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.     

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.     

Rapid Detection of Virulence-Associated Genes in Environmental Strains of Vibrio cholerae by Multiplex PCR

Vibrio cholerae, the causative agent of cholera is ubiquitously distributed in aquatic environment particularly in coastal waters, estuaries, and rivers. In the present investigation, a multiplex PCR assay was developed for the detection of virulence-associated genes (rtxA, tcpA, ctxA, hlyA, and sto) in environmental isolates of V. cholerae. A total of 90 strains isolated from different environmental sources were screened for the presence of virulence-associated genes. Our results showed that this method represents a simple, cost effective, and robust tool for rapid detection of virulence-associated genes. This multiplex PCR can be used for examining prevalence of virulence-associated genes and hence will be useful for better understanding of epidemiology of environmental V. cholerae.     

Molecular Characterization of Vibrio cholerae O139 Bengal Isolated from Water and the Aquatic Plant Eichhornia crassipes in the River Ganga, Varanasi, India

A collection of ten strains of Vibrio cholerae O139, comprising six isolates from Eichhornia crassipes, two from water of the River Ganga, and one each from a well and a hand pump, were characterized. All the strains carried the CTX genetic element (ctxA, zot, and ace) except for the st gene and carried structural and regulatory genes for toxin-coregulated pilus (tcpA, tcpI, and toxR), adherence factor (ompU), and accessory colonization factor (acfB); all produced cholera toxin (CT). These strains were resistant to trimethoprim, sulfamethoxazole, streptomycin, and to the vibriostatic agent pteridine. Results obtained by ribotyping and enterobacterial repetitive intergenic consensus sequence-PCR fingerprint analysis indicate that multiple clones of toxigenic-pathogenic V. cholerae O139 were present in the aquatic environment.     

Putative Virulence Traits and Pathogenicity of Vibrio cholerae Non-O1, Non-O139 Isolates from Surface Waters in Kolkata, India

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (≥100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.     

Molecular characterization of Vibrio cholerae outbreak strains with altered El Tor biotype from southern India

Forty-four Vibrio cholerae isolates collected over a 7-month period in Chennai, India in 2004 were characterized for gene traits, antimicrobial susceptibility and genomic fingerprints. All 44 isolates were identified as O1 El Tor Ogawa, positive for various toxigenic and pathogenic genes viz. ace, ctxB, hlyA, ompU, ompW, rfbO1, rtx, tcpA, toxR and zot. Nucleotide sequencing revealed the presence of cholera toxin B of classical biotype in all the El Tor isolates, suggesting infection of isolates by classical CTXΦ. Antibiogram analysis showed a broad-spectrum antibiotic resistance that was also confirmed by the presence of resistant genes in the genomes. All isolates contained a class 1 integron and an SXT constin. However, isolates were sensitive to chloramphenicol and tested negative for the chloramphenicol resistant gene suggesting a deletion in SXT constin. Fingerprinting analysis of isolates by ERIC- and Box PCR revealed similar DNA patterns indicating the clonal dissemination of a single predominant V. cholerae O1 strain throughout the 2004 outbreak in Chennai.     

Virulence profile and clonal relationship among the <Emphasis Type="Italic">Vibrio cholerae</Emphasis> isolates from ground and surface water in a cholera endemic area during rainy season

All the V. cholerae non-O1, non-O139 isolates from ground and surface water samples collected during the rainy season (rainfall contributes significantly in the spread of cholera) contained ompW and a regulatory toxR gene, while many others possessed accessory cholera toxin (ace), hemolysin (hlyA) and outer membrane protein (ompU) genes. All the isolates lacked ctxAB, tcp, zot, rfbO1 and rfbO139 genes. The strains could be grouped into two main clusters colligating the isolates from ground water and surface water samples. The results suggest that surface water harbors various virulent V. cholerae strains that contaminate the ground water due to rain or poor hygienic practices, and result in the emergence of new toxigenic strains for cholera.     

Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products

A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.     

Genetic characterization of toxigenic Vibrio cholerae non-O1/non-O139 strains, isolated in the Middle Asia

Here, we report on the characterization of 22 clinical toxigenic V. cholerae non-O1/non-O139 strains isolated in the Middle Asia (Uzbekistan) in 1971–1990. PCR analysis has revealed that these strains contain the main virulence genes such as ctxA, zot, ace (CTXφ); rstC (RS1φ); tcpA, toxT, aldA (pathogenicity island VPI), but they lack both pandemic islands VSP-I and VSP-II specific to epidemic strains of O1 serogroup of El Tor biotype and O139 serogroup. Only two of the twenty two toxigenic strains have tcpA gene of El Tor type, one strain has tcpA gene of classical type, while nineteen other strains carry a new variant of this gene, designated as tcpA ^uzb. Nucleotide sequences analysis of virulence genes in toxigenic V. cholerae non-O1/non-O139 strains from Uzbekistan showed that they differ significantly from the sequences of these genes in epidemic O1 and O139 strain indicating that they belong to a separate line of evolution of virulent V. cholerae strains. For the first time it is shown that V. cholerae non-O1/non-O139 toxigenic strains of different serogroups may belong to the same clone.     

Quadruplex Real-Time PCR Assay for Detection and Identification of Vibrio cholerae O1 and O139 Strains and Determination of Their Toxigenic Potential

Vibrio cholerae is a natural inhabitant of the aquatic environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the cholera toxin gene (ctxA), the hemolysin gene (hlyA), O1-specific rfb, and O139-specific rfb, was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of V. cholerae and 31 strains of non-V. cholerae organisms. The analytical sensitivity for detection of toxigenic V. cholerae O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify V. cholerae and reliably predict the toxigenic potential of strains detected.Occasional outbreaks and pandemics caused by the bacterium Vibrio cholerae indicate that cholera is still a global threat to public health (1, 2, 6, 13, 14). The disease may become life-threatening if appropriate therapy is not undertaken quickly. Of the more than 200 serogroups of V. cholerae that have been identified (28), two serogroups, O1 and O139, cause epidemic and pandemic cholera (14), whereas non-O1, non-O139 serogroups are associated only with sporadic, isolated outbreaks of diarrhea (3, 23). O1 and O139 strains are also categorized as toxin-producing and non-toxin-producing strains. The toxin-producing strains cause life-threatening secretory diarrhea, while the non-toxin-producing isolates elicit only mild diarrhea. These differences among the serogroups of V. cholerae demand rapid diagnostic tests capable of both distinguishing O1 and O139 from other serogroups and differentiating toxin-producing from nonproducing isolates (20).PCR has become a molecular alternative to culture, microscopy, and biochemical testing for the identification of bacterial species (27). Many PCR methods have been developed for characterization of serogroups (O1 and/or O139), biotypes, and the toxigenic potential of V. cholerae strains (7, 11, 15, 19, 21, 22, 24-26). However, these conventional PCR methods require gel electrophoresis for product analysis and are therefore not suitable for routine use due to the risk of carryover contamination, low throughput, and intensive labor.Real-time PCR allows detection of amplification product accumulation through fluorescence intensity changes in a closed-tube setting, which is faster and more sensitive than conventional PCR and has become increasingly popular in clinical microbiology laboratories. Moreover, when multicolor fluorophore-labeled probes and/or melting curve analysis is used, multiplex real-time PCR can be designed to simultaneously detect many different target genes in a single reaction tube (8). So far, the majority of published real-time PCR assays for V. cholerae detect no more than two genes simultaneously (4, 8, 18), which precludes their use for simultaneous serogroup and toxin status determination. Recent reports show that multiplex real-time PCR greatly improves specificity and sensitivity for the detection of V. cholerae through either melting curve analysis (9) or using differently fluorophore-labeled probes (10).In the present work, we report the development of a quadruplex real-time PCR assay that enables simultaneous serogroup differentiation and toxigenic potential detection. By using four different fluorophore-labeled probes, which target hlyA, O1-specfic rfb, O139-specific rfb, and ctxA, the quadruplex assay can reveal whether the target is an O1, O139, or non-O1/non-O139 strain and whether the bacterium detected is capable of producing toxins. We report that by alleviating primer dimer formation by use of a homotag-assisted nondimer system (HANDS) (5), we were able to retain the analytical sensitivity of uniplex PCR and successfully differentiated serogroups and toxigenic potentials from aquatic animal and environmental samples.     

Genetic Relatedness of Clinical and Environmental Vibrio cholerae Isolates Based on Triple Housekeeping Gene Analysis

Sequence analysis of dnaE, hlyA, and asd housekeeping genes were used to determine the genetic relatedness of our collection of Vibrio cholerae isolated from patients and surface waters over a 5-year period in Iran. The results showed 41, 17, and 9 variable sites throughout the sequenced fragments of dnaE (837 bp), hlyA (495 bp), and asd (295 bp), respectively. The results from sequence typing showed that all our clinical isolates were grouped in the same cluster. Eleven genotypes were identified among the environmental isolates. One environmental isolate was found to be in close genetic relatedness with our clinical isolates. One V. cholerae isolate showed a single-locus variant in the dnaE. For each of the studied genetic loci 10, 7, and 7 sequence types were observed for dnaE, hlyA, and asd, respectively. Only asd sequence analysis could make the distinction between the classical and El Tor isolates which emphasizes on selection of housekeeping locus with better discrimination power for analysis of different groups of isolates. Overall, the results indicated that surface waters in Tehran are a pool of non-toxigenic V. cholerae strains which are rarely related to clinical toxigenic isolates. In addition, our results verified that housekeeping gene sequence analysis could be a suitable approach for determination of the relatedness between clinical and environmental V. cholerae isolates.     

Molecular Characterization of Vibrio cholerae O1 Reveals Continuous Evolution of Its New Variants in India

Vibrio cholerae, the causing agent of cholera is still a major health challenge in most of the developing countries. In this study, V. cholerae strains collected from different cholera outbreaks in India over a period of past 7 years were found to have various toxigenic, pathogenic and regulatory genes viz. ctxAB, zot, tcp, hlyA, ace, ompU, ompW, rfbO1, toxT and toxR. The biotype specific genes rstR and rtxC revealed the El Tor biotype in majority of the isolates. However, variants among the isolates were found having genotype of both the biotypes. Sequencing of ctxB gene revealed the presence of altered ctxB of classical biotype with additional variations in isolates of 2007. Mismatch amplification mutation assay PCR also confirmed the isolates belonging to classical biotype. Antibiogram of the isolates revealed resistance for nalidixic acid, co-trimoxazole, streptomycin, and polymyxin B and susceptibility for tetracycline among most of the isolates from India. However, V. cholerae isolates from a recent outbreak in Eastern India were resistant to tetracycline. The study corroborated the continuous emergence and wide-spread of multidrug resistant El Tor variant strains in the Indian subcontinent.     

Non-O1/Non-O139 Vibrio cholerae Carrying Multiple Virulence Factors and V. cholerae O1 in the Chesapeake Bay,Maryland

Non-O1/non-O139 Vibrio cholerae inhabits estuarine and coastal waters globally, but its clinical significance has not been sufficiently investigated, despite the fact that it has been associated with septicemia and gastroenteritis. The emergence of virulent non-O1/non-O139 V. cholerae is consistent with the recognition of new pathogenic variants worldwide. Oyster, sediment, and water samples were collected during a vibrio surveillance program carried out from 2009 to 2012 in the Chesapeake Bay, Maryland. V. cholerae O1 was detected by a direct fluorescent-antibody (DFA) assay but was not successfully cultured, whereas 395 isolates of non-O1/non-O139 V. cholerae were confirmed by multiplex PCR and serology. Only a few of the non-O1/non-O139 V. cholerae isolates were resistant to ampicillin and/or penicillin. Most of the isolates were sensitive to all antibiotics tested, and 77 to 90% carried the El Tor variant hemolysin gene hlyA_ET, the actin cross-linking repeats in toxin gene rtxA, the hemagglutinin protease gene hap, and the type 6 secretion system. About 19 to 21% of the isolates carried the neuraminidase-encoding gene nanH and/or the heat-stable toxin (NAG-ST), and only 5% contained a type 3 secretion system. None of the non-O1/non-O139 V. cholerae isolates contained Vibrio pathogenicity island-associated genes. However, ctxA, ace, or zot was present in nine isolates. Fifty-five different genotypes showed up to 12 virulence factors, independent of the source of isolation, and represent the first report of both antibiotic susceptibility and virulence associated with non-O1/non-O139 V. cholerae from the Chesapeake Bay. Since these results confirm the presence of potentially pathogenic non-O1/non-O139 V. cholerae, monitoring for total V. cholerae, regardless of serotype, should be done within the context of public health.     

Genome structure and origin of nontoxigenic strains of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> of El Tor biovar with different epidemiological significance

Intraspecies genetic differentiation of nontoxigenic strains of Vibrio cholerae of El Tor biovar containing one of the key pathogenicity genes, tcpA, is studied along with the phylogenetic relationships between these strains and toxigenic isolates. Comparative analysis of the whole genome nucleotide sequences demonstrates for the first time that ctxA^–tcpA⁺ strains vary considerably and can be clustered into two separate groups, the CTX_φ–RS1_φ⁺VPI⁺VSP⁺/CTX_φ–RS1_φ–VPI⁺VSP⁺ isolates and the CTX_φ–RS1_φ–VPI⁺VSP^– isolates, differing in their epidemiological significance. In the course of model experiments, it is established that nontoxigenic potentially epidemic CTX_φ–RS1_φ⁺VPI⁺VSP⁺/CTX_φ–RS1_φ–VPI⁺VSP⁺ isolates are derivatives of toxigenic strains. The results of whole genome SNP analysis of 35 Vibrio cholerae strains confirm these data and indicate genetic remoteness of nontoxigenic CTX_φ–RS1_φ–VPI⁺VSP^– strains both from the potentially epidemic strains and from the toxigenic isolates. It is found that the genomes of the CTX_φ–RS1_φ–VPI⁺VSP^– strains contain unique SNPs which are characteristic of them alone. The new data on the structure of the genome of nontoxigenic strains with different epidemiological significance may be further used for their genetic differentiation.     

Antimicrobial susceptibility and molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from cholera outbreaks in Chennai

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.     

RNA Colony Blot Hybridization Method for Enumeration of Culturable Vibrio cholerae and Vibrio mimicus Bacteria

A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. cholerae in the environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.Vibrio cholerae is autochthonous to the aquatic environment, but some strains produce enterotoxins and are capable of causing epidemics of the human disease cholera. Strains of V. cholerae are classified by their O antigen, with over 210 serogroups recognized to date. Seven cholera pandemics have occurred since 1832: while microbiologic data on the earlier pandemics are not available, the last two are known to have been caused by strains within serogroup O1, with the major pathogenic factor being production of cholera toxin. The genes encoding cholera toxin and other pathogenic factors have been shown to reside in a mobile genetic element of phage origin, designated CTXΦ (20).Standard microbiologic methods for isolation of V. cholerae present in natural waters rely primarily on a method originally developed for clinical diagnosis, namely, enrichment in alkaline peptone water, followed by subculture on selective media and confirmation using selected biochemical and immunological tests (7). The alkaline nature of the enrichment broth allows differential multiplication of Vibrio species but renders this method inappropriate for enumeration. PCR methods and oligonucleotide hybridization have been used to detect and enumerate toxigenic V. cholerae bacteria (3, 11, 12, 14, 15, 21). These methods typically rely on amplification of or hybridization to pathogenic markers, such as O1/O139 wbe, tcpA, and ctxA DNA sequences.However, occasional localized outbreaks of cholera have been caused by non-O1, non-O139 V. cholerae, which may be toxigenic or nontoxigenic. Conversely, many environmental V. cholerae O1 strains isolated from areas of endemicity do not harbor ctx genes (9). It has also been shown that CTXΦ is capable of lysogenic conversion of strains that are CTXΦ negative (20). Additionally, the cholera toxin (CTX) prophage has also been detected in clinical strains of V. mimicus, and V. mimicus has been proposed as a natural reservoir for CTXΦ (2). Furthermore, ecological studies of V. cholerae are often hampered by the fact that toxigenic strains represent only a small percentage of the total V. cholerae population in the environment, especially in areas where cholera is not endemic. These facts underline the need for a method of detection of the total number of V. cholerae bacteria present in environmental samples.The many copies of 16S rRNA molecules in each V. cholerae cell offer appropriate targets for species-specific enumeration. In this study, the probe Vchomim1276, previously described by Heidelberg et al. (4-6), was employed in an RNA colony blot hybridization protocol. The specificity and sensitivity of the probe were tested using type strains and environmental and clinical isolates. The method was evaluated using laboratory microcosms to which cells of V. cholerae were added, and the protocol was used to enumerate V. cholerae bacteria in samples collected from ponds in a region of cholera endemicity in Bangladesh.     

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.     

Distribution of Virulence-Associated Genes and Genetic Relationships in Non-O1/O139 Vibrio cholerae Aquatic Isolates from China

Non-O1/O139 Vibrio cholerae is naturally present in aquatic ecosystems and has been linked with cholera-like diarrhea and local outbreaks. The distribution of virulence-associated genes and genetic relationships among aquatic isolates from China are largely unknown. In this study, 295 aquatic isolates of V. cholerae non-O1/O139 serogroups from different regions in China were investigated. Only one isolate was positive for ctxB and harbored a rare genotype; 10 (3.4%) isolates carried several types of rstR sequences, eight of which carried rare types of toxin-coregulated pili (tcpA). Furthermore, 16 (5.4%) isolates carried incomplete (with partial open reading frames [ORFs]) vibrio seventh pandemic island I (VSP-I) or VSP-II clusters, which were further classified as 11 novel types. PCR-based analyses revealed remarkable variations in the distribution of putative virulence genes, including mshA (95.6%), hlyA (95.3%), rtxC (89.8%), rtxA (82.7%), IS1004 (52.9%), chxA (30.2%), SXT (15.3%), type III secretion system (18.0%), and NAG-ST (3.7%) genes. There was no correlation between the prevalence of putative virulence genes and that of CTX prophage or TCP genes, whereas there were correlations among the putative virulence genes. Further multilocus sequence typing (MLST) placed selected isolates (n = 70) into 69 unique sequence types (STs), which were different from those of the toxigenic O1 and O139 counterparts, and each isolate occupied a different position in the MLST tree. The V. cholerae non-O1/O139 aquatic isolates predominant in China have high genotypic diversity; these strains constitute a reservoir of potential virulence genes, which may contribute to evolution of pathogenic isolates.     

Direct Detection of Vibrio cholerae and ctxA in Peruvian Coastal Water and Plankton by PCR

Seawater and plankton samples were collected over a period of 17 months from November 1998 to March 2000 along the coast of Peru. Total DNA was extracted from water and from plankton grouped by size into two fractions (64 μm to 202 μm and >202 μm). All samples were assayed for Vibrio cholerae, V. cholerae O1, V. cholerae O139, and ctxA by PCR. Of 50 samples collected and tested, 33 (66.0%) were positive for V. cholerae in at least one of the three fractions. Of these, 62.5% (n = 32) contained V. cholerae O1; ctxA was detected in 25% (n = 20) of the V. cholerae O1-positive samples. None were positive for V. cholerae O139. Thus, PCR was successfully employed in detecting toxigenic V. cholerae directly in seawater and plankton samples and provides evidence for an environmental reservoir for this pathogen in Peruvian coastal waters.     

Discrepancies in identification of Vibrio cholerae strains as members of Aeromonadaceae and Enterobacteriaceae by automated microbial identification system

Aims: Incidental observation of a discrepancy in identification of Vibrio cholerae prompted a study to understand the ability of an automated microbial identification system to identify this important pathogen. Methods and Results: Twenty clinical isolates of V. cholerae showing difference in genetic profiles by random amplified polymorphic DNA (RAPD) fingerprinting, serologically confirmed as O1, and showing presence of ctxA and tcpA genes in PCR were subjected to analysis by Vitek 2 Compact automated identification system for identification. Vitek 2 Compact detected 10 of 20 isolates correctly, whereas the remaining 10 were identified as various members of Aeromonadaceae and Enterobacteriaceae. Conclusions: Our results indicate that Vitek 2 Compact automated microbial system does not always identify V. cholerae strains correctly. Significance and Impact of Study: These observations should create awareness among end users about possible misidentifications by automated systems and encourage simultaneous use of serology and/or PCR for correct identification at least for V. cholerae, which is one of the most important enteric pathogens.