Isolation of Vibrio cholerae serotype O1 from the eastern oyster, Crassostrea virginica.

Two strains of Vibrio cholerae serotype O1 Inaba were isolated from eastern oysters, Crassostrea virginica, collected from estuarine waters in Florida during April 1980. The oyster meats and waters from which the oysters were collected had low fecal coliform counts, and the area had no prior evidence of sewage contamination.     

Non-O1/Non-O139 Vibrio cholerae Carrying Multiple Virulence Factors and V. cholerae O1 in the Chesapeake Bay,Maryland

Non-O1/non-O139 Vibrio cholerae inhabits estuarine and coastal waters globally, but its clinical significance has not been sufficiently investigated, despite the fact that it has been associated with septicemia and gastroenteritis. The emergence of virulent non-O1/non-O139 V. cholerae is consistent with the recognition of new pathogenic variants worldwide. Oyster, sediment, and water samples were collected during a vibrio surveillance program carried out from 2009 to 2012 in the Chesapeake Bay, Maryland. V. cholerae O1 was detected by a direct fluorescent-antibody (DFA) assay but was not successfully cultured, whereas 395 isolates of non-O1/non-O139 V. cholerae were confirmed by multiplex PCR and serology. Only a few of the non-O1/non-O139 V. cholerae isolates were resistant to ampicillin and/or penicillin. Most of the isolates were sensitive to all antibiotics tested, and 77 to 90% carried the El Tor variant hemolysin gene hlyA_ET, the actin cross-linking repeats in toxin gene rtxA, the hemagglutinin protease gene hap, and the type 6 secretion system. About 19 to 21% of the isolates carried the neuraminidase-encoding gene nanH and/or the heat-stable toxin (NAG-ST), and only 5% contained a type 3 secretion system. None of the non-O1/non-O139 V. cholerae isolates contained Vibrio pathogenicity island-associated genes. However, ctxA, ace, or zot was present in nine isolates. Fifty-five different genotypes showed up to 12 virulence factors, independent of the source of isolation, and represent the first report of both antibiotic susceptibility and virulence associated with non-O1/non-O139 V. cholerae from the Chesapeake Bay. Since these results confirm the presence of potentially pathogenic non-O1/non-O139 V. cholerae, monitoring for total V. cholerae, regardless of serotype, should be done within the context of public health.     

Distribution of Vibrio cholerae O1 antigen biosynthesis genes among O139 and other non-O1 serogroups of Vibrio cholerae

The organization and distribution of the genes responsible for O antigen biosynthesis in various serogroups of Vibrio cholerae were investigated using several DNA probes derived from various regions of the genes responsible for O1 antigen biosynthesis. Based on the reactivity pattern of the probes against the various serogroups, the cluster of genes responsible for the O1 antigen biosynthesis could be broadly divided into six groups, designated as class 1-6. The class 3 cluster of genes corresponding to gmd to wbeO, wbeT and a part of wbeU was specific for only the O1 serogroup. The other cluster of genes (class 1, 2, 4-6) reacted with other serogroups of V. cholerae. These data indicate that serotype conversion in V. cholerae does not depend on a simple mutational event but may involve horizontal gene transfer not only between V. cholerae strains but also between V. cholerae and species other than V. cholerae.     

A serogroup of non-O1 Vibrio cholerae possessing the Inaba antigen of Vibrio cholerae O1

A serogroup of non-O1 Vibrio cholerae, tentatively named Hakata, possessing the C (Inaba) factor but not the B (Ogawa) and A factors of V. cholerae O1 is described. Strains of this serogroup were isolated from river and estuarine waters and from frozen shrimps.     

A serogroup of non-O1 Vibrio cholerae possessing the Inaba antigen of Vibrio cholerae O1

A serogroup of non-O1 Vibrio cholerae , tentatively named Hakata, possessing the C (Inaba) factor but not the B (Ogawa) and A factors of V. cholerae O1 is described. Strains of this serogroup were isolated from river and estuarine waters and from frozen shrimps.     

Occurrence and Expression of Luminescence in Vibrio cholerae

Several species of the genus Vibrio, including Vibrio cholerae, are bioluminescent or contain bioluminescent strains. Previous studies have reported that only 10% of V. cholerae strains are luminescent. Analysis of 224 isolates of non-O1/non-O139 V. cholerae collected from Chesapeake Bay, MD, revealed that 52% (116/224) were luminescent when an improved assay method was employed and 58% (130/224) of isolates harbored the luxA gene. In contrast, 334 non-O1/non-O139 V. cholerae strains isolated from two rural provinces in Bangladesh yielded only 21 (6.3%) luminescent and 35 (10.5%) luxA⁺ isolates. An additional 270 clinical and environmental isolates of V. cholerae serogroups O1 and O139 were tested, and none were luminescent or harbored luxA. These results indicate that bioluminescence may be a trait specific for non-O1/non-O139 V. cholerae strains that frequently occur in certain environments. Luminescence expression patterns of V. cholerae were also investigated, and isolates could be grouped based on expression level. Several strains with defective expression of the lux operon, including natural K variants, were identified.     

Screening of aquatic samples for Vibrio cholerae serotype O1 by a dot-blot method and a latex agglutination test.

A dot-blot, enzyme-linked immunosorbent method and a latex agglutination test were studied for their abilities to detect Vibrio cholerae serotype O1 in aquatic samples by testing artificially contaminated water as well as samples from natural potential sources. Water samples were preenriched with alkaline peptone and then enriched with Monsur peptone water. For the dot-blot test, enriched cultures of organisms in a small portion of the Monsur peptone water were transferred to a polyvinylidene difluoride membrane with a microfiltration apparatus. The enzyme-linked immunosorbent assay was performed by using biotin-labeled antibodies and avidin-biotin-peroxidase complex; brown dots developed in the wells that contained serotype O1 vibrios. Latex agglutination tests were performed by mixing 1 drop of the culture in Monsur with 1 drop of reagent coated with monoclonal antibody specific for antigen A. The sensitivities and specificities of the methods were compared with those of the colony-blot method, which identified individual colonies of V. cholerae O1 in mixed bacterial cultures on isolation media. Our results indicate that the dot-blot method is as sensitive as the colony-blot method and is useful for screening for V. cholerae serotype O1 even in specimens that are heavily contaminated with non-O1 vibrios.     

Worldwide Occurrence of Integrative Conjugative Element Encoding Multidrug Resistance Determinants in Epidemic Vibrio cholerae O1

In the last decades, there has been an increase of cholera epidemics caused by multidrug resistant strains. Particularly, the integrative and conjugative element (ICE) seems to play a major role in the emergence of multidrug resistant Vibrio cholerae. This study fully characterized, by whole genome sequencing, new ICEs carried by multidrug resistant V. cholerae O1 strains from Nigeria (2010) (ICEVchNig1) and Nepal (1994) (ICEVchNep1). The gene content and gene order of these two ICEs are the same, and identical to ICEVchInd5, ICEVchBan5 and ICEVchHai1 previously identified in multidrug resistant V. cholerae O1. This ICE is characterized by dfrA1, sul2, strAB and floR antimicrobial resistance genes, and by unique gene content in HS4 and HS5 ICE regions. Screening for ICEs, in publicly available V. cholerae genomes, revealed the occurrence and widespread distribution of this ICE among V. cholerae O1. Metagenomic analysis found segments of this ICE in marine environments far from the direct influence of the cholera epidemic. Therefore, this study revealed the epidemiology of a spatio-temporal prevalent ICE in V. cholerae O1. Its occurrence and dispersion in V. cholerae O1 strains from different continents throughout more than two decades can be indicative of its role in the fitness of the current pandemic lineage.     

Viability of the nonculturable Vibrio cholerae O1 and O139

Vibrio cholerae is capable of transforming into a viable but nonculturable (VBNC) state, and, in doing so, undergoes alteration in cell morphology. In the study reported here, Vibrio cholerae O1 and O139 cells were maintained in laboratory microcosms prepared with 1% Instant Ocean and incubated at 4 degrees C, i.e., conditions which induce the VBNC state. Cells were fixed at different stages during entry into the VBNC state and, when no growth was detectable on solid or in liquid media, the ultrastructure of these cells was examined, using both transmission and scanning electron microscopy. As shown in earlier studies, the cells became smaller in size and changed from rod to ovoid or coccoid morphology, with the central region of the cells becoming compressed and surrounded by denser cytoplasm. Because the coccoid morphology, indicative of the VBNC state is common for Vibrio cholerae in the natural environment, as well as in starved cells (Baker et al., 1983; Hood et al., 1986) viability of the coccoid, viable but nonculturable cell was investigated. The percentage of coccoid (VBNC) cells showing metabolic activity and retention of membrane integrity was monitored using direct fluorescence staining (LIVE/DEAD BacLight Bacterial Viability kit), with 75 to 90% of the viable but nonculturable coccoid cells found to be metabolically active by this test. Furthermore, the proportion of actively respiring cells, using the redox dye, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC), relative to total cells, the latter determined by DAPI staining, ranged from 10 to 50%. VBNC coccoid cells retained the antigenic determinants of Vibrio cholerae O1 and O139, respectively, evidenced by positive reaction with monoclonal fluorescent antibody. Viability was further established by susceptibility of the VBNC cells to chlorine, copper sulfate, zinc sulfate, and formaldehyde. Since retention of cell membrane integrity is a determining characteristic of viable cells, DNA was extracted from VBNC cells in microcosms maintained for two months and for one year. Conservation of cholera toxin and toxin-associated genes, ctxA, toxR, tcpA, and zot in chromosomal DNA of VBNC cells was demonstrated using PCR and employing specific primers. It is concluded that not only do VBNC V cholerae O1 and O139 retain viability up to one year, but genes associated with pathogenicity are retained, along with chromosomal integrity.     

Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

Effect of alum on free-living and copepod-associated Vibrio cholerae O1 and O139.

The effects of alum [KAl(SO4)2] on free-living and copepod-associated Vibrio cholerae O1 and O139 were investigated by using plate counts and immunofluorescence direct viable counting (DVC). Growth of alum-treated cells in 0.5/1000 Instant Ocean seawater was inhibited, i.e., no growth was obtained on Luria-Bertani (LB) agar or thiosulfate-citrate-bile salt-sucrose (TCBS) agar. However, a significant number of the inhibited cells maintained viability, as measured by DVC. In comparison, a significant number of V. cholerae organisms associated with zooplankton, most of which were crustacean copepods, were viable but nonculturable, with only a small number of cells retaining culturability on LB and TCBS agar. Both DVC and viable plate counts (CFU) were significantly greater for V. cholerae O1 and O139 associated with zooplankton than for V. cholerae in water alone, i.e., without copepods. It is concluded that alum is an effective coagulant but not an effective killing agent for V. cholerae and that association with copepods offers protection for V. cholerae O1 and O139 against alum and chlorine treatments.     

Development of hyperfimbriated strains of Vibrio cholerae O1

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR vector. These clones were sequenced. The fimA sequences were found to be identical between V cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

Filamentous Phage fs1 of Vibrio cholerae O139

Filamentous phage, fs1, was obtained from Vibrio cholerae O139. The lysogenized strains produced a large amount of fs1 phage in the culture supernatant. This phage was previously reported as novel fimbriae of that organism. The genome of the phage was a 6.5 kb single-stranded DNA. The capsid of fs1 consists of a small molecule peptide (about 2.5 kDa).     

Incidence of Vibrio cholerae from estuaries of the United States West Coast.

The incidence of Vibrio cholerae in shellfish, sediment, and waters of California, Oregon, and Washington was determined during the summer of 1984. Samples from 24 distinct estuaries were analyzed qualitatively. V. cholerae non-O1 was found in 23 estuaries and in 44.6% of the 529 samples examined. V. cholerae O1 Inaba was isolated from water samples in Morro Bay, Calif. Vibrio mimicus was found in 2.3% of the samples. Cholera enterotoxin was not found in cell-free filtrates of the 100 isolates tested in the Y-1 mouse adrenal cell assay, but heat-labile cytotoxic activity was observed with 3% of the isolates.     

Vibrio cholerae O139 bacteriophages

Cholera bacteriophages have been isolated from 27 lysogenic cultures of V. cholerae O139. As shown the pages under study belong to two morphological groups A1 and F1 and serological types II and XII. The use of prophage typing and the sensitivity test to specific phage made it possible to differentiate V. cholerae strains, serogroup O139.     

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.     

Immunogenicity of Vibrio cholerae O1 Fimbriae in Animal and Human Cholera

Parenteral immunization with either formalin-fixed whole cells of the fimbriate Bgd17 strain or purified fimbriae protected against Vibrio cholerae O1 infection in rabbits, independent of biotype and serotype. Parenteral immunization of adult rabbits with purified fimbriae prior to V. cholerae O1 challenge resulted in a reduction of 2 to 3 orders of magnitude in the number of bacteria recovered from the small intestines of immunized rabbits in comparison to non-immunized controls. IgG and IgA antibodies against fimbrillin of V. cholerae O1 were detected in the convalescent sera of patients with cholera; however, little fimbrial antigen was detected in the commercially available cholera vaccines when examined by polyclonal and monoclonal antibodies against fimbriae. These data suggest that fimbrial hemagglutinin is a major adhesin of V. cholerae O1 and that parenteral immunization with fimbriae generates a specific immune response in the gut that may serve as one means of mitigating subsequent V. cholerae O1 gut infection.     

Pili of Vibrio cholerae Widely Distributed in Serogroup O1 Strains

The distribution of Vibrio cholerae O1 pili consisting of 16 kDa subunit protein (16K-pili) was examined by Western blotting, using 211 strains from various origins and specific anti-16K-pili sera. The 16 kDa protein was detected in all 211 strains. The pili were purified from 3 El Tor and 3 classical strains, and characterized by hemagglutination and inhibition tests. All purified pili were hemagglutinative. However, the hemagglutinating activity of classical pili disappeared after exposure to 5 M urea and the agglutination induced by the classical pili was inhibited by D -mannose, alpha-methylmannoside, D -glucose and N-acetylglucosamine. On the contrary, El Tor pili were resistant to these sugars and urea.