Molecular evolution of the seventh-pandemic clone of Vibrio cholerae and its relationship to other pandemic and epidemic V. cholerae isolates.

Genetic variation and molecular evolution within the seventh-pandemic clone of Vibrio cholerae O1 and its relationship to other V. cholerae isolates were examined by studying 58 clinical isolates that were epidemiologically unassociated and isolated from patients in different countries over 62 years (1931 to 1993). The sample consisted of 45 isolates from the seventh cholera pandemic (1961 to the present), 3 from the sixth pandemic, 3 from sporadic El Tor outbreaks prior to the seventh pandemic, 2 from the U.S. Gulf Coast, and 5 O139 Bengal isolates. Ribotyping detected 11 polymorphic restriction sites within the seventh-pandemic isolates and showed major differences in ribotypes in comparison with sixth- and pre-seventh-pandemic isolates. O139 isolates were very similar to isolates from the start of the seventh pandemic, differing at only two sites. The majority of seventh-pandemic isolates fall into two groups, the first present from 1961 to the present and found only in Asia and the second arising in 1966 and spreading worldwide. Both groups underwent change over time, allowing a provisional estimate for the nucleotide substitution rate within the seventh pandemic clone.     

Genetic markers of epidemic strains of Vibrio cholerae

In this review new data on the key pathogenicity genes of V. cholerae are presented. As shown on the basis of the analysis of the latest information on the structure of the genomes of different V. cholerae strains, structural genes ctxAB coding cholera toxin may not serve as the only marker of epidemically dangerous strains. More complete and reliable information for the evaluation of the epidemic potential of V. cholerae isolated from the environment may be obtained by the simultaneous detection of 4 genetic markers: genes ctxAB, tcpA and hap coding, respectively, cholera toxin, toxin-corregulated adhesion pili and soluble hemagglutinin/protease, as well as regulatory virulence gene toxR.     

Survival of Vibrio cholerae and Escherichia coli in estuarine waters and sediments.

In in vitro estuarine water and sediment chambers, the survival of Vibrio cholerae and Escherichia coli was determined by plate counting and direct counting techniques. V. cholerae strains included environmental, clinical, and serotype O1 and non-O1 isolates, whereas E. coli strains included ATCC 25922 and a freshly cultured human isolate. Recovery of V. cholerae varied significantly with incubation temperature. Growth and extended periods of survival occurred in sterile sediments, sterile waters, and nonsterile waters, but not in nonsterile sediments. In contrast to V. cholerae, viable cells of E. coli decreased rapidly in both sterile and nonsterile estuarine waters. Direct counts revealed that E. coli cells were intact in the estuarine water, but attempts to resuscitate them were unsuccessful. The data suggest that V. cholerae survives better in estuarine waters than E. coli. The results may explain the recent observations that V. cholerae levels do not correlate well with fecal coliform concentrations in estuarine waters. Furthermore, the results add increasing evidence to support the theory that V. cholerae is an autochthonous bacterium in estuaries.     

Trophic regulation of Vibrio cholerae in coastal marine waters

Cholera disease, caused by the bacterium Vibrio cholerae, afflicts hundreds of thousands worldwide each year. Endemic to aquatic environments, V. cholerae's proliferation and dynamics in marine systems are not well understood. Here, we show that under a variety of coastal seawater conditions V. cholerae remained primarily in a free-living state as opposed to attaching to particles. Growth rates of free-living V. cholerae (micro: 0.6-2.9 day(-1)) were high (similar to reported values for the bacterial assemblages; 0.3-2.5 day(-1)) particularly in phytoplankton bloom waters. However, these populations were subject to heavy grazing-mortality by protozoan predators. Thus, grazing-mortality counterbalanced growth, keeping V. cholerae populations in check. Net population gains were observed under particularly intense bloom conditions when V. cholerae proliferated, overcoming grazing pressure terms in part via rapid growth (> 4 doublings day(-1)). Our results show V. cholerae is subject to protozoan control and capable of utilizing multiple proliferation pathways in the marine environment. These findings suggest food web effects play a significant role controlling this pathogen's proliferation in coastal waters and should be considered in predictive models of disease risk.     

Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count.

A field trial to enumerate Vibrio cholerae O1 in aquatic environments in Bangladesh was conducted, comparing fluorescent-antibody direct viable count with culture detection by the most-probable-number index. Specificity of a monoclonal antibody prepared against the O1 antigen was assessed and incorporated into the fluorescence staining method. All pond and water samples yielded higher counts of viable V. cholerae O1 by fluorescent-antibody direct viable count than by the most-probable-number index. Fluorescence microscopy is a more sensitive detection system than culture methods because it allows the enumeration of both culturable and nonculturable cells and therefore provides more precise monitoring of microbiological water quality.     

Vibrio cholerae (non-O1) isolated from California coastal waters.

Nineteen strains of Vibrio cholerae non-O1 were isolated from five separate marine sites along the Santa Cruz County coast. This environmental study was initiated after a human case of non-O1 cholera-like diarrhea was acquired endemically.     

Groups of the species Vibrio cholerae having different epidemic importance

Subdivision of V. cholerae 01 into toxins on the basis of the whole complex of signs characteristic of this species does not make it possible to judge on their epidemic importance and to use the data on identification of V. cholerae for solving practical problems. Classification of V. cholerae by their capacity for producing toxin (choleragen and hemolysin of type 1, subtype beta) removes these difficulties.     

Molecular typing of epidemic and nonepidemic Vibrio cholerae isolates and differentiation of V. cholerae and V. mimicus isolates by PCR-single-strand conformation polymorphism analysis

AIMS: To examine the utility of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis to differentiate epidemic and nonepidemic Vibrio cholerae isolates as well as to differentiate V. cholerae and Vibrio mimicus isolates. METHODS AND RESULTS: By both PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis of groEL-I on chromosome 1 and groEL-II on chromosome 2, V. cholerae isolates gave distinct profiles compared with V. mimicus isolates. In addition, PCR-SSCP analysis of groEL-I and groEL-II could differentiate between V. cholerae epidemic and nonepidemic isolates. Interestingly, the relationships among strains based on groEL-I from chromosome 1 and groEL-II from chromosome 2 were congruent with each other, highlighting the conserved evolutionary history of both chromosomes in this species. CONCLUSIONS: PCR-SSCP is a powerful typing technique, which has the ability to differentiate V. cholerae and V. mimicus isolates. The epidemic V. cholerae O1/O139 serogroup isolates represent a clonal complex distinct from non-O1/non-O139 isolates that can be identified by PCR-SSCP analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the effectiveness of using reliable molecular typing methods and in particular PCR-SSCP, to identify genetic variation among V. cholerae and V. mimicus isolates.     

Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters.

Vibrio cholerae serogroup O1, the causative agent of cholera, is capable of surviving in aquatic environments for extended periods and is considered an autochthonous species in estuarine and brackish waters. These environments contain numerous elements that may affect its ecology. The studies reported here examined physical interactions between V. cholerae O1 and natural plankton populations of a geographical region in Bangladesh where cholera is an endemic disease. Results showed that four of five clinical V. cholerae O1 strains and endogenous bacterial flora were attached preferentially to zooplankton molts (exuviae) rather than to whole specimens. One strain attached in approximately equal numbers to both exuviae and whole specimens. V. cholerae O1 also attached to several phytoplankton species. The results show that V. cholerae O1 can bind to diverse plankton species collected from an area where cholera is an endemic disease, with potentially significant effects on its ecology.     

Differences among marine and hospital strains of Vibrio cholerae during Peruvian epidemic

During a period of 18 months of an epidemic of Vibrio cholerae, cultures from 450 samples of fish, shellfish and seawater were isolated. The highest frequencies of occurrence observed were 5.2% in fish from inshore waters, 3.9% in marine snails, and 1.8% in mussels and crabs. No incidents were isolated from cultures of fish in the open seas or cultures from frozen shrimp. Cultures of marine origin were compared with cultures from hospitalized patients, and these revealed marked serological and toxigenic differences. Marine strains were mainly non-O1 V. cholerae, non toxigenic. We presume fishing off-shore not to be the cause of this outbreak. However, marine species from contaminated waters could contain toxigenic V. cholerae remaining viable and potentially pathogenic. Methods used were more sensitive and specific for detecting marine strains. In this paper the need to use more specific methods is discussed.     

Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count

A field trial to enumerate Vibrio cholerae O1 in aquatic environments in Bangladesh was conducted, comparing fluorescent-antibody direct viable count with culture detection by the most-probable-number index. Specificity of a monoclonal antibody prepared against the O1 antigen was assessed and incorporated into the fluorescence staining method. All pond and water samples yielded higher counts of viable V. cholerae O1 by fluorescent-antibody direct viable count than by the most-probable-number index. Fluorescence microscopy is a more sensitive detection system than culture methods because it allows the enumeration of both culturable and nonculturable cells and therefore provides more precise monitoring of microbiological water quality.     

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.     

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.     

The sodium cycle. III. Vibrio alginolyticus resembles Vibrio cholerae and some other vibriones by flagellar motor and ribosomal 5S-RNA structures

An electron microscopic study of the basal bodies of the Vibrio albinolyticus flagellum revealed a four-disc structure. The diameters of the two discs localized closer to the cytoplasmic membrane proved to be about 2-fold shorter than those of the two others. In this respect the basal body of V. alginolyticus resembles very much that of V. cholerae described by Ferris and co-workers. The sequence of the V. alginolyticus ribosomal 5S-RNA showed that it is similar to those of V. cholerae, V. harveyi and some other vibriones. On the basis of the 5S-RNA sequences, a dendrogram of prokaryotes is presented. It confirmed the suggestion that V. alginolyticus is a typical representative of Vibrionaceae rather than a 'monster' greatly differing from other vibriones. Possible evolutionary relation of various bacterial species possessing the primary Na+ pumps is discussed.     

Ecology of Vibrio species, including Vibrio cholerae, in natural waters of Kent, England

The distribution of Vibrio species in water from two sites in Kent was studied between 1978 and 1980. They were counted by a most probable number technique using alkaline peptone water for enrichment followed by plating onto thiosulphate citrate bile salt sucrose agar, or by direct plating of water onto the same agar. In a freshwater stream both upstream and downstream from a human sewage works outfall V. metschnikovii was the predominant Vibrio. Vibrio anguillarum was isolated sporadically. Non-O1 serovars of V. cholerae occurred only twice. At the other site in a ditch containing static, brackish water, non-O1 V. cholerae (highest number 400 cfu/ml) was observed as present only from May to November. Vibrio anguillarum was isolated throughout the sampling period. The presence of non-O1 V. cholerae in both sites was not dependent on the input of human sewage. The hypothesis that non-toxigenic V. cholerae can survive and multiply in water was tested in the ditch by the use of submersible chambers constructed of polycarbonate membranes and Plexiglass. The seasonal incidence of non-O1 V. cholerae in the brackish water site could be explained by the multiplication of the organism when the water temperature exceeded 9°C. It was concluded that strains of V. cholerae that are unable to produce cholera toxin are indigenous to static brackish water environments and the possibility that this applies to toxigenic strains as well should be investigated.     

Growth of Vibrio cholerae O1 in red tide waters off California

Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of V. cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with V. cholerae or the 0.6- micro m-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day(-1) (4.2 day(-1) +/- 3.9) for V. cholerae and 0.1 to 9.7 day(-1) (2.2 +/- 2.8 day(-1)) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V. cholerae in seawater. Under the conditions tested, free-living V. cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10(4) cell ml(-1)) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V. cholerae in disease propagation and prevention during phytoplankton blooms.     

Growth response of Vibrio cholerae and other Vibrio spp. to cyanobacterial dissolved organic matter and temperature in brackish water

Environmental control of growth and persistence of vibrios in aquatic environments is poorly understood even though members of the genus Vibrio are globally important pathogens. To study how algal-derived organic matter and temperature influenced the abundance of different Vibrio spp., Baltic Sea microcosms inoculated with Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus and native bacterioplankton, were exposed to different temperatures (12-25 degrees C) and amended with dissolved organic matter from Nodularia spumigena (0-4.2 mg C L(-1)). Vibrio abundance was monitored by culture-dependent and molecular methods. Results suggested that Vibrio populations entered a viable but nonculturable state during the incubations. Abundance of Vibrio spp. and total bacterioplankton were orders of magnitude higher in microcosms amended with organic matter compared with reference microcosms. Vibrio cholerae abundances ranged from 0.9 to 1.9 x 10(5) cells mL(-1) in treatments amended with 4.2 mg C L(-1). Vibrio cholerae abundance relative to total bacterioplankton and other Vibrio spp. also increased >10-fold. In addition, V. vulnificus abundance increased in mesocosms with the highest organic matter addition (0.9-1.8 x 10(4) cells mL(-1)). Temperature alone did not significantly affect abundances of total bacterioplankton, total Vibrio spp. or individual Vibrio populations. By contrast, cyanobacterial-derived organic matter represented an important factor regulating growth and abundance of V. cholerae and V. vulnificus in brackish waters.     

Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters

Vibrio cholerae serogroup O1, the causative agent of cholera, is capable of surviving in aquatic environments for extended periods and is considered an autochthonous species in estuarine and brackish waters. These environments contain numerous elements that may affect its ecology. The studies reported here examined physical interactions between V. cholerae O1 and natural plankton populations of a geographical region in Bangladesh where cholera is an endemic disease. Results showed that four of five clinical V. cholerae O1 strains and endogenous bacterial flora were attached preferentially to zooplankton molts (exuviae) rather than to whole specimens. One strain attached in approximately equal numbers to both exuviae and whole specimens. V. cholerae O1 also attached to several phytoplankton species. The results show that V. cholerae O1 can bind to diverse plankton species collected from an area where cholera is an endemic disease, with potentially significant effects on its ecology.