Phenotypic and Genotypic Characterization Vibrio cholerae O139 of Clinical and Aquatic Isolates in China

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.     

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.     

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.     

非O1群、非O139群霍乱弧菌致败血症1例报道

对襄阳市中心医院分离的2株疑似霍乱弧菌进行鉴定及药物敏感性试验。利用MicroScan WalkAway 40鉴定仪进行生化鉴定及药物敏感性试验,玻片凝集法确定血清型别,PCR扩增16SrRNA保守区基因并将产物进行测序分析。此2株疑似霍乱菌株O1群及O139群霍乱弧菌诊断血清均不凝集,16SrRNA扩增产物测序Blast比对分析与数据库中霍乱弧菌相似性达100％,药敏结果显示对氨苄西林、庆大霉素、环丙沙星、阿米卡星、氯霉素、复方新诺明（SXT）、四环素均敏感。该病例为非O1、非O139群霍乱弧菌导致的败血症,可能经胃肠道途径传播。     

Population Structure and Evolution of Non-O1/Non-O139 Vibrio cholerae by Multilocus Sequence Typing

Pathogenic non-O1/non-O139 Vibrio cholerae strains can cause sporadic outbreaks of cholera worldwide. In this study, multilocus sequence typing (MLST) of seven housekeeping genes was applied to 55 non-O1/non-O139 isolates from clinical and environmental sources. Data from five published O1 isolates and 17 genomes were also included, giving a total of 77 isolates available for analysis. There were 66 sequence types (STs), with the majority being unique, and only three clonal complexes. The V. cholerae strains can be divided into four subpopulations with evidence of recombination among the subpopulations. Subpopulations I and III contained predominantly clinical strains. PCR screening for virulence factors including Vibrio pathogenicity island (VPI), cholera toxin prophage (CTXΦ), type III secretion system (T3SS), and enterotoxin genes (rtxA and sto/stn) showed that combinations of these factors were present in the clinical isolates with 85.7% having rtxA, 51.4% T3SS, 31.4% VPI, 31.4% sto/stn (NAG-ST) and 11.4% CTXΦ. These factors were also present in environmental isolates but at a lower frequency. Five strains previously mis-identified as V. cholerae serogroups O114 to O117 were also analysed and formed a separate population with V. mimicus. The MLST scheme developed in this study provides a framework to identify sporadic cholera isolates by genetic identity.     

Prevalence of Cholera Toxin Genes (ctxA and zot) among Non-O1/O139 Vibrio cholerae Strains from Newport Bay, California

The examination of 137 non-O1/O139 Vibrio cholerae isolates from Newport Bay, California, indicated the presence of diverse genotypes and a temporal succession. Unexpectedly, the cholera toxin gene (ctxA) was found in 17% of the strains, of which one-third were also positive for the zot gene. This suggests that ctxA is prevalent in the region of nonepidemicity and is likely to have an environmental origin.     

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.     

Distribution of Vibrio cholerae O1 antigen biosynthesis genes among O139 and other non-O1 serogroups of Vibrio cholerae

The organization and distribution of the genes responsible for O antigen biosynthesis in various serogroups of Vibrio cholerae were investigated using several DNA probes derived from various regions of the genes responsible for O1 antigen biosynthesis. Based on the reactivity pattern of the probes against the various serogroups, the cluster of genes responsible for the O1 antigen biosynthesis could be broadly divided into six groups, designated as class 1-6. The class 3 cluster of genes corresponding to gmd to wbeO, wbeT and a part of wbeU was specific for only the O1 serogroup. The other cluster of genes (class 1, 2, 4-6) reacted with other serogroups of V. cholerae. These data indicate that serotype conversion in V. cholerae does not depend on a simple mutational event but may involve horizontal gene transfer not only between V. cholerae strains but also between V. cholerae and species other than V. cholerae.     

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.     

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.     

Scanning the Landscape of Genome Architecture of Non-O1 and Non-O139 Vibrio cholerae by Whole Genome Mapping Reveals Extensive Population Genetic Diversity

Historically, cholera outbreaks have been linked to V. cholerae O1 serogroup strains or its derivatives of the O37 and O139 serogroups. A genomic study on the 2010 Haiti cholera outbreak strains highlighted the putative role of non O1/non-O139 V. cholerae in causing cholera and the lack of genomic sequences of such strains from around the world. Here we address these gaps by scanning a global collection of V. cholerae strains as a first step towards understanding the population genetic diversity and epidemic potential of non O1/non-O139 strains. Whole Genome Mapping (Optical Mapping) based bar coding produces a high resolution, ordered restriction map, depicting a complete view of the unique chromosomal architecture of an organism. To assess the genomic diversity of non-O1/non-O139 V. cholerae, we applied a Whole Genome Mapping strategy on a well-defined and geographically and temporally diverse strain collection, the Sakazaki serogroup type strains. Whole Genome Map data on 91 of the 206 serogroup type strains support the hypothesis that V. cholerae has an unprecedented genetic and genomic structural diversity. Interestingly, we discovered chromosomal fusions in two unusual strains that possess a single chromosome instead of the two chromosomes usually found in V. cholerae. We also found pervasive chromosomal rearrangements such as duplications and indels in many strains. The majority of Vibrio genome sequences currently in public databases are unfinished draft sequences. The Whole Genome Mapping approach presented here enables rapid screening of large strain collections to capture genomic complexities that would not have been otherwise revealed by unfinished draft genome sequencing and thus aids in assembling and finishing draft sequences of complex genomes. Furthermore, Whole Genome Mapping allows for prediction of novel V. cholerae non-O1/non-O139 strains that may have the potential to cause future cholera outbreaks.     

Viability of the nonculturable Vibrio cholerae O1 and O139

Vibrio cholerae is capable of transforming into a viable but nonculturable (VBNC) state, and, in doing so, undergoes alteration in cell morphology. In the study reported here, Vibrio cholerae O1 and O139 cells were maintained in laboratory microcosms prepared with 1% Instant Ocean and incubated at 4 degrees C, i.e., conditions which induce the VBNC state. Cells were fixed at different stages during entry into the VBNC state and, when no growth was detectable on solid or in liquid media, the ultrastructure of these cells was examined, using both transmission and scanning electron microscopy. As shown in earlier studies, the cells became smaller in size and changed from rod to ovoid or coccoid morphology, with the central region of the cells becoming compressed and surrounded by denser cytoplasm. Because the coccoid morphology, indicative of the VBNC state is common for Vibrio cholerae in the natural environment, as well as in starved cells (Baker et al., 1983; Hood et al., 1986) viability of the coccoid, viable but nonculturable cell was investigated. The percentage of coccoid (VBNC) cells showing metabolic activity and retention of membrane integrity was monitored using direct fluorescence staining (LIVE/DEAD BacLight Bacterial Viability kit), with 75 to 90% of the viable but nonculturable coccoid cells found to be metabolically active by this test. Furthermore, the proportion of actively respiring cells, using the redox dye, 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC), relative to total cells, the latter determined by DAPI staining, ranged from 10 to 50%. VBNC coccoid cells retained the antigenic determinants of Vibrio cholerae O1 and O139, respectively, evidenced by positive reaction with monoclonal fluorescent antibody. Viability was further established by susceptibility of the VBNC cells to chlorine, copper sulfate, zinc sulfate, and formaldehyde. Since retention of cell membrane integrity is a determining characteristic of viable cells, DNA was extracted from VBNC cells in microcosms maintained for two months and for one year. Conservation of cholera toxin and toxin-associated genes, ctxA, toxR, tcpA, and zot in chromosomal DNA of VBNC cells was demonstrated using PCR and employing specific primers. It is concluded that not only do VBNC V cholerae O1 and O139 retain viability up to one year, but genes associated with pathogenicity are retained, along with chromosomal integrity.     

Filamentous Phage fs1 of Vibrio cholerae O139

Filamentous phage, fs1, was obtained from Vibrio cholerae O139. The lysogenized strains produced a large amount of fs1 phage in the culture supernatant. This phage was previously reported as novel fimbriae of that organism. The genome of the phage was a 6.5 kb single-stranded DNA. The capsid of fs1 consists of a small molecule peptide (about 2.5 kDa).     

Relatedness of Vibrio cholerae O1/O139 Isolates from Patients and Their Household Contacts,Determined by Multilocus Variable-Number Tandem-Repeat Analysis

The genetic relatedness of Vibrio cholerae O1/O139 isolates obtained from 100 patients and 146 of their household contacts in Dhaka, Bangladesh, between 2002 and 2005 was assessed by multilocus variable-number tandem-repeat analysis. Isolate genotypes were analyzed at five loci containing tandem repeats. Across the population, as well as within households, isolates with identical genotypes were clustered in time. Isolates from individuals within the same household were more likely to have similar or identical genotypes than were isolates from different households, but even within a household, isolates from different individuals often had different genotypes. When household contacts were sampled regularly for 3 weeks after the illness of the household index patient, isolates with genotypes related to the index patient appeared in contacts, on average, ∼3 days after the index patient, while isolates with unrelated genotypes appeared in contacts ∼6 days after. Limited data revealed that multiple isolates from the same individual collected within days of each other or even from a single stool sample may have identical, similar, or unrelated genotypes as well. Our results demonstrate that genetically related V. cholerae strains cluster in local outbreaks but also suggest that multiple distinct strains of V. cholerae O1 may circulate simultaneously within a household.Vibrio cholerae is the etiologic agent of cholera, a secretory diarrheal disease with a high mortality rate in humans if untreated (25). Serogroups of V. cholerae, a motile, Gram-negative, curved rod, can be defined serologically by the O side chain of the lipopolysaccharide (LPS) component of the outer membrane (9). V. cholerae is found in a variety of forms in aquatic ecosystems (41, 42), and more than 200 different serogroups have been isolated, mostly from environmental sources (45). However, the vast majority of V. cholerae strains that cause the clinical disease cholera belong to serogroup O1 or O139 (37, 42). V. cholerae O1, the historical agent of epidemic and pandemic cholera and the current leading cause of cholera both globally and in Bangladesh (42), is classified into two major biotypes, classical and El Tor (44), and two major serotypes, Ogawa and Inaba (48). The current global pandemic is caused by V. cholerae O1 El Tor. A second pathogenic serogroup, O139, emerged in the Bengal region in 1992 by horizontal transfer of new LPS biosynthesis-encoding genes into the El Tor biotype (1, 4). This new serogroup continues to cocirculate with El Tor V. cholerae O1 serotypes Ogawa and Inaba as a cause of disease in humans, although it accounts for a smaller proportion of all cholera now than in its first years of circulation (16, 20). Recently, comparative genomics has revealed an extensive amount of lateral gene transfer between strains, suggesting that genomic classification may be an alternative to serogrouping for classifying pathogenic V. cholerae strains (11).Toxigenic V. cholerae may be present in environmental sources in regions of endemicity and emerge, often seasonally, to cause cholera in humans (12, 18). Once an outbreak has begun, organisms from one infected individual are more infectious for the next individual, a property termed hyperinfectivity, and these forms may be able to pass directly from human to human through fecal-oral contamination (35). However, because vibrio organisms are difficult to isolate from implicated environmental or domestic water sources (28, 29), little is known about the diversity of V. cholerae in inocula that cause human infection.Established laboratory methods for differentiating V. cholerae strains, apart from serogrouping and serotyping, include rRNA restriction fragment length polymorphism (ribotyping), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). These methods, however, have a limited capacity to differentiate between pathogenic V. cholerae strains, as clinical isolates are relatively genetically monomorphic. For instance, V. cholerae O1 comprises approximately 30 ribotypes (39); however, only a few ribotypes are common in clinical isolates, ribotypes evolve slowly, and all isolates of a given pathogenic V. cholerae serotype in a local area over a period of multiple years often belong to a single ribotype (8, 14, 17). In a broad sampling of 154 V. cholerae isolates from Bangladesh and worldwide over several decades, only 15 ribotypes were identified, and of these, many were found in nonpathogenic environmental isolates only; only five ribotypes were associated with the V. cholerae O1 El Tor biotype that currently predominates as the cause of clinical disease, while pathogenic isolates of serogroup O139 were indistinguishable from each other by ribotype (19).PFGE, in which restriction endonuclease digestion of genomic DNA generates mutation-sensitive banding patterns, is often more sensitive than ribotyping in detecting strain variation (7, 34, 51) and detects extensive genetic variation within nonpathogenic V. cholerae serogroups (3, 46). However, PFGE types change slowly and are useful primarily for distinguishing between strains in different pandemics or between different continental branches of those pandemics. In an analysis of 180 mostly western-hemisphere isolates (7), PFGE differences had developed from a prior pandemic strain over the 30 years since its arrival in Latin America, but a new strain that had been causing disease for 2 years still had only a single PFGE type across the 64 isolates analyzed. Similarly, in a Japanese study (2), although 19 PFGE types were identified among O1 isolates, the majority of the domestic isolates, along with several imported isolates, belonged to a single PFGE type.Further differentiation between V. cholerae isolates is achievable by MLST, which characterizes isolates by internal DNA sequences in selected housekeeping genes (32). Nevertheless, epidemic strains also cluster tightly in this typing scheme (5, 32) and the method has been useful primarily for determining relationships between nontoxigenic strains (36) or for linking regional outbreaks (which typically appear monoclonal by these methods) with the pandemic strain responsible (5, 33).Although these methods have distinguished major pandemic clones from other nonpathogenic human and environmental isolates of V. cholerae, the near clonality of pathogenic O1 and O139 strains means that established methods may not provide sufficiently robust differentiation of these genetically similar pathogenic strains to answer important epidemiological questions. Therefore, there is a need for other methods that can distinguish among clinical O1 and O139 isolates and track the epidemiology of outbreaks in a restricted geographic area on a shorter time scale.Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is one method that may be useful for differentiating between pathogenic V. cholerae O1 and O139 strains that would be indistinguishable by other techniques (15). This method examines short repeating DNA segments at various locations in the genome that can vary in number at each location and uses the number of repeats at each varying locus as a fingerprint to distinguish between isolates.Escherichia coli is the paradigm organism for demonstrating the value of the MLVA method. Noller et al. (38) showed that E. coli O157 isolates that were indistinguishable by MLST could be distinguished to some extent by PFGE but that MLVA distinguished between isolates that had the same PFGE type and did so in a manner consistent with the known epidemiology of the isolates (38a). In addition, machine-scored VNTR assays have been demonstrated to be robust and portable and to discriminate clearly between isolates by using relatively few loci, therefore limiting the effect of compounding genotyping errors (6).For V. cholerae, five VNTR loci have been identified (15), and the initial application of MLVA at those loci has demonstrated distinct populations of clinical isolates of V. cholerae in different geographic regions within Bangladesh and India (23, 47). Predominant isolates in each of two rural Bangladeshi regions varied gradually over a time scale of months to years (47), and isolates collected from India over a 15-year period varied widely, with individual MLVA types clustering in time and place—some with widespread dissemination and others with limited local occurrence only (23). MLVA has also been used to classify hybrid and altered V. cholerae variants and to demonstrate their genetic distance from the pandemic El Tor strain (10). Use of the MLVA method for epidemiologic study of cholera requires that V. cholerae VNTR alleles remain reasonably stable during bacterial replication in patients or in laboratory culture after isolation. Some degree of stability of two of the five loci used in V. cholerae MLVA has been demonstrated previously by serial passage in vitro through four overnight cultures (15). In this study, we used MLVA to examine V. cholerae O1 and O139 isolates obtained from infected patients and their household contacts—including multiple isolates from the same individual and isolates from multiple individuals within the same household—in a large city where cholera is endemic.     

Characterization of enteropathogenic Vibrio cholerae non O1/O139 isolated in Uzbekistan

The results of the serotyping of 244 V. cholerae non O1/O139 cultures isolated from patients in Uzbekistan in 2000 and 2001 are presented. All isolates were studied by the method of molecular probing and in the polymerase chain reaction for the presence of virulence genes and for sensitivity to phages ctx+, ctx- and hemolytic activity. The use of monoreceptor O-sera O2-O83 made it possible to determine vibrios of 32 serogroups with the dominating role in the etiology of acute enteric diseases belonging to serogroups O18, O62, O82, O37. Genes ctx AB were detected in none of the isolates, 5 of them contained gene tcp A. A group of cultures, sensitive to phage ctx+ and belonging mainly to enteropathogenic serogroups, was detected.     

Genomic and Phenotypic Characterization of Vibrio cholerae Non-O1 Isolates from a US Gulf Coast Cholera Outbreak

Between November 2010, and May 2011, eleven cases of cholera, unrelated to a concurrent outbreak on the island of Hispaniola, were recorded, and the causative agent, Vibrio cholerae serogroup O75, was traced to oysters harvested from Apalachicola Bay, Florida. From the 11 diagnosed cases, eight isolates of V. cholerae were isolated and their genomes were sequenced. Genomic analysis demonstrated the presence of a suite of mobile elements previously shown to be involved in the disease process of cholera (ctxAB, VPI-1 and -2, and a VSP-II like variant) and a phylogenomic analysis showed the isolates to be sister taxa to toxigenic V. cholerae V51 serogroup O141, a clinical strain isolated 23 years earlier. Toxigenic V. cholerae O75 has been repeatedly isolated from clinical cases in the southeastern United States and toxigenic V. cholerae O141 isolates have been isolated globally from clinical cases over several decades. Comparative genomics, phenotypic analyses, and a Caenorhabditis elegans model of infection for the isolates were conducted. This analysis coupled with isolation data of V. cholerae O75 and O141 suggests these strains may represent an underappreciated clade of cholera-causing strains responsible for significant disease burden globally.     

Seasonal Cholera Caused by Vibrio cholerae Serogroups O1 and O139 in the Coastal Aquatic Environment of Bangladesh

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.     

Pili of a Vibrio cholerae O139

The pili of a strain of Vibrio cholerae O139 were purified and characterized. They were morphologically, electrophoretically and immunologically indistinguishable from the pili with 16 kDa subunit protein of V. cholerae O1. All 22 strains of V. cholerae O139 examined possessed the pili. The pili were different in hemagglutination inhibition pattern from V. cholerae O1 16K pili.     

gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental,Stool, and Historical Continuous Plankton Recorder Samples

The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 10² genomic units/l for drinking and seawater samples, 10¹ genomic units/g for sediment and 10² genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.