Cell surface characteristics of environmental and clinical isolates of Vibrio cholerae non-O1.

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.     

Genetic diversity of clinical and environmental isolates of Vibrio cholerae determined by amplified fragment length polymorphism fingerprinting

Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years. To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed. Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V. cholerae serogroup O1, O139, and non-O1, O139 isolates. Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain. However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains. This result confirmed that clinical O1 and O139 strains are genetically closely related. On the other hand, AFLP analyses of restriction enzyme ApaI- and TaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains. Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic. A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India. Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates. Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates. Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart. Both strains were distinct from the O1 seventh pandemic strain. Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study. These results suggest that although a single clone of pathogenic V. cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V. cholerae strains that appear to have been derived locally from environmental O1 or non-O1 strains.     

Distribution of genes for virulence and ecological fitness among diverse Vibrio cholerae population in a cholera endemic area: tracking the evolution of pathogenic strains

The pathogenic strains of Vibrio cholerae that cause acute enteric infections in humans are derived from environmental nonpathogenic strains. To track the evolution of pathogenic V. cholerae and identify potential precursors of new pathogenic strains, we analyzed 324 environmental or clinical V. cholerae isolates for the presence of diverse genes involved in virulence or ecological fitness. Of 251 environmental non-O1, non-O139 strains tested, 10 (3.9%) carried the toxin coregulated pilus (TCP) pathogenicity island encoding TCPs, and the CTX prophage encoding cholera toxin, whereas another 10 isolates carried the TCP island alone, and were susceptible to transduction with CTX phage. Most V. cholerae O1 and O139 strains carried these two major virulence determinants, as well as the Vibrio seventh pandemic islands (VSP-1 and VSP-2), whereas 23 (9.1%) non-O1, non-O139 strains carried several VSP island genes, but none carried a complete VSP island. Conversely, 30 (11.9%) non-O1, non-O139 strains carried type III secretion system (TTSS) genes, but none of 63 V. cholerae O1 or O139 strains tested were positive for TTSS. Thus, the distribution of major virulence genes in the non-O1, non-O139 serogroups of V. cholerae is largely different from that of the O1 or O139 serogroups. However, the prevalence of putative accessory virulence genes (mshA, hlyA, and RTX) was similar in all strains, with the mshA being most prevalent (98.8%) followed by RTX genes (96.2%) and hlyA (94.6%), supporting more recent assumptions that these genes imparts increased environmental fitness. Since all pathogenic strains retain these genes, the epidemiological success of the strains presumably depends on their environmental persistence in addition to the ability to produce major virulence factors. Potential precursors of new pathogenic strains would thus require to assemble a combination of genes for both ecological fitness and virulence to attain epidemiological predominance.     

Incidence and toxigenicity of Vibrio cholerae in a freshwater lake during the epidemic of cholera caused by serogroup O139 Bengal in Calcutta, India

Abstract The extent of contamination of a freshwater lake with Vibrio cholerae 0139 Bengal and the toxigenicity of all the V. cholerae isolates recovered during the period of the study were examined during and after an explosive outbreak of 0139 cholera in Calcutta. Strains biochemically characterized as V. cholerae could be isolated throughout the period of study examined from the freshwater lake samples. Most probable number of V. cholerae belonging to the 0139 serogroup in surface waters was 3 to 4 per 100 ml during major part of the study but isolation of this serogroup from sediment and plankton samples was infrequent. Of the total of 150 strains recovered, 23 (15.3%) agglutinated with the 0139 antiserum while the remaining belonged to the non-O1 non-O139 serogroups. None of the strains agglutinated with the O1 antiserum. All the 23 strains of V. cholerae O139 produced cholera toxin while 7.9% of the 127 non-O1 non-O139 strains also produced cholera toxin. Resistance to ampilicillin, furazolidone and streptomycin was encountered among strains belonging to both V. cholerae O139 and V. cholerae non-O1 non-O139 strains, but the percentage of resistant strains in the former was much higher than in the latter. During this cholera epidemic, possibly due to the introduction of large numbers of toxigenic V. cholerae such as the O139 serogroup, there was an increase in the number of toxigenic vibrios among the innocuous aquatic residents. This presumably occured through genetic exchange and, if substantiated, could play an important role in the re-emergence of epidemics.     

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

The sixth and seventh cholera pandemics are due to independent clones separately derived from environmental, nontoxigenic, non-O1 Vibrio cholerae.

The DNA sequences of the asd genes from 45 isolates of Vibrio cholerae (19 clinical O1 isolates, 2 environmental nontoxigenic O1 isolates, and 24 isolates with different non-O1 antigens) were determined. No differences were found within either sixth- or seventh-pandemic isolates; however, variation was found between the two forms and among the non-O1 isolates. O139 isolates had sequences identical to those of seventh-pandemic isolates. Phylogenetic trees with Vibrio mimicus as the outgroup suggest that the sixth-pandemic, seventh-pandemic, and U.S. Gulf isolates are three clones that have evolved independently from different lineages of environmental, nontoxigenic, non-O1 V. cholerae isolates. There is evidence for horizontal transfer of O antigen, since isolates with nearly identical asd sequences had different O antigens, and isolates with the O1 antigen did not cluster together but were found in different lineages. We also found evidence for recombination events within the asd gene of V. cholerae. V. cholerae may have a higher level of genetic exchange and a lower level of clonality than species such as Salmonella enterica and Escherichia coli.     

Difference of Phenotype and Genotype Between Human and Environmental: Isolated Vibrio cholerae in Surabaya,Indonesia

Cholera due to Vibrio cholerae has been spreading worldwide, although the reports focusing on Indonesian V. cholerae are few. In this study, in order to investigate how V. cholerae transmitted to human from environment. We extended an epidemiological report that had investigated the genotype of V. cholerae isolated from human pediatric samples and environmental samples. We examined 44 strains of V. cholerae isolated from pediatric diarrhea patients and the environment such as shrimps or oysters collected in three adjacent towns in Surabaya, Indonesia. Susceptibilities were examined for 11 antibiotics. Serotype O1 or O139 genes and pathogenic genes including cholera toxin were detected. Multi-locus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC)-PCR were also performed to determine genetic diversity of those isolates. Serotype O1 was seen in 17 strains (38.6%) with all pathogenic genes among 44 isolates. Other isolates were non-O1/non-O139 V. cholerae. Regarding antibiotic susceptibilities, those isolates from environmental samples showed resistance to ampicillin (11.4%), streptomycin (9.1%) and nalidixic acid (2.3%) but those isolates from pediatric stools showed no resistance to those 3 kinds of antibiotics. MLST revealed sequence type (ST) 69 in 17 strains (38.6%), ST198 in 3 strains (6.8%) and non-types in 24 strains (54.5%). All the ST69 strains were classified to O1 type with more than 95% similarity by ERIC-PCR, including all 6 (13.6%) isolates from environmental samples with resistance to streptomycin. In conclusion, V. cholerae O1 ST69 strains has been clonally spreading in Surabaya, exhibiting pathogenic factors and antibiotic resistance to streptomycin, especially in the isolates from environment.     

Genotypes associated with virulence in environmental isolates of Vibrio cholerae

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.     

Genomic and phenotypic diversity of coastal Vibrio cholerae strains is linked to environmental factors

Studies of Vibrio cholerae diversity have focused primarily on pathogenic isolates of the O1 and O139 serotypes. However, autochthonous environmental isolates of this species routinely display more extensive genetic diversity than the primarily clonal pathogenic strains. In this study, genomic and metabolic profiles of 41 non-O1/O139 environmental isolates from central California coastal waters and four clinical strains are used to characterize the core genome and metabolome of V. cholerae. Comparative genome hybridization using microarrays constructed from the fully sequenced V. cholerae O1 El Tor N16961 genome identified 2,787 core genes that approximated the projected species core genome within 1.6%. Core genes are almost universally present in strains with widely different niches, suggesting that these genes are essential for persistence in diverse aquatic environments. In contrast, the dispensable genes and phenotypic traits identified in this study should provide increased fitness for certain niche environments. Environmental parameters, measured in situ during sample collection, are correlated to the presence of specific dispensable genes and metabolic capabilities, including utilization of mannose, sialic acid, citrate, and chitosan oligosaccharides. These results identify gene content and metabolic pathways that are likely selected for in certain coastal environments and may influence V. cholerae population structure in aquatic environments.     

Antibodies (IgG) to lipopolysaccharide of Vibrio cholerae O1 mediate protection through inhibition of intestinal adherence and colonisation in a mouse model

An antiserum raised against purified lipopolysaccharide (LPS) of a Vibrio cholerae O1 strain (Co366) induced passive protection against challenge with the parent as well as other O1 organisms but not against O139 or non-O1/non-O139 organisms. A considerable level of protection against O1 strains was also observed with the IgG fraction of the antiserum which inhibited intestinal adherence and colonisation. The monovalent Fab(IgG) fragment, on the other hand, showed only a low level of protection. Interestingly, purified LPS failed to inhibit intestinal colonisation by the parent strain (Co366), thereby suggesting that the cell surface LPS moieties of vibrios may not be directly involved in the colonisation process. It may be concluded that the anti-LPS antibodies induce passive protection through microagglutination and/or immobilisation of vibrios which do not allow the organisms to adhere to and colonise the intestine.     

Incidence of Vibrio cholerae and related vibrios in a coastal lagoon and seawater influenced by lake discharges along an annual cycle

Most probable numbers of Vibrio cholerae and related vibrios were determined in Albufera Lake, Valencia, Spain, and in coastal waters under the influence of the lake discharges over the course of an annual cycle. The influence of temperature, kind of water, and characteristics of the different sampling sites on the numbers of vibrios recovered was evaluated. Maximum recovery of vibrios reached 10(3)/ml in both types of waters analyzed. V. cholerae numbers reached 10(3)/ml in the lake and 10(2) in one of the coastal sites. Frequently during the warm season, all vibrios isolated were identified as V. cholerae. Occasionally, no V. cholerae was recovered. The recovery of vibrios was significantly influenced by the temperature of the water and the type of water analyzed. Most of the V. cholerae isolates were included in Heiberg groups I and II, and nearly 50% of the strains used chitin as sole carbon source. Indole was not produced by 100% of the strains. All strains tested were non-O1 serovars.     

Incidence of Vibrio cholerae and related vibrios in a coastal lagoon and seawater influenced by lake discharges along an annual cycle.

Most probable numbers of Vibrio cholerae and related vibrios were determined in Albufera Lake, Valencia, Spain, and in coastal waters under the influence of the lake discharges over the course of an annual cycle. The influence of temperature, kind of water, and characteristics of the different sampling sites on the numbers of vibrios recovered was evaluated. Maximum recovery of vibrios reached 10(3)/ml in both types of waters analyzed. V. cholerae numbers reached 10(3)/ml in the lake and 10(2) in one of the coastal sites. Frequently during the warm season, all vibrios isolated were identified as V. cholerae. Occasionally, no V. cholerae was recovered. The recovery of vibrios was significantly influenced by the temperature of the water and the type of water analyzed. Most of the V. cholerae isolates were included in Heiberg groups I and II, and nearly 50% of the strains used chitin as sole carbon source. Indole was not produced by 100% of the strains. All strains tested were non-O1 serovars.     

Biotype-specific tcpA genes in Vibrio cholerae

Abstract The tcpA gene, encoding the structural subunit of the toxin-coregulated pilus, has been isolated from a variety of clinical isolates of Vibrio cholerae , and the nucleotide sequence determined. Strict biotype-specific conservation within both the coding and putative regulatory regions was observed, with important differences between the El Tor and classical biotypes. V. cholerae O139 Bengal strains appear to have El Tor-type tcpA genes. Environmental O1 and non-O1 isolates have sequences that bind an E1 Tor-specific tcpA DNA probe and that are weakly and variably amplified by tcpA -specific polymerase chain reaction primers, under conditions of reduced stringency. The data presented allow the selection of primer pairs to help distinguish between clinical and environmental isolates, and to distinguish El Tor (and Bengal) biotypes from classical biotypes from classical biotypes of V. cholerae . While the role of TcpA in cholera vaccine preparations remains unclear, the data strongly suggest that TcpA-containing vaccines directed at O1 strains need include only the two forms of TcpA, and that such vaccines directed at (O139) Bengal strains should include the TcpA of El Tor biotype.     

Biofilm acts as a microenvironment for plankton-associated Vibrio cholerae in the aquatic environment of Bangladesh

The role of biofilm as a microenvironment of plankton-associated Vibrio cholerae was investigated using plexiglass as a bait. A total of 72 biofilm samples were tested using culture, direct fluorescent antibody (DFA) and molecular techniques following standard procedures. Culturable V. cholerae (smooth and rugose variants) were isolated from 33% of the samples. V. cholerae O1 were detected by FA technique throughout the year except April and June. All V. cholerae O1 isolates were positive for tcpA, ctxA and ace genes while V. cholerae non-O1, non-O139 isolates lacked these genes. V. cholerae O1 (both Inaba and Ogawa) strains had identical ribotype pattern (R1), but V. cholerae non-O1, non-O139 had different ribotype patterns. All V. cholerae O1 strains were resistant to vibrio-static compound (O/129). All V. cholerae O1 except one were resistant to trimethoprime-sulphamethoxazole, streptomycin, nalidixic acid and furazolidone but sensitive to ciprofloxacin, and tetracycline. This study indicates that plexiglass can act as a bait to form biofilm, a microenvironment that provides shelter for plankton containing V. cholerae in the aquatic environment of Bangladesh.     

O139霍乱弧菌生物学特性研究

１９９２年以来,许多国家和地区先后暴发了Ｏ１３９霍乱大流行。本文从微生物学和分子遗传学的角度对来自不同地区的四株Ｏ１３９霍乱弧菌的生物学特性进行了研究。结果表明四株Ｏ１３９霍乱弧菌均呈典型弧形、单端单鞭毛,培养要求不高、耐碱,固体平板上菌落呈不透明。电镜下显示有菌毛、荚膜结构。有较广的抗生素敏感谱及霍乱Ｈｅｉｂｅｒｇ氏Ⅰ群的糖发酵能力。ＤＮＡＧ＋ＣＭＯＬ％测定值均在霍乱弧菌范围之内且数值接近。质粒图谱检测发现四株中有三株含有一个４．１０ＭＤａ大小的质粒,而另一株不含质粒。Ｏ１３９霍乱弧菌的生物学特性大多数与Ｏ１群菌相似,两者重大的区别在于Ｏ１３９菌具荚膜结构。     

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.     

Diversity of DNA sequences among Vibrio cholerae O1 and non-O1 isolates detected by whole-cell repetitive element sequence-based polymerase chain reaction

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.     

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.     

High Prevalence of Vibrio cholerae Non-O1 Carrying Heat-Stable-Enterotoxin-Encoding Genes among Vibrio Isolates from a Temperate-Climate River Basin of Central Italy

Vibrio spp. of clinical interest from the Arno River basin (Tuscany, Italy) were investigated in this study. Vibrios were isolated from 70% of water samples. Vibrio cholerae non-O1 was the most prevalent species (82% of isolates), followed by Vibrio mimicus (10%) and Vibrio metschnikovii (8%). Recovery of vibrios was correlated with temperature, pH, and various indicators of municipal pollution. None of the 150 Vibrio isolates carried ctx-related genomic sequences, whereas 18 (14.6%) of the 123 V. cholerae non-O1 isolates and 1 (6.7%) of the 15 V. mimicus isolates carried sto alleles. These findings indicate that considerable circulation of sto-positive vibrios may occur in temperate-climate freshwater environments.     

Effects of temperature and salinity on Vibrio cholerae growth.

Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars O1 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (25%) and a temperature of 10 degrees C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.