Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah

Vibrio cholerae was isolated from cloacal swabs and freshly voided feces collected from 20 species of aquatic birds in Colorado and Utah during 1986 and 1987. About 17% (198 of 1,131) fecal specimens collected from July 1986 through August 1987 contained the organism. Both O1 and non-O1 V. cholerae strains were isolated from the fecal specimens. Isolates from eight birds (representing five species) agglutinated in O group 1 antiserum. Supernatants of broth cultures from three isolates which typed as V. cholerae O1 serotype Ogawa gave reactions typical of cholera toxin when tested on Y-1 mouse adrenal cell cultures. Several serovars of non-O1 V. cholerae were isolated from the fecal specimens; serovar 22 was the most prevalent type. All non-O1 isolates were cytotoxic to Y-1 mouse adrenal cells. Only non-O1 V. cholerae was detected in water samples collected from the habitat of the birds. The results of this study suggest that aquatic birds serve as carriers and disseminate V. cholerae over a wide area.     

Isolation of non-O1 Vibrio cholerae serovars from surface waters in western Colorado.

Non-O1 Vibrio cholerae was isolated from rivers, creeks, washes, irrigation canals, and ditches in western Colorado during the summer of 1985. The organism occurred in fresh water (less than or equal to 5 mmol of Na+ per liter) as well as in water of higher salinity (approximately equal to 17 mmol per liter). Sixteen serovars of non-O1 V. cholerae were Sixteen serovars of non-O1 V. cholerae were identified among the environmental isolates. All of the isolates were cytotoxic to Y-1 mouse adrenal cells.     

Isolation of non-O1 Vibrio cholerae serovars from surface waters in western Colorado

Non-O1 Vibrio cholerae was isolated from rivers, creeks, washes, irrigation canals, and ditches in western Colorado during the summer of 1985. The organism occurred in fresh water (less than or equal to 5 mmol of Na+ per liter) as well as in water of higher salinity (approximately equal to 17 mmol per liter). Sixteen serovars of non-O1 V. cholerae were Sixteen serovars of non-O1 V. cholerae were identified among the environmental isolates. All of the isolates were cytotoxic to Y-1 mouse adrenal cells.     

Isolation of Candida albicans and halophilic Vibrio spp. from aquatic birds in Connecticut and Florida.

Halophilic vibrios were recovered from feces of six types of aquatic birds (gulls, pelicans, Canada geese, swans, egrets, cormorants) from Connecticut and/or Florida shorelines. Candida albicans was isolated from gulls and Canada geese in Connecticut and from gulls and cormorants in Florida.     

Isolation of Candida albicans and halophilic Vibrio spp. from aquatic birds in Connecticut and Florida

Halophilic vibrios were recovered from feces of six types of aquatic birds (gulls, pelicans, Canada geese, swans, egrets, cormorants) from Connecticut and/or Florida shorelines. Candida albicans was isolated from gulls and Canada geese in Connecticut and from gulls and cormorants in Florida.     

Serogroup conversion of Vibrio cholerae in aquatic reservoirs

The environmental reservoirs for Vibrio cholerae are natural aquatic habitats, where it colonizes the chitinous exoskeletons of copepod molts. Growth of V. cholerae on a chitin surface induces competence for natural transformation, a mechanism for intra-species gene exchange. The antigenically diverse O-serogroup determinants of V. cholerae are encoded by a genetically variable biosynthetic cluster of genes that is flanked on either side by chromosomal regions that are conserved between different serogroups. To determine whether this genomic motif and chitin-induced natural transformation might enable the exchange of serogroup-specific gene clusters between different O serogroups of V. cholerae, a strain of V. cholerae O1 El Tor was co-cultured with a strain of V. cholerae O139 Bengal within a biofilm on the same chitin surface immersed in seawater, and O1-to-O139 transformants were obtained. Serogroup conversion of the O1 recipient by the O139 donor was demonstrated by comparative genomic hybridization, biochemical and serological characterization of the O-antigenic determinant, and resistance of O1-to-O139 transformants to bacteriolysis by a virulent O1-specific phage. Serogroup conversion was shown to have occurred as a single-step exchange of large fragments of DNA. Crossovers were localized to regions of homology common to other V. cholerae serogroups that flank serogroup-specific encoding sequences. This result and the successful serogroup conversion of an O1 strain by O37 genomic DNA indicate that chitin-induced natural transformation might be a common mechanism for serogroup conversion in aquatic habitats and for the emergence of V. cholerae variants that are better adapted for survival in environmental niches or more pathogenic for humans.     

Isolation of Vibrio cholerae O139 synonym Bengal from the aquatic environment in Bangladesh: implications for disease transmission.

Currently, Bangladesh is experiencing an epidemic of acute watery diarrhea caused by Vibrio cholerae O139. Surface waters were collected and cultured for vibrious following enrichment. Twelve percent (11 of 92) of samples yielded V. cholerae O139, and all of them were positive for cholera toxin. The data suggest that V. cholerae O139 is easily culturable from surface water samples.     

Simple procedure for rapid identification of Vibrio cholerae from the aquatic environment

Biochemical tests commonly used to screen for Vibrio cholerae in environmental samples were evaluated, and we found that a combination of alkaline peptone enrichment followed by streaking on thiosulfate citrate bile salts sucrose agar and testing for arginine dihydrolase activity and esculin hydrolysis was an effective rapid technique to screen for aquatic environmental V. cholerae. This technique provided 100% sensitivity and > or =70% specificity.     

Isolation of Vibrio cholerae Serotype Ogawa from a Florida Estuary

Vibrio cholerae serotype Ogawa was recently isolated from the estuarine waters of Apalachicola Bay, Fla., in areas that are subject to consistent fecal contamination and in areas that are remote from any apparent source of contamination. The significance of these organisms in the environment has not been determined.     

Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments.

A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore) as well as some strains of Vibrio anguillarum deficient in the anguibactin-mediated system. The chromatographic analyses and absorption spectra of siderophores extracted from culture supernatants suggest that vibriobactin may be produced by the strains examined. Interestingly, some strains also produced hydroxamate-type compounds, as determined by chemical tests, and were able to promote the growth of an aerobactin-deficient strain of Escherichia coli. These results were confirmed by the absorption spectra and chromatographic analyses of the culture extracts. The synthesis of iron-regulated outer membrane proteins in representative strains was also examined. The molecular sizes of the main induced proteins ranged from 70 to 78 kilodaltons. These results indicate that several iron uptake mechanisms which could be involved in environmental survival and pathogenicity are present in environmental V. cholerae non-O1 strains.     

Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae

Multidrug-resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an attempt to identify a homologue of the Escherichia coli TolC in V . cholerae , we isolated a DNA fragment (pVC) that enabled an E . coli tolC mutant to grow in the presence of 0.05% deoxycholate (DOC). However, other TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins respectively. The translational products of these two ORFs correlated closely with the molecular weights of the predicted proteins. The deduced amino acid sequences of ORF1 and ORF2 showed a high degree of similarity with conserved regions of the E . coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E . coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V . cholerae isogenic mutant defective in ORF2 was constructed by gene replacement. Characterization of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results suggest that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V . cholerae . In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.     

Toxigenicity and toxin genes of Vibrio cholerae 01 isolated from an artificial aquatic environment

A simple experiment was carried out to examlne the effect of varlous physicochemical conditions on toxigenicity and toxin genes of Vibrio cholerae 01 lsolated from an artificial aquatic environment. All isolated strains, tested by tissue culture assay, DNA-DNA hybridization and ELISA, were cytotoxic to Vero cells, did not lose their toxin genes and continued to produce cholera toxin. These results are consistent with the hypothesis that V. cholerae can survive in the environment without losing potential pathogenicity.     

Isolation of Non-O1 Vibrio cholerae Serovars from Oregon Coastal Environments

Water, sediment, and shellfish from three Oregon estuaries were cultured for pathogenic Vibrio species. Non-O1 serovars of V. cholerae were the most common pathogenic Vibrio species recovered. Non-O1 V. cholerae were isolated from all three estuaries sampled, covering an area of about 170 miles along the Oregon coast. Non-O1 V. cholerae were isolated from water and sediment, but not shellfish, at temperatures ranging from 11 to 19°C and salinities of 2.3 to 26‰. Sixteen isolates representing 12 different non-O1 serovars were identified, while four non-O1 V. cholerae isolates failed to react with any of the 54 antisera tested. These results indicate that non-O1 V. cholerae serovars can be found over a large geographic area and under a variety of environmental conditions. These organisms are apparently an autochthonous component of these estuarine microbial communities.     

Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan

The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after HindIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance.     

Toxigenic Vibrio cholerae in the aquatic environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

Isolation of the R'his plasmids of Vibrio cholerae

V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.     

Isolation and characterization of the Vibrio cholerae recA gene

A 3.6-kilobase PstI fragment was isolated from a Vibrio cholerae chromosomal DNA library and shown to encode RecA-like activity in complementation studies with Escherichia coli recA mutants. Although DNA hybridization experiments failed to detect any homology between the E. coli and V. cholerae recA genes, hyperimmune antiserum produced against purified E. coli RecA protein recognized epitopes shared by the V. cholerae protein. The V. cholerae chromosomal fragments, when cloned and transferred to E. coli, provided the missing recA functions, including resistance to the alkylating agent methyl methanesulfonate, resistance to UV irradiation, and promotion of homologous recombination in Hfr mating experiments.