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.     

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.     

Occurrence of Vibrio parahaemolyticus, V. cholerae, and V. vulnificus in Norwegian Blue Mussels (Mytilus edulis)

Vibrio parahaemolyticus, V. cholerae, and V. vulnificus were isolated from 10.3%, 1.0%, and 0.1% of 885 blue mussel samples, respectively. Four of the samples contained trh(+) V. parahaemolyticus, while no tdh-positive isolates were detected. The V. cholerae isolates were non-O:1/non-O:139 serotypes and were ctxA negative.     

Environment and virulence factors of Vibrio cholerae strains isolated in Argentina

AIMS: To determine the presence of Vibrio cholerae in different areas of Argentina in three sample types, to determine the composition of planktonic communities in areas at which this pathogen was detected and to characterize the virulence properties and antimicrobial resistance of the recovered environmental isolates. METHODS AND RESULTS: Water and plankton samples were collected in marine, brackish and freshwater environments. Vibrio cholerae non-O1, non-O139 was isolated in 36.1% of the samples analysed. The micro-organism was detected in freshwater but not in marine or brackish samples. No relationship was found between isolation of V. cholerae and presence of any species of plankton. All the isolates presented very similar virulence profiles by PCR, lacking ctxA and tcpA El Tor and containing hlyA (98.7%), rtxA (99.0%), toxR (98.7%) and stn-sto (1.9%). Resistance to ampicillin was found in both Tucumán (21%) and Buenos Aires isolates (45%). CONCLUSIONS: We identified two geographic areas in Argentina where V. cholerae was present: freshwaters of the rivers from Tucumán and the Río de la Plata. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of V. cholerae strains in the environment, carrying both virulence factors and resistance to antimicrobial agents, highlight the need for a continuous and active surveillance of this pathogen.     

Incidence of toxigenic vibrios in foods available in Taiwan

A total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V. fluvialis I, V. fluvialis II, V. parahaemolyticus, V. mimicus, Aeromonas caviae, A. hydrophila, A. sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. The Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V. cholerae, V. fluvialis I and V. fluvialis II isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-1 V. cholerae (2.07%) and two V. parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods.     

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.     

Incidence of toxigenic vibrios in foods available in Taiwan.

A total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V. fluvialis I, V. fluvialis II, V. parahaemolyticus, V. mimicus, Aeromonas caviae, A. hydrophila, A. sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. The Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V. cholerae, V. fluvialis I and V. fluvialis II isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-O1 V. cholerae (2.07%) and two V. parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods.     

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.     

Identification of virulence factors in Vibrio cholerae isolated from Iraq during the 2007-2009 outbreak

Thousands of people were infected with Vibrio cholerae during the outbreak in Iraq in 2007-2009. Vibrio cholerae was shown to be variable in its content of virulence determinants and in its antibiotic sensitivity. This study was designed to isolate and characterize clinical and environmental V.?cholerae isolates and to determine antibiotic sensitivity, enzyme and toxin production, and the presence of virulence genes. Eighty clinical and five environmental bacterial isolates were collected and diagnosed by subjecting them to microscopic, biochemical, serological, and molecular analysis. The results revealed that 55% of clinical isolates belonged to the Inaba serotype, 32.5% to the Ogawa serotypes, and 12.5% to the Non-O1 serotype. All environmental V.?cholerae isolates belonged to the Non-O1 serotype. All environmental isolates were sensitive to all examined antimicrobial agents, while all clinical isolates showed a high sensitivity (100%) to ampicillin, gentamicin, cephalothin, tetracycline, erythromycin, and ciprofloxacin, and a high resistance (97.5%) to co-trimoxazole, nalidixic acid, and chloramphenicol. It was found that all V.?cholerae (O1) isolates were resistant to the Vibrio static O129 and all Non-O1 V.?cholerae isolates were sensitive to the Vibrio static O129. All clinical and environmental isolates produced hemolysin (100%) and lecithinase (100%), while they showed various production rates of protease (90% of clinical and 60% of environmental) and lipase (50% of clinical and 20% of environmental). The ompW gene was amplified in all the clinical and environmental V.?cholerae isolates, but not in other related and nonrelated bacteria. Multiplex PCR analysis showed that the toxR gene was amplified in all clinical and environmental isolates, while ctxA, ctxB, tcpA genes were amplified only in clinical (O1) isolates. This study indicates the differences in the production of some enzymes and toxins and in the content of virulence genes between clinical and environmental isolates in Iraq during the outbreak (2007-2009).     

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.     

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.     

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.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah

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

Isolation of 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.     

Occurrence, diversity, and pathogenicity of halophilic Vibrio spp. and non-O1 Vibrio cholerae from estuarine waters along the Italian Adriatic coast.

The occurrence, diversity, and pathogenicity of Vibrio spp. were investigated in two estuaries along the Italian Adriatic coast. Vibrio alginolyticus was the predominant species, followed by Vibrio parahaemolyticus, non-O1 Vibrio cholerae, and Vibrio vulnificus. By using a biochemical fingerprinting method, all isolates were grouped into nine phenotypes with similarity levels of 75 to 97.5%. The production of toxins capable of causing cytoskeleton-dependent changes was detected in a large number of Vibrio strains. These findings indicate a significant presence of potentially pathogenic Vibrio strains along the Adriatic coast.     

Occurrence of Vibrio cholerae serotype O1 in Maryland and Louisiana estuaries.

Vibrio cholerae serotype O1 has been isolated from Chesapeake Bay in Maryland and estuaries and sewers in Louisiana. The occurrence of V. cholerae O1 in the aquatic environment in the absence of human disease suggests that this organism survives and multiples in the natural environment.     

Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.     

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.     

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.