Comparison of genome structures of vibrios,bacteria possessing two chromosomes

Vibrios are gram-negative gamma-proteobacteria which are ubiquitous in marine and estuarine environments. Recently, we demonstrated that some, if not all, Vibrio species have two circular chromosomes. The whole genome sequence of Vibrio cholerae N16961 has been reported. In this study, we constructed a physical and genetic map of the genome of Kanagawa phenomenon-positive Vibrio parahaemolyticus strain KX-V237 and compared it with those of V. parahaemolyticus AQ4673 and V. cholerae N16961. The genome of KX-V237 comprised two circular chromosomes (3.3 and 1.9 Mb), similar to the structure of the AQ4673 genome. The relative positions of the genes on the genomes were well conserved in the two strains, but a large inversion on the large chromosomes, probably symmetric around the replication origin, was suggested. Although the sizes of the large chromosomes of KX-V237 and V. cholerae N16961 were similar, the sizes of the small chromosomes were very different. Unlike N16961, the superintegron of KX-V237 was located on the large chromosome. Comparison of the genetic maps of the chromosomes of KX-V237 and V. cholerae N16961 revealed that most of the open reading frames (ORFs) present on the large chromosome of the V. cholerae strain had homologues on the large chromosome of the V. parahaemolyticus strain and that most of the ORFs on the small chromosome of N16961 were present on the small chromosome of KX-V237. The difference in the orders of the ORFs on the chromosomes of N16961 and KX-V237 implies that numerous and frequent genetic exchanges have occurred intrachromosomally rather than interchromosomally.     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

Detection and identification of Vibrio species using whole-cell protein pattern analysis

Outbreaks of foodborne diseases associated with Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae frequently occur in countries having a dietary habit of raw seafood consumption. For rapid identification of different Vibrio species involved in foodborne diseases, whole-cell protein pattern analysis for 13 type strains of 12 Vibrio species was performed using SDS-PAGE analysis. Pathogenic Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus, V. fluvialis, and V. mimicus were included in the 12 Vibrio species used in this study. Each of the 12 Vibrio species showed clearly specific band patterns of its own. Two different strains of V. parahaemolyticus showed two different SDS-PAGE wholecell protein patterns, giving the possibility of categorizing isolated strains in the same V. parahaemolyticus species into two subgroups. The 36 Vibrio isolates collected from sushi restaurants in Busan were all identified as V. parahaemolyticus by comparing their protein patterns with those of Vibrio type strains. The identified isolates were categorized into two different subgroups of V. parahaemolyticus. The whole-cell protein pattern analysis by SDS-PAGE can be used as a specific, rapid, and simple identification method for Vibrio spp. involved in foodborne diseases at the subspecies level.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains.

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.     

Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling.

A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis. A total of 90.9% of V. tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands. In contrast, the V. tapetis strains showed a low degree of similarity with six reference Vibrio species tested. All V. tapetis strains harbored a large plasmid of 74.5 kb. This plasmid was not detected in any of the other Vibrio species. In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V. tapetis possessed the same cleavage pattern. The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb. The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V. tapetis strains belonged to the same ribotype when the enzyme PvuII was used.     

Sequence Analyses of Type IV Pili from Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus

Bacterial surface structures called pili have been studied extensively for their role as possible colonization factors. Most sequenced Vibrio genomes predict a variety of pili genes in these organisms, including several types of type IV pili. In particular, the mannose-sensitive hemagglutinin (MSHA) and the PilA pili, also known as the chitin-regulated pilus (ChiRP), are type IVa pili commonly found in Vibrio genomes and have been shown to play a role in the colonization of Vibrio species in the environment and/or host tissue. Here, we report sequence comparisons of two type IVa pilin subunit genes, mshA and pilA, and their corresponding amino acid sequences, for several strains from the three main human pathogenic Vibrio species, V. cholerae, V. parahaemolyticus, and V. vulnificus. We identified specific groupings of these two genes in V. cholerae, whereas V. parahaemolyticus and V. vulnificus strains had no apparent allelic clusters, and these genes were strikingly divergent. These results were compared with other genes from the MSHA and PilA operons as well as another Vibrio pili from the type IVb group, the toxin co-regulated pilus (TCP) from V. cholerae. Our data suggest that a selective pressure exists to cause these strains to vary their MSHA and PilA pilin subunits. Interestingly, V. cholerae strains possessing TCP have the same allele for both mshA and pilA. In contrast, V. cholerae isolates without TCP have polymorphisms in their mshA and pilA sequences similar to what was observed for both V. parahaemolyticus and V. vulnificus. This data suggests a possible linkage between host interactions and maintaining a highly conserved type IV pili sequence in V. cholerae. Although the mechanism underlying this intriguing diversity has yet to be elucidated, our analyses are an important first step towards gaining insights into the various aspects of Vibrio ecology.     

Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China

AIMS: To investigate the distribution of the virulence of two Vibrio species among different strains obtained from the mariculture systems on the coast of Guangdong in China and the correlation between the virulence strains and the virulence genes among Vibrio alginolyticus. METHODS: Besides three strains, 72 V. alginolyticus strains and seven Vibrio parahaemolyticus strains were examined by PCR or semi-nested PCR for the virulence genes (tlh, trh, tdh, toxR, toxRS, ctxA, VPI). Additionally, the virulence of 18 V. alginolyticus strains was tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulence genes homologous to those in the V. parahaemolyticus and Vibrio cholerae are widely distributed among V. alginolyticus and V. parahaemolyticus in the coastal mariculture systems in Guangdong, China. Some of the V. alginolyticus strains are pathogenic to aquatic animals, and might have derived their virulence genes from V. parahaemolyticus or V. cholerae, representing a possible reservoir of these genes. However, there is no correlation between presence and absence of the virulence genes used to investigate V. alginolyticus and its virulent strains. In this report, we also show that tlh is distributed among V. alginolyticus.     

Construction of a Helicobacter pylori genome map and demonstration of diversity at the genome level.

Genomic DNA from 30 strains of Helicobacter pylori was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with NotI and NruI. The genome sizes of the strains ranged from 1.6 to 1.73 Mb, with an average size of 1.67 Mb. By using NotI and NruI, a circular map of H. pylori UA802 (1.7 Mb) which contained three copies of 16S and 23S rRNA genes was constructed. An unusual feature of the H. pylori genome was the separate location of at least two copies of 16S and 23S rRNA genes. Almost all strains had different PFGE patterns after NotI and NruI digestion, suggesting that the H. pylori genome possesses a considerable degree of genetic variability. However, three strains from different sites (the fundus, antrum, and body of the stomach) within the same patient gave identical PFGE patterns. The genomic pattern of individual isolates remained constant during multiple subcultures in vitro. The reason for the genetic diversity observed among H. pylori strains remains to be explained.     

Genetic transformation of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae non O-1 with plasmid DNA by electroporation

An electroporation procedure for the plasmid-mediated transformation of the genus Vibrio was performed, as part of an effort to develop recombinant DNA techniques for genetic manipulation of the genus Vibrio. Vibrio parahaemolyticus, V. alginolyticus, and V. cholerae non O-1 (9 different strains) were transformed with 3 vector plasmids (pACYC184, pHSG398, and pBR325). The efficiency of transformation was highly dependent on three parameters: the concentration of plasmid DNA; the strength of the electric field; and the combination of plasmid DNA and recipient strain. The drug-resistance genes on the vector plasmid were expressed in the Vibrio strains.     

Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.     

Polyphasic Taxonomy of the Genus Vibrio: Polynucleotide Sequence Relationships Among Selected Vibrio Species

Polynucleotide relationships among selected Vibrio species were examined by means of deoxyribonucleic acid (DNA) reassociation reactions and chromatography on hydroxyapatite. Relative levels of intraspecific DNA duplex formation (V. cholerae-V. cholerae and V. parahaemolyticus-V. parahaemolyticus) were found to be high at 60 C (>80%), and only minimally reduced at 75 C. Interspecific DNA duplexes between V. cholerae DNA and that of the non-cholera vibrios also exhibited high relative levels of formation at 60 C (>80%) and, with one exception, were only slightly reduced at 75 C. The thermal stability of these duplexes formed at 60 or 75 C was virtually identical to that of homologous V. cholerae DNA duplexes. The degree of reassociation and the thermal stability of V. cholerae-non-cholera vibrio DNA duplexes suggests relatively little evolutionary divergence in these organisms. In all other interspecific DNA reassociation reactions, only low levels of DNA duplex formation were noted at 60 C (<25%), and these were drastically reduced (>50%) at 75 C. The degree of nucleotide sequence divergence indicated by these reactions suggests that these Vibrio species are not significantly related to V. cholerae or V. parahaemolyticus. Reassociation reactions between V. cholerae DNA and the DNA of V. parahaemolyticus indicated these species were not significantly related to each other.     

Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy

The members of the genus Vibrio include harmless aquatic strains as well as strains capable of causing epidemics of cholera. Diarrhoea caused by Vibrio cholerae is attributed to cholerae enterotoxin (CT) codified by the ctx operon and regulated by a number of virulence genes such as toxT, toxR and toxS. Fifty-two Vibrio strains were isolated from different aquatic environments in and around Sardinia and searched by PCR for the presence of ctxA, zot, ace, toxR, toxS, toxT, tcpA and vpi virulence genes in the genomes of the isolates. The toxR operon was found in 27 Vibrio alginolyticus strains out of 42 analysed, in three out of four V. cholerae non-O1 strains and in three Vibrio parahaemolyticus isolates. A positive amplification for the virulence pathogenic island (vpi) was produced by five V. alginolyticus strains. Finally, the ace expected amplification fragment was found in two V. alginolyticus isolates whereas the amplification with zot primers produced the expected fragment in one V. alginolyticus isolate. Differentiation of these strains with a PCR fingerprinting technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern. Although most Vibrio species are considered non-pathogenic or only potentially harmful to humans, the finding of V. cholerae virulence genes in other members of the genus Vibrio, and the recent reports of the creation and evolution of pandemic strains of V. cholerae, may give a new perspective to the significance of these results.     

On the origin and function of an insertion element VPaI-1 specific to post-1995 pandemic Vibrio parahaemolyticus strains

Since 1995, new virulent strains of Vibrio parahaemolyticus have emerged and spread throughout the world. These "pandemic" strains have four strain specific genomic islands (GIs), which are considered to be potential factors of the pandemicity. We investigated the origin and function of 24 genes in the so-called VPaI-1, one of the four GIs, by searching homologs in various species in Bacteria and Archaea. Of these 24 genes, two are found only in Vibrio vulnificus CMCP6 and Shewanella sp. MR-7. The genomic segment (- 8 kb) encompassing the two genes shows the synteny among the three species. Since many of the Shewanella species can grow at 4 degrees C, these two genes may be candidates of adaptation to temperature stress. Further, we found a candidate for a swarming gene, which is reported as the V. cholerae virulence gene. Based on these findings, we hypothesized the emergence of pandemicity and discuss the mechanism for how these strains spread throughout the world.     

Polynucleotide sequence relationships among Japanese and American strains of Vibrio parahaemolyticus

Polynucleotide sequence relationships between two reference Vibrio parahaemolyticus strains isolated from Japanese and American gastroenteritis patients were investigated by use of (32)P-DNA/DNA reassociation in free solution. In addition, these strains were similarly compared with 22 other strains of estuarine and marine vibrios, including 11 strains previously identified as V. parahaemolyticus (2 Japanese, 1 of unknown location, and 8 American strains obtained from diverse geographical locations and sources in North America), 3 strains of V. alginolyticus, and 8 of Vibrio spp. Deoxyribonucleic acid (DNA) from the Japanese and American gastroenteritis isolates showed high relative levels of intraspecific duplex formation (92 to 93%) when reassociated, reciprocally, at 60 C. Heterologous DNA duplexes exhibited thermal elution midpoint [Tm(e)] values comparable to those obtained from homologous duplexes (88.0) when thermally eluted from hydroxyapatite, thus indicating high base-pair complementarity. Other V. parahaemolyticus strains showed DNA homologies of 85% or greater, with correspondingly high Tm(e) values (86.0 to 88.0) for the heteroduplexes formed. DNA of two of three V. alginolyticus strains (ATCC 17749 and 166-70) was 55 to 60% homologous to reference V. parahaemolyticus DNA preparations; Vibrio sp. strain 5144 (originally classified as V. parahaemolyticus biotype 2 and subsequently as V. alginolyticus strain 5144) showed only 24 to 26% DNA homology to the same reference DNA. These data provide evidence that Vibrio sp. strain 5144 is genetically distinct from the other V. alginolyticus strains used in this study. Three bioluminescent strains thought to be closely related to V. parahaemolyticus demonstrated only 24 to 31% DNA homology to the reference V. parahaemolyticus DNA. These data firmly establish the existence in some Atlantic and Gulf Coast estuaries of organisms genetically very similar to V. parahaemolyticus, the causative agent of "shirasu" food poisoning in Japan.     

Environmental Vibrio spp., isolated in Mozambique, contain a polymorphic group of integrative conjugative elements and class 1 integrons

Circulation of mobile genetic elements linked to drug resistance spread was studied in Vibrio strains isolated from surface urban water (river and sea) and shellfish samples in 2002-2003 in Maputo, Mozambique. Class 1 integrons and integrating conjugative elements (ICE) were investigated by PCR and mating experiments in strains of major health interest: 10 Vibrio cholerae, six Vibrio parahaemolyticus, two Vibrio alginolyticus and one Vibrio fluvialis. Resistance to at least two antibiotics (predominantly beta-lactams) was detected in all the strains, with additional resistances to sulfamethoxazole, spectinomycin, streptomycin and/or trimethoprim. Class 1 integrons contributed partially to the expression of drug resistance and were found in five isolates: four V. cholerae (blaP1 cassette, one strain also contained the dfrA15 cassette) and one V. alginolyticus (aadA2 cassette). ICEs, apparently devoid of resistance genes, were found in eight V. cholerae, three V. parahaemolyticus and one V. fluvialis isolates. A wide variability was observed by molecular characterization of ICEs. Five ICEs were included in the SXT/R391 family and seven ICEs were not classified. Our results indicate that the SXT/R391 family and related ICEs comprise a large class of polymorphic genetic elements widely circulating in environmental Vibrio strains in Africa, beside those evidently linked to drug resistance in clinical isolates.     

Seasonal distribution of facultatively enteropathogenic vibrios (Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus) in the freshwater of the Elbe River at Hamburg

Between June 1981 and December 1982 the incidence of Vibrio cholerae, V. mimicus and V. parahaemolyticus was determined at two sampling sites on the Elbe River at Hamburg. A total of 183 strains was isolated from 147 water samples. Of these, 107 belonged to non-01 V. cholerae (ten strains producing a cholera-like enterotoxin); 33 were identified as V. mimicus , including two enterotoxin producers; 42 strains were Kanagawa-negative cultures of V. parahaemolyticus ; and one was V. fluvialis. The highest incidence was observed from June to September with about 10² organisms/1. Halophilic vibrios, less than five organisms/1, were detectable during the period June/July to October. The vibrio incidence was not influenced by the numbers of aerobic heterotrophic bacteria, coliforms or faecal bacteria. In general water temperature correlated with the seasonal variation. Thus, a temperature rise over 10° to 20°C was followed by a distinct increase in vibrio numbers. Of 14 chemical parameters only chloride concentration might have had an influence on the seasonal variation. It is concluded that the three Vibrio species are indigenous organisms of the Elbe River.     

Seasonal distribution of facultatively enteropathogenic vibrios (Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus) in the freshwater of the Elbe River at Hamburg

Between June 1981 and December 1982 the incidence of Vibrio cholerae, V. mimicus and V. parahaemolyticus was determined at two sampling sites on the Elbe River at Hamburg. A total of 183 strains was isolated from 147 water samples. Of these, 107 belonged to non-01 V. cholerae (ten strains producing a cholera-like enterotoxin); 33 were identified as V. mimicus, including two enterotoxin producers; 42 strains were Kanagawa-negative cultures of V. parahaemolyticus; and one was V. fluvialis. The highest incidence was observed from June to September with about 10(2) organisms/l. Halophilic vibrios, less than five organisms/l, were detectable during the period June/July to October. The vibrio incidence was not influenced by the numbers of aerobic heterotrophic bacteria, coliforms or faecal bacteria. In general water temperature correlated with the seasonal variation. Thus, a temperature rise over 10 degrees to 20 degrees C was followed by a distinct increase in vibrio numbers. Of 14 chemical parameters only chloride concentration might have had an influence on the seasonal variation. It is concluded that the three Vibrio species are indigenous organisms of the Elbe River.     

Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by bacteriophage susceptibility patterns.

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the O and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter-and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agar-digesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.     

Occurrence and potential pathogenesis of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden

During the summer of 2006, several wound infections - of which three were fatal - caused by Vibrio cholerae were reported from patients who had been exposed to water from the Baltic Sea. Before these reports, we initiated a sampling project investigating the occurrence of potential human pathogenic V. cholerae, Vibrio vulnificus and Vibrio parahaemolyticus in The Sound between Sweden and Denmark. The Blue mussel (Mytilus edulis) was used as an indicator to follow the occurrence of vibrios over time. Molecular analyses showed high frequencies of the most potent human pathogenic Vibrio spp.; 53% of mussel samples were positive for V. cholerae (although none were positive for the cholera toxin gene), 63% for V. vulnificus and 79% for V. parahaemolyticus (of which 47% were tdh(+) and/or trh(+)). Viable vibrios were also isolated from the mussel meat and screened for virulence by PCR. The mortality of eukaryotic cells when exposed to bacteria was tested in vivo, with results showing that the Vibrio strains, independent of species and origin, were harmful to the cells. Despite severe infections and several deaths, no report on potential human pathogenic vibrios in this area had been published before this study.