Search

Cholera is an acute watery, diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage is present. In this study, we used an

Vibrio vulnificus is the leading cause of death in the United States associated with the consumption of raw seafood, particularly oysters. In epidemiological studies, primary septicemia and inflammation-mediated septic shock caused by V . vulnificus is strongly associated with liver disease, often in the context of chronic alcohol abuse. The present study was undertaken to determine whether clinical biomarkers of liver function or cellular oxidative stress are associated with peripheral blood mononuclear cell inflammatory cytokine responses to V . vulnificus . Levels of interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha elicited in response to V . vulnificus and measured in cell supernatants were not associated with the liver biomarkers aspartate aminotransferase (AST) or alanine aminotransferase (ALT) or the AST/ALT ratio. In contrast, reduced glutathione (GSH) levels were associated with the release of all four cytokines (IL-1β [ R 2 = 0.382; P = 0.006], IL-6 [ R 2 = 0.393; P = 0.005], IL-8 [ R 2 = 0.487; P = 0.001], and TNF-α [ R 2 = 0.292; P = 0.021]). Those individuals with below-normal GSH levels produced significantly less proinflammatory cytokines in response to V . vulnificus . We hypothesize that persons with markers for cellular oxidative stress have increased susceptibility to V . vulnificus septicemia.

Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using Tn phoA mutagenesis, we identified a V . vulnificus CPS locus, which included an upstream ops element, a wza gene ( wza Vv ), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wza gene product is required for transport of CPS to the cell surface in Escherichia coli . Polar transposon mutations in wza Vv eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza Vv was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans . Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in wza Vv and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V . vulnificus .

Vibrio cholerae can switch from a smooth to a wrinkled or rugose colony phenotype characterized by the secretion of a polysaccharide that enables the bacteria to survive harsh environmental conditions. In order to understand the genetic basis of rugosity, we isolated Tn phoA -induced stable, smooth mutants of two O1 El Tor rugose strains and mapped the insertion sites in several of the mutants using a modified Y-adapter PCR technique. One of the Tn phoA insertions was mapped to the first gene of the vps region that was previously shown to encode the rugose polysaccharide biosynthesis cluster. Three insertions were mapped to a previously unknown hlyA -like gene, also in the vps region. Five other insertions were found in loci unlinked to the vps region: (i) in the epsD gene (encodes the “secretin” of the extracellular protein secretion apparatus), (ii) in a hydG -like gene (encodes a ς 54 -dependent transcriptional activator similar to HydG involved in labile hydrogenase production in Escherichia coli , (iii) in a gene encoding malic acid transport protein upstream of a gene similar to yeiE of E. coli (encodes a protein with similarities to LysR-type transcriptional activators), (iv) in dxr (encodes 1-deoxy- d -xylulose 5-phosphate reductoisomerase), and (v) in the intergenic region of lpd and odp (encode enzymes involved in the pyruvate dehydrogenase complex formation). These data suggest the involvement of a complex regulatory network in rugose polysaccharide production and highlight the general utility of the Y-adapter PCR technique described here for rapid mapping of transposon insertion sites.

The DNA sequence of the O-antigen biosynthesis cluster ( wbf ) of a recently emergent pathogen, Vibrio cholerae serogroup O139, has been determined. Here we report the sequence of the genes downstream of the O139 wbfX gene and analysis of the genes flanking the wbf gene cluster in other serogroups. The gene downstream of wbfX , designated rjg (right junction gene), is predicted to be not required for O-antigen biosynthesis but appears to be a hot spot for DNA rearrangements. Several variants of the rjg gene (three different insertions and a deletion) have been found in other serogroups. DNA dot blot analysis of 106 V. cholerae strains showed the presence of the left and right junction genes, gmhD and rjg , respectively, in all strains. Further, these genes mapped to a single I- Ceu I fragment in all 21 strains analyzed by pulsed-field gel electrophoresis, indicating a close linkage. The insertion sequence element IS 1358 , found in both O1 and O139 wb * regions, is present in 61% of the strains tested; interestingly, where present, it is predominantly linked to the wb * region. These results indicated a cassette-like organization of the wb * region, with the conserved genes ( gmhD and rjg ) flanking the divergent, serogroup-specific wb * genes and IS 1358 . A similar organization of the wb * region in other serogroups raises the possibility of the emergence of new pathogens by homologous recombination via the junction genes.

Vibrio cholerae NRT36S is a non-cholera toxin-producing, non-O1 strain that causes diarrhea in volunteers. The genome of NRT36S was sequenced to create a draft containing 174 contigs plus the superintegron region. Our analysis of the draft genome revealed several putative toxin genes and colonization factors. Besides confirming the existence of nonagglutinable heat-stable toxin, we also identified the genes for a type three secretion system, a putative exotoxin, two different RTX toxins, and four pilus systems.

We sequenced a 705-bp fragment of the recA gene from 113 Vibrio cholerae strains and closely related species. One hundred eighty-seven nucleotides were phylogenetically informative, 55 were phylogenetically uninformative, and 463 were invariant. Not unexpectedly, Vibrio parahaemolyticus and Vibrio vulnificus strains formed out-groups; we also identified isolates which resembled V. cholerae biochemically but which did not cluster with V. cholerae . In many instances, V. cholerae serogroup designations did not correlate with phylogeny, as reflected by recA sequence divergence. This observation is consistent with the idea that there is horizontal transfer of O-antigen biosynthesis genes among V. cholerae strains.