The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

Genome sequencing reveals unique mutations in characteristic metabolic pathways and the transfer of virulence genes between V. mimicus and V. cholerae

Vibrio mimicus, the species most similar to V. cholerae, is a microbe present in the natural environmental and sometimes causes diarrhea and internal infections in humans. It shows similar phenotypes to V. cholerae but differs in some biochemical characteristics. The molecular mechanisms underlying the differences in biochemical metabolism between V. mimicus and V. cholerae are currently unclear. Several V. mimicus isolates have been found that carry cholera toxin genes (ctxAB) and cause cholera-like diarrhea in humans. Here, the genome of the V. mimicus isolate SX-4, which carries an intact CTX element, was sequenced and annotated. Analysis of its genome, together with those of other Vibrio species, revealed extensive differences within the Vibrionaceae. Common mutations in gene clusters involved in three biochemical metabolism pathways that are used for discrimination between V. mimicus and V. cholerae were found in V. mimicus strains. We also constructed detailed genomic structures and evolution maps for the general types of genomic drift associated with pathogenic characters in polysaccharides, CTX elements and toxin co-regulated pilus (TCP) gene clusters. Overall, the whole-genome sequencing of the V. mimicus strain carrying the cholera toxin gene provides detailed information for understanding genomic differences among Vibrio spp. V. mimicus has a large number of diverse gene and nucleotide differences from its nearest neighbor, V. cholerae. The observed mutations in the characteristic metabolism pathways may indicate different adaptations to different niches for these species and may be caused by ancient events in evolution before the divergence of V. cholerae and V. mimicus. Horizontal transfers of virulence-related genes from an uncommon clone of V. cholerae, rather than the seventh pandemic strains, have generated the pathogenic V. mimicus strain carrying cholera toxin genes.     

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.     

Variation in epitopes of the B subunit of Vibrio cholerae non-O1 and Vibrio mimicus cholera toxins.

Monoclonal antibodies reacting with the B subunit of Vibrio cholerae O1 strain 569B cholera toxin (CT-B) were used to identify unique and common epitopes of V. cholerae non-O1 and Vibrio mimicus CT-B. Vibrio cholerae non-O1 strains produced CT-B showing three monoclonal antibody reaction patterns (epitypes), which corresponded with epitypes described previously for V. cholerae O1 classical biotype CT-B (CT1), El Tor biotype CT-B (CT2), and a unique V. cholerae non-O1 CT-B (CT3), which lacked an epitope located in or near the GM1 ganglioside binding site of 569B CT-B. Vibrio mimicus CT-B was immunologically indistinguishable from 569B CT-B. These and previous results define six epitopes on 569B CT-B, and a fourth epitope in or near the GM1 ganglioside binding site.     

A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139

Abstract The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae , in addition to cholera toxin, was investigated in selected strains of V. mimicus and the new pandemic V. cholerae non-O1 serotype O139. The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V. cholerae O1 zot sequence. PCR amplification of genomic DNAs of both cholera toxin gene ( ctx ) positive and ctx⁻ strains of V. mimicus detected the presence of zot gene. An Acc -I- Eco RV V. cholerae zot gene fragment designed to overlap PCR products was used as a probe. Southern hybridization studies confirmed that the PCR fragments from V. mimicus and V. cholerae O139 were strongly homologous to the V. cholerae O1 zot gene. The zot gene was found with 3 to 5 strains of V. mimicus of which only one strain harbored the ctx gene. The presence of a zot gene in ctx⁻ toxigenic V. mimicus indicates a possible role of ZOT in the toxigenicity of this species. We conclude that, in addition to ctx, V. mimicus and V. cholerae O139 have the potential to produce ZOT.     

Distribution of virulence-associated genes in Vibrio mimicus isolates from clinical and environmental origins

Distribution of virulence-associated genes in Vibrio mimicus was studied including the toxin genes ctxA, tdh, st and vmh and the genes necessary for regulation of toxin production, toxR, toxS, toxT, tcpA and tcpP. Approximately half of clinical V. mimicus isolates possessed one or more genes encoding V. cholerae enterotoxic factors such as ctxA, tdh and st. All of the clinical and environmental isolates possessed vmh encoding V. mimicus hemolysin (VMH). The ctxA encoding cholera toxin was detected in only 2 strains, 5% of the clinical isolates. Furthermore, there were very few strains possessing tcpP and toxT needed for the expression of ctxA. These results may suggest that VMH is a more important pathogenic factor than well recognized toxins such as cholera toxin (CT) in V. mimicus infection.     

Lipopolysaccharide composition and virulence properties of clinical and environmental strains of Vibrio fluvialis and Vibrio mimicus.

Vibrio mimicus strains W-26768 (stool isolate) and N-1301 (environmental isolate) and Vibrio fluvialis strains AA-18239 (stool isolate) and M-940 (environmental isolate) were studied for virulence properties and lipopolysaccharide composition. All four strains were hydrophobic, produced cytotoxin, adhered to HeLa cells and showed mannose-sensitive agglutination of guinea pig erythrocyte. The strains were negative for enterotoxin production and were mostly susceptible to the common antibiotics. The environmental and clinical isolates of both species were antigenically unrelated to each other. Lipopolysaccharide antigen analysis showed that O-antigen polysaccharides of two strains of V. fluvialis and two strains of V. mimicus differed with respect to the sugar components. Only LPS from V. mimicus W-26768 showed the presence of an unusual sugar, 3,6-dideoxy-3-acetamido-hexose. The sugar compositions of these V. fluvialis and V. mimicus strains differed from those of previously reported Japanese isolates. These differences probably reflect differences in the serogroup of strains.     

Toxin profiles of Vibrio cholerae non-O1 from environmental sources in Calcutta, India.

A collection of Vibrio cholerae non-O1 isolated from the aquatic environs of Calcutta, a cholera-hyperendemic area, were examined for the production of cholera toxin (CT), Shiga-like toxins (Vero toxins), heat-stable enterotoxin, and hemolysins. Two (0.5%) V. cholerae non-O1 isolates produced CT. The DNA from both these isolates also hybridized with a DNA probe containing sequences encoding the A subunit of CT. None of the strains produced Shiga-like toxins or heat-stable enterotoxin. Hemolytic activity was observed in 89.7% of the strains, of which 36.1% exhibited biological activity in the suckling mouse. However, none of them produced a hemolysin that cross-reacted with the thermostable direct hemolysin of Vibrio parahaemolyticus. It appears from this study that a small percentage of environmental V. cholerae non-O1 strains do possess the potential for causing cholera-like diarrhea.     

Toxin profiles of Vibrio cholerae non-O1 from environmental sources in Calcutta, India

A collection of Vibrio cholerae non-O1 isolated from the aquatic environs of Calcutta, a cholera-hyperendemic area, were examined for the production of cholera toxin (CT), Shiga-like toxins (Vero toxins), heat-stable enterotoxin, and hemolysins. Two (0.5%) V. cholerae non-O1 isolates produced CT. The DNA from both these isolates also hybridized with a DNA probe containing sequences encoding the A subunit of CT. None of the strains produced Shiga-like toxins or heat-stable enterotoxin. Hemolytic activity was observed in 89.7% of the strains, of which 36.1% exhibited biological activity in the suckling mouse. However, none of them produced a hemolysin that cross-reacted with the thermostable direct hemolysin of Vibrio parahaemolyticus. It appears from this study that a small percentage of environmental V. cholerae non-O1 strains do possess the potential for causing cholera-like diarrhea.     

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.     

PCR-based identification of Vibrio cholerae and the closely related species Vibrio mimicus using the large chromosomal ori sequence of Vibrio cholerae

The bacterial chromosomal replication origin (ori) sequences are a highly conserved essential genetic element. In this study, the large chromosomal replication origin sequence of Vibrio cholerae (oriCIVC) has been targeted for identification of the organism, including the biotypes of serogroup O1. The oriCIVC sequence-based PCR assay specifically amplified an 890 bp fragment from all the V. cholerae strains examined. A point mutation in the oriCIVC sequence of the classical biotype of O1 serogroup led to the loss of a BglII site, which was utilized for differentiation from El Tor vibrios. Interestingly, the PCR assay amplified a similarly sized ori segment, designated as oriCIVM, from V. mimicus strains, but failed to produce any amplicon with other strains. Cloning and sequencing of the oriCIVM revealed high sequence similarity (96%) with oriCIVC. The results indicate that V. mimicus is indeed very closely related to V. cholerae. In addition, the BglII restriction fragment length polymorphism (RFLP) between oriCIVM and oriCIVC sequences allowed us to differentiate the two species. The ori sequence-based PCR-RFLP assay developed in this study appears to be a useful method for rapid identification and differentiation of V. cholerae and V. mimicus strains, as well as for the delineation of classical and El Tor biotypes of V. cholerae O1.     

Characterization of enterotoxin and soluble hemagglutinin from Vibrio mimicus: identity with V. cholerae O1 toxin and hemagglutinin

Abstract Eight strains of Vibrio mimicus isolated from patients with diarrhoea in Bangladesh were all found to produce an extracellular toxin identical to cholera toxin produced by Vibrio cholerae O1 bacteria, with regard to subunit structure and immunological properties. Like cholera toxin, but in contrast to heat-labile enterotoxin from Escherichia coli most of the toxin from V. mimicus was found extracellularly and was proteolytically 'nicked' in its A subunit. This may relate to the finding that V. mimicus also produced an extracellular hemagglutinin which was immunologically indistinguishable from the soluble hemagglutinin/nicking protease of V. cholerae O1.     

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.     

Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.     

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。     

Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.