In addition, the expression of chloratoxin (Ctx-ß) and inflammatory cytokines such as TNF-α, IL-1ß, and IL-8 were evaluated by real-time polymerase chain reaction (RT-PCR). The rate of apoptosis was also evaluated by Annexin V-PI flow cytometry. Bacterial attachment and Ctx-ß expression were significantly reduced in the co-culture group compared to the Vibrio cholerae-exposed Caco-2. IL-6 and PGE2 secretion increased in the co-culture group. NO, was also slightly reduced in exposure to Vibrio cholerae. An elevated level of bacterial internalization was observed in the co-culture group compared to the Caco-2 cells leading to an increase in the expression of pro-inflammatory cytokines. The rate of apoptosis was also increased significantly. Cell-to-cell contact of AD-MSCs and Caco-2 promoted inflammatory responses and disruption of the epithelium barrier by enhancing bacterial invasion. This may be due to the high expression of surface matrix metalloproteinases on MSCs.

A Systems Biology Approach Identifies B Cell Maturation Antigen (BCMA) as a Biomarker Reflecting Oral Vaccine Induced IgA Antibody Responses in Humans.

Vaccines against enteric diseases could improve global health. Despite this, only a few oral vaccines are currently available for human use. One way to facilitate such vaccine development could be to identify a practical and relatively low cost biomarker assay to assess oral vaccine induced primary and memory IgA immune responses in humans. Such an IgA biomarker assay could complement antigen-specific immune response measurements, enabling more oral vaccine candidates to be tested, whilst also reducing the work and costs associated with early oral vaccine development. With this in mind, we take a holistic systems biology approach to compare the transcriptional signatures of peripheral blood mononuclear cells isolated from volunteers, who following two oral priming doses with the oral cholera vaccine Dukoral®, had either strong or no vaccine specific IgA responses. Using this bioinformatical method, we identify TNFRSF17, a gene encoding the B cell maturation antigen (BCMA), as a candidate biomarker of oral vaccine induced IgA immune responses. We then assess the ability of BCMA to reflect oral vaccine induced primary and memory IgA responses using an ELISA BCMA assay on a larger number of samples collected in clinical trials with Dukoral® and the oral enterotoxigenic Escherichia coli vaccine candidate ETVAX. We find significant correlations between levels of BCMA and vaccine antigen-specific IgA in antibodies in lymphocyte secretion (ALS) specimens, as well as with proportions of circulating plasmablasts detected by flow cytometry. Importantly, our results suggest that levels of BCMA detected early after primary mucosal vaccination may be a biomarker for induction of long-lived vaccine specific memory B cell responses, which are otherwise difficult to measure in clinical vaccine trials. In addition, we find that ALS-BCMA responses in individuals vaccinated with ETVAX plus the adjuvant double mutant heat-labile toxin (dmLT) are significantly higher than in subjects given ETVAX only. We therefore propose that as ALS-BCMA responses may reflect the total vaccine induced IgA responses to oral vaccination, this BCMA ELISA assay could also be used to estimate the total adjuvant effect on vaccine induced-antibody responses, independently of antigen specificity, further supporting the usefulness of the assay.

Vibrio cholerae FeoB hydrolyzes ATP and GTP in vitro in the absence of stimulatory factors.

Feo is the most widely conserved system for ferrous iron transport in prokaryotes, and it is important for virulence in some pathogens. However, its mechanism of iron transport is not fully understood. In this study, we used full-length Vibrio cholerae FeoB (VcFeoB) as a model system to study whether its enzymatic activity is affected by regulatory factors commonly associated with FeoB proteins from other species or with G-proteins that have homology to FeoB. VcFeoB showed a higher rate of hydrolysis of both ATP and GTP than its N-terminal domain alone; likewise, ions such as K+ and Fe2+ did not modulate its nucleotide hydrolysis. We also showed that the three V. cholerae Feo proteins (FeoA, FeoB, and FeoC) work in a 1 : 1 : 1 molar ratio in vivo. Although both FeoA and FeoC are required for Feo-mediated iron transport, neither of these proteins affected the VcFeoB NTPase rate. These results are consistent with an active transport mechanism independent of stimulatory factors and highlight the importance of using full-length FeoB proteins as a reliable proxy to study Feo-mediated iron transport in vitro.

Synthesis of an acyl-acyl carrier protein synthetase inhibitor to study fatty acid recycling.

Fatty acids are essential to most organisms and are made endogenously by the fatty acid synthase (FAS). FAS is an attractive target for antibiotics and many inhibitors are in clinical development. However, some gram-negative bacteria harbor an enzyme known as the acyl-acyl carrier protein synthetase (AasS), which allows them to scavenge fatty acids from the environment and shuttle them into FAS and ultimately lipids. The ability of AasS to recycle fatty acids may help pathogenic gram-negative bacteria circumvent FAS inhibition. We therefore set out to design and synthesize an inhibitor of AasS and test its effectiveness on an AasS enzyme from Vibrio harveyi, the most well studied AasS to date, and from Vibrio cholerae, a pathogenic model. The inhibitor C10-AMS [5'-O-(N-decanylsulfamoyl)adenosine], which mimics the tightly bound acyl-AMP reaction intermediate, was able to effectively inhibit AasS catalytic activity in vitro. Additionally, C10-AMS stopped the ability of Vibrio cholerae to recycle fatty acids from media and survive when its endogenous FAS was inhibited with cerulenin. C10-AMS can be used to study fatty acid recycling in other bacteria as more AasS enzymes continue to be annotated and provides a platform for potential antibiotic development.

The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP.

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in "cis-mediated" style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

A comprehensive review of therapeutic approaches available for the treatment of cholera.

OBJECTIVES: The oral rehydration solution is the most efficient method to treat cholera; however, it does not interfere in the action mechanism of the main virulence factor produced by Vibrio cholerae, the cholera toxin (CT), and this disease still stands out as a problem for human health worldwide. This review aimed to describe therapeutic alternatives available in the literature, especially those related to the search for molecules acting upon the physiopathology of cholera. KEY FINDINGS: New molecules have offered a protection effect against diarrhoea induced by CT or even by infection from V. cholerae. The receptor regulator cystic fibrosis channel transmembrane (CFTR), monosialoganglioside (GM1), enkephalinase, AMP-activated protein kinase (AMPK), inhibitors of expression of virulence factors and activators of ADP-ribosylarginine hydrolase are the main therapeutic targets studied. Many of these molecules or extracts still present unclear action mechanisms. CONCLUSIONS: Knowing therapeutic alternatives and their molecular mechanisms for the treatment of cholera could guide us to develop a new drug that could be used in combination with the rehydration solution.

Repositioning of a mucolytic drug to a selective antibacterial against Vibrio cholerae.

Drug repositioning, the approach to explore existing drugs for use in new therapeutic indications, has emerged as an alternative drug development strategy. In this study, we found that a mucolytic drug, N-acetylcysteine (NAC) showed antibacterial activity against Vibrio cholerae. NAC can provide acid stress that selectively inhibited the growth of V. cholerae among other bacterial pathogens. To address the antibacterial mechanism of NAC against V. cholerae, six acr (acetylcys-teine-resistant) mutants were isolated from 3,118 random transposon insertion clones. The transposon insertion sites of the six mutants were mapped at the five genes. All these mutants did not display NAC resistance under acidic conditions, despite their resistance to NAC under alkaline conditions, indicating that the NAC resistance directed by the acr mutations was independent of the unusual pH-sensitivity of V. cholerae. Furthermore, all these mutants displayed attenuated virulence and reduced biofilm formation, suggesting that the acr genes are required for pathogenesis of V. cholerae. This study validates the relevance of drug repositioning for antibacterials with new modes of action and will provide an insight into a novel antibacterial therapy for V. cholerae infections to minimize side effects and resistance emergence.

Cyclic nucleotides, gut physiology and inflammation.

Misregulation of gut function and homeostasis impinges on the overall well-being of the entire organism. Diarrheal disease is the second leading cause of death in children under 5 years of age, and globally, 1.7 billion cases of childhood diarrhea are reported every year. Accompanying diarrheal episodes are a number of secondary effects in gut physiology and structure, such as erosion of the mucosal barrier that lines the gut, facilitating further inflammation of the gut in response to the normal microbiome. Here, we focus on pathogenic bacteria-mediated diarrhea, emphasizing the role of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in driving signaling outputs that result in the secretion of water and ions from the epithelial cells of the gut. We also speculate on how this aberrant efflux and influx of ions could modulate inflammasome signaling, and therefore cell survival and maintenance of gut architecture and function.

Vibrio cholera and V. parahaemolyticus coinfection in an oncology patient with gastroenteritis.

Vibrio-related gastroenteritis in the United States is mostly associated with the consumption of raw or improperly cooked seafood. We describe a case of a stage IV lung adenocarcinoma patient who became ill after eating crab while visiting Upstate New York. Molecular testing and culture confirmed a coinfection with V. parahaemolyticus and a nontoxigenic strain V. cholera.

El Tor Biotype Vibrio cholerae Activates the Caspase-11-Independent Canonical Nlrp3 and Pyrin Inflammasomes.

Vibrio cholerae is a Gram-negative enteropathogen causing potentially life-threatening cholera disease outbreaks, for which the World Health Organization currently registers 2-4 million cases and ~100.000 cholera-associated deaths annually worldwide. Genomic Vibrio cholerae research revealed that the strains causing this ongoing cholera pandemic are members of the El Tor biotype, which fully replaced the Classical biotype that caused former cholera pandemics. While both of these biotypes express the characteristic Cholera Toxin (CT), the El Tor biotype additionally expresses the accessory toxins hemolysin (hlyA) and multifunctional auto-processing repeat-in-toxin (MARTX). Previous studies demonstrated that the Classical biotype of Vibrio cholerae triggers caspase-11-dependent non-canonical inflammasome activation in macrophages following CT-mediated cytosolic delivery of LPS. In contrast to the Classical biotype, we here show that El Tor Vibrio cholerae induces IL-1ß maturation and secretion in a caspase-11- and CT-independent manner. Instead, we show that El Tor Vibrio cholerae engages the canonical Nlrp3 inflammasome for IL-1ß secretion through its accessory hlyA toxin. We further reveal the capacity of this enteropathogen to engage the canonical Pyrin inflammasome as an accessory mechanism for IL-1ß secretion in conditions when the pro-inflammatory hlyA-Nlrp3 axis is blocked. Thus, we show that the V. cholerae El Tor biotype does not trigger caspase-11 activation, but instead triggers parallel Nlrp3- and Pyrin-dependent pathways toward canonical inflammasome activation to induce IL-1ß-mediated inflammatory responses. These findings further unravel the complex inflammasome activating mechanisms that can be triggered when macrophages face the full arsenal of El Tor Vibrio cholerae toxins, and as such increase our understanding of host-pathogen interactions in the context of the Vibrio cholerae biotype associated with the ongoing cholera pandemic.

Serotype-shifting gene rfbT is a direct transcriptional target of cAMP receptor protein (CRP) in V. cholerae O1.

Ogawa and Inaba are two main serotypes of O1 V. cholerae and alternate among cholera epidemics. The rfbT gene encodes a methyltransferase and is required for Ogawa serotype. The Inaba serotype is the consequence of genetic alterations in rfbT gene which results in loss-of-function enzyme product. However, the expression and regulation of rfbT has not been understood yet. Here we demonstrated that a global regulator, cAMP receptor protein (CRP), positively regulates rfbT transcription through a non-canonical CRP binding site (CBS) in its promoter region. This finding is supported by the analyses of rfbT mRNA abundance, rfbT-lacZ fusions and electrophoretic mobility shift assay (EMSA). The analyses of rfbT mRNA level in wild type (WT), Δcrp, and lower or higher level of cAMP backgrounds revealed that CRP is required for rfbT expression in response to intracellular cAMP level. Subsequent rfbT-lacZ fusions and EMSA collectively displayed that cAMP-CRP complex transcriptionally activates rfbT through directly binding to CBS in rfbT promoter region. Consistently, serological microagglutination test showed that crp deletion resulted in at least 4-fold decrease in titer of Ogawa serum compared to its WT. These results expanded our knowledge of understanding the genetic determinants and probable regulatory mechanism of V. cholerae O1 serotype shift between Ogawa and Inaba.

Bacteriemia por Vibrio cholerae no-O1/no-O139 que porta una región homóloga a la isla de patogenicidad VpaI-7 / Bacteremia caused by Vibrio cholerae non-O1/non-O139 carrying a region homologous to pathogenicity island VpaI-7

Resumen Presentamos un caso de bacteriemia por Vibrio cholerae no-O1/ no-O139 en una mujer de 81 años con un cuadro de dolor abdominal, fiebre, vómitos, diarrea, coluria e ictericia, mientras visitaba una zona rural sin acceso a agua potable. La identificación se realizó por la técnica de espectrometría de masa MALDI-TOF, confirmándose una cepa no toxigénica no-O1/no-139. La caracterización molecular del aislado demostró la ausencia del gen de la toxina del cólera (CTX), y pilus TCP; sin embargo, presentó cinco de los seis genes de virulencia presentes en la isla de patogenicidad homóloga denominada VPaI-7 del V. parahaemolyticus (vcs N2+, vcs C2+, vcs V2+,toxR-, vspD+, T vopF+). Además, el aislado presentó los genes de virulencia hylA y rtxA. Este es el primer caso reportado en Chile de una cepa clínica de V. cholerae no-O1, no-O139 aislada de hemocultivos portador de un segmento homólogo de la isla de patogenicidad denominada VPaI-7 de V. parahaemolyticus, el cual codifica para un sistema de secreción tipo III (TTSS), que probablemente contribuye a su virulencia.

We report a case of V. cholerae non-O1 / non-O139 bacteremia in an 81-year-old woman with abdominal pain, fever, vomiting, liquid stools, choluria and jaundice, while visiting a rural area without access to potable water. The identification was made by the MALDI-TOF mass spectrometry technique and subsequently the non-toxigenic non-O1 / non-139 strain was confirmed in the national reference laboratory. The molecular characterization demonstrated the absence of the cholera toxin gene (CTX), and the TCP pilus, however, presented 5 of 6 virulence genes present in an island of homologous pathogenicity named VPaI-7 of V. parahaemolyticus (vcs N2 +, vcs C2 +, vcs V2 +, toxR-, vspD +, T vopF +) and in addition it was positive for hylAy rtxA virulence genes recognized outside the island. This is the first case reported in Chile of a clinical strain of V. cholerae non-O1, non-O139 isolated from blood culture that carries in its genome a homologous segment of the pathogenicity island named VPaI-7 of V. parahaemolyticus, which codifies for a type III secretion system (TTSS) that probably contributes to his virulence.

Vibrio cholerae derived outer membrane vesicles modulate the inflammatory response of human intestinal epithelial cells by inducing microRNA-146a.

The small intestinal epithelium of Vibrio cholerae infected patients expresses the immunomodulatory microRNAs miR-146a and miR-155 at acute stage of disease. V. cholerae release outer membrane vesicles (OMVs) that serve as vehicles for translocation of virulence factors including V. cholerae cytolysin (VCC). The aim was to investigate whether OMVs, with and/or without VCC-cargo could be responsible for induction of microRNAs in intestinal epithelial cells and thereby contribute to immunomodulation. Polarized tight monolayers of T84 cells were challenged with OMVs of wildtype and a VCC deletion mutant of the non-O1/non-O139 (NOVC) V. cholerae strain V:5/04 and with soluble VCC. OMVs, with and without VCC-cargo, caused significantly increased levels of miR-146a. Increase was seen already after 2 hours challenge with OMVs and persisted after 12 hours. Challenge with soluble VCC caused significant increases in interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), CCL20, IL-1ß, and IRAK2 mRNA levels while challenge with OMVs did not cause increases in expression levels of any of these mRNAs. These results suggest that V. cholerae bacteria release OMVs that induce miR-146a in order to pave the way for colonization by reducing the strength of an epithelial innate immune defence reaction and also preventing inflammation in the mucosa that factors like VCC can evoke.

Hypermutation-induced in vivo oxidative stress resistance enhances Vibrio cholerae host adaptation.

Bacterial pathogens are highly adaptable organisms, a quality that enables them to overcome changing hostile environments. For example, Vibrio cholerae, the causative agent of cholera, is able to colonize host small intestines and combat host-produced reactive oxygen species (ROS) during infection. To dissect the molecular mechanisms utilized by V. cholerae to overcome ROS in vivo, we performed a whole-genome transposon sequencing analysis (Tn-seq) by comparing gene requirements for colonization using adult mice with and without the treatment of the antioxidant, N-acetyl cysteine. We found that mutants of the methyl-directed mismatch repair (MMR) system, such as MutS, displayed significant colonization advantages in untreated, ROS-rich mice, but not in NAC-treated mice. Further analyses suggest that the accumulation of both catalase-overproducing mutants and rugose colony variants in NAC- mice was the leading cause of mutS mutant enrichment caused by oxidative stress during infection. We also found that rugose variants could revert back to smooth colonies upon aerobic, in vitro culture. Additionally, the mutation rate of wildtype colonized in NAC- mice was significantly higher than that in NAC+ mice. Taken together, these findings support a paradigm in which V. cholerae employs a temporal adaptive strategy to battle ROS during infection, resulting in enriched phenotypes. Moreover, ΔmutS passage and complementation can be used to model hypermuation in diverse pathogens to identify novel stress resistance mechanisms.

Environmental surveillance for Vibrio cholerae in selected households' water storage systems in Accra Metropolitan Area (AMA) prior to the 2014 cholera outbreak in Accra, Ghana.

Cholera is a global public health problem with high endemicity in many developing countries in Africa. In 2014, Ghana experienced its largest epidemic with more than 20,000 cases and 200 deaths; most of it occurred in the Accra Metropolitan Area (AMA). Ghana's disease surveillance system is mainly clinically based and focused on case detection and management. Environmental exploration for the etiological agents is missing from the surveillance strategy. This study therefore assessed the occurrence of toxigenic Vibrio cholerae in water storage systems in selected high risk areas in the AMA area prior to the 2014 outbreak. Three hundred twenty water samples from 80 households' water storage systems were analyzed for toxigenic Vibrio cholerae using the bacterial culture method. Presumptive V. cholerae was isolated from 83.8% of households' water storage systems. The viable cells ranged from 1 to 1400 CFU/100 ml. Vibrio cholerae O1 serotype was isolated from five households in Old Fadama, one household in Shiabu, and one household in Bukom in the month of May and a similar trend was observed for the months of June and July. The presence of Vibro cholerae in the water storage vessels used for drinking confirms the need to consider environmental surveillance for toxigenic Vibro cholerae particularly in high-risk areas to strengthen the existing surveillance system.