Vibrio cholerae O139 persists in Dhaka, Bangladesh since 1993.

BACKGROUND: After a multi-country Asian outbreak of cholera due to Vibrio cholerae serogroup O139 which started in 1992, it is rarely detected from any country in Asia and has not been detected from patients in Africa. METHODOLOGY/PRINCIPAL FINDINGS: We extracted surveillance data from the Dhaka and Matlab Hospitals of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) to review trends in isolation of Vibrio cholerae O139 in Bangladesh. Data from the Dhaka Hospital is a 2% sample of > 100,000 diarrhoeal patients treated annually. Data from the Matlab Hospital includes all diarrhoeal patients who hail from the villages included in the Matlab Health and Demographic Surveillance System. Vibrio cholerae O139 was first isolated in Dhaka in 1993 and had been isolated every year since then except for a gap between 2005 and 2008. An average of thirteen isolates was detected annually from the Dhaka Hospital during the last ten years, yielding an estimated 650 cases annually at this hospital. During the last ten years, cases due to serogroup O139 represented 0.47% of all cholera cases; the others being due to serogroup O1. No cases with serogroup O139 were identified at Matlab since 2006. Clinical signs and symptoms of cholera due to serogroup O139 were similar to cases due to serogroup O1 though more of the O139 cases were not dehydrated. Most isolates of O139 remained sensitive to tetracycline, ciprofloxacin, and azithromycin, but they became resistant to erythromycin starting in 2009. CONCLUSIONS/SIGNIFICANCE: Cholera due to Vibrio cholerae serogroup O139 continues to cause typical cholera in Dhaka, Bangladesh.

Diagnostic techniques for rapid detection of Vibrio cholerae O1/O139.

Cholera caused by the toxigenic Vibrio cholerae is still a major public health problem in many countries. This disease is mainly due to poor sanitation, hygiene and consumption of unsafe water. Several recent epidemics of cholera showed its increasing intensity, duration and severity of the illness. This indicates an urgent need for effective management and preventive measures in controlling the outbreaks and epidemics. In preventing and spread of epidemic cholera, rapid diagnostic tests (RDTs) are useful in screening suspected stool specimens, water/food samples. Several RDTs developed recently are considered as investigative tools in confirming cholera cases, as the culture techniques are difficult to establish and/or maintain. The usefulness of RDTs will be more at the point-of-care facilities as it helps to make appropriate decisions in the management of outbreaks or epidemiological surveillance by the public health authorities. Apart from RDTs, several other tests are available for the direct detection of either V. cholerae or its cholera toxin. Viable but non-culturable (VBNC) state of V. cholerae poses a great challenge in developing RDTs. The aim of this article is to provide an overview of current knowledge about RDT and other techniques with reference to their status and future potentials in detecting cholera/V. cholerae.

Development of a loop-mediated isothermal amplification assay for Vibrio cholerae O1 and O139.

A loop-mediated isothermal amplification assay was developed. It was designed for recognizing Vibrio cholerae O1/O139, where atpA, rfbN, and wfbR genes were adopted. The assay specifically detected the target with sensitivities of 5-67 copies per reaction in 1 h. The assay will aid rapid detection of the cholera bacterium.

[Vibrio cholerae NO-O1/NO-O139 bacteremia in a cirrhotic patient. First case report in Peru and literatura review]. / Bacteremia por Vibrio cholerae NO-O1/NO-O139 en un paciente cirrótico. Primer reporte de caso en el Perú y revisión de la literatura.

Non-O1, non-O139 Vibrio cholerae (NOVC) strains are an uncommon cause of gastroenteritis. However, they have been recently associated with severe extraintestinal infections in immunocompromised hosts. Among them, bacteremia in cirrhotic patients is noteworthy. We present the case of a 58-year-old woman with cirrhosis that developed septic shock, multiple organ failure and died four days after admission. Blood cultures yielded Gram-negative rods identified as Vibrio cholerae. Further serogrouping by slide agglutination and a negative PCR for ctxA gen confirmed the strain to be NOVC. Antimicrobial susceptibility testing showed sensitivity to ampicillin, chloramphenicol, tetracycline and ciprofloxacin; and resistance to trimethoprim-sulfamethoxazole. To the best of our knowledge, this is first report in Peru, described in the Hospital Nacional Dos de Mayo, of NOVC bacteremia.

Potential application of SMART II for Vibrio cholerae O1 and O139 detection in ship's ballast water.

Ballast water is used to safely stabilize and operate shipping vessels worldwide, in a multitude of aquatic settings, including inland, coastal and open oceans. However, ballast water may pose ecological, public health, and/or economic problems as it may serve as vehicles of transmission of microorganisms. Current ballast water regulations include limits of Escherichia coli, Enterococcus spp. and toxigenic Vibrio cholerae. Several United States Environmental Protection Agency approved standard operating protocols (SOPs) exist for detection of E. coli and Enterococci, yet none exists for V. cholerae. Current V. cholerae detection methods include colony blot hybridization, direct fluorescent antibody test (DFA), and/or polymerase chain reaction (PCR), which can be time consuming and difficult to perform. This study utilizes Cholera SMART II to determine its potential use in detection of V. cholerae. Validation of this method would help provide quick and accurate analysis for V. cholerae in ballast discharge waters in the field.

Bacteremia por Vibrio cholerae NO-O1/NO-O139 en un paciente cirrótico: Primer reporte de caso en el Perú y revisión de la literatura / Vibrio cholerae NO-O1/NO-O139 bacteremia in a cirrhotic patient: First case report in Peru and literatura review

Vibrio cholerae serogrupo NO-O1/NO-O139 (VCNO) es causa infrecuente de gastroenteritis. Sin embargo, se le asocia a infección extra-intestinal severa en huéspedes inmunocomprometidos, y entre ellas, la bacteremia en pacientes con cirrosis hepática es digna de mención. A continuación, presentamos el caso de una mujer de 58 años, con el diagnóstico de cirrosis hepática de fondo, que desarrolló progresivamente choque séptico, disfunción orgánica múltiple y desenlace fatal al cuarto día de su admisión. Los resultados obtenidos post mortem, de los hemocultivos previamente tomados, aislaron bacilos gram negativos compatibles con Vibrio cholerae. Posteriormente, se identificó el serogrupo NO-O1/NO-O139, a través de aglutinación en placa y PCR negativo para el gen ctxA. El antibiograma mostró susceptibilidad conservada a ampicilina, cloranfenicol, tetraciclina y ciprofloxacino, con resistencia al trimetoprim-sulfametoxazol. El presente caso, descrito en el Hospital Nacional Dos de Mayo, es hasta la fecha, el primer reporte de bacteremia VCNO en el Perú.

Non-O1, non-O139 Vibrio cholerae (NOVC) strains are an uncommon cause of gastroenteritis. However, they have been recently associated with severe extraintestinal infections in immunocompromised hosts. Among them, bacteremia in cirrhotic patients is noteworthy. We present the case of a 58-year-old woman with cirrhosis that developed septic shock, multiple organ failure and died four days after admission. Blood cultures yielded Gram-negative rods identified as Vibrio cholerae. Further serogrouping by slide agglutination and a negative PCR for ctxA gen confirmed the strain to be NOVC. Antimicrobial susceptibility testing showed sensitivity to ampicillin, chloramphenicol, tetracycline and ciprofloxacin; and resistance to trimethoprim-sulfamethoxazole. To the best of our knowledge, this is first report in Peru, described in the Hospital Nacional Dos de Mayo, of NOVC bacteremia.

Development and evaluation of an up-converting phosphor technology-based lateral flow assay for the rapid, simultaneous detection of Vibrio cholerae serogroups O1 and O139.

Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL-1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL-1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera.

Plasma and Mucosal Immunoglobulin M, Immunoglobulin A, and Immunoglobulin G Responses to the Vibrio cholerae O1 Protein Immunome in Adults With Cholera in Bangladesh.

Background: Cholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera. Methods: We probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay. Results: Overall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O-specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate-2-oxoglutarate aminotransferase. Conclusions: This study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine.

Characterization and Genetic Variation of Vibrio cholerae Isolated from Clinical and Environmental Sources in Thailand.

Cholera is still an important public health problem in several countries, including Thailand. In this study, a collection of clinical and environmental V. cholerae serogroup O1, O139, and non-O1/non-O139 strains originating from Thailand (1983 to 2013) was characterized to determine phenotypic and genotypic traits and to investigate the genetic relatedness. Using a combination of conventional methods and whole genome sequencing (WGS), 78 V. cholerae strains were identified. WGS was used to determine the serogroup, biotype, virulence, mobile genetic elements, and antimicrobial resistance genes using online bioinformatics tools. In addition, phenotypic antimicrobial resistance was determined by the minimal inhibitory concentration (MIC) test. The 78 V. cholerae strains belonged to the following serogroups O1: (n = 44), O139 (n = 16) and non-O1/non-O139 (n = 18). Interestingly, we found that the typical El Tor O1 strains were the major cause of clinical cholera during 1983-2000 with two Classical O1 strains detected in 2000. In 2004-2010, the El Tor variant strains revealed genotypes of the Classical biotype possessing either only ctxB or both ctxB and rstR while they harbored tcpA of the El Tor biotype. Thirty O1 and eleven O139 clinical strains carried CTXϕ (Cholera toxin) and tcpA as well four different pathogenic islands (PAIs). Beside non-O1/non-O139, the O1 environmental strains also presented chxA and Type Three Secretion System (TTSS). The in silico MultiLocus Sequence Typing (MLST) discriminated the O1 and O139 clinical strains from other serogroups and environmental strains. ST69 was dominant in the clinical strains belonging to the 7th pandemic clone. Non-O1/non-O139 and environmental strains showed various novel STs indicating genetic variation. Multidrug-resistant (MDR) strains were observed and conferred resistance to ampicillin, azithromycin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim and harboured variants of the SXT elements. For the first time since 1986, the presence of V. cholerae O1 Classical was reported causing cholera outbreaks in Thailand. In addition, we found that V. cholerae O1 El Tor variant and O139 were pre-dominating the pathogenic strains in Thailand. Using WGS and bioinformatic tools to analyze both historical and contemporary V. cholerae circulating in Thailand provided a more detailed understanding of the V. cholerae epidemiology, which ultimately could be applied for control measures and management of cholera in Thailand.

[TYPING OF VIBRIO CHOLERAE NON O1/NON O139 STRAINS, ISOLATED IN ROSTOV REGION IN 2014].

AIM: Comparative study of antibiotics resistance and VNTR-typing of Vibrio cholerae non O1/ non O139 strains, isolated on the territory of Rostov region in 2014. MATERIALS AND METHODS: Antibioticogramms of strains were determined by serial dilution method in dense nutrient medium according to MG 4.2.2495-09 (2009). Pheno-, sero- and VNTR-typing was carried out by conventional-methods. RESULTS: The studied strains belonged to V. cholerae species, did not agglutinate with O1 and O139 sera, were atoxigenic hemolysis-positive, did not contain genes of cholera toxin and toxin-coregulating pili of adhesion, contained genes of hemagglutinin/protease, protease PrtV, collagenase, cytotonic factor Cef, outer membrane protein-OmpW, tol- and -vps-clusters, regulatory genes toxR and hapR. Antibioticogramms of the strains have shown the presence of cultures, resistant to ampicillin, ceftazidime-furazolidone, trimethoprim/sulfamethoxazole with intermediate resistance to streptomycin, kanamycin, gentamycin, amikacin, netilmicin, Approximately 20% of isolates had multiple drug resistance. Data of VNTR- and genotyping confirmed a possibility of water transmission route of the infection. CONCLUSION: Execution of monitoring of cultures from environmental samples is necessary for timely detection of genetic characteristics, antibiotics resistance.

Notes from the Field: Ongoing Cholera Outbreak - Kenya, 2014-2016.

On January 6, 2015, a man aged 40 years was admitted to Kenyatta National Hospital in Nairobi, Kenya, with acute watery diarrhea. The patient was found to be infected with toxigenic Vibrio cholerae serogroup O1, serotype Inaba. A subsequent review of surveillance reports identified four patients in Nairobi County during the preceding month who met either of the Kenya Ministry of Health suspected cholera case definitions: 1) severe dehydration or death from acute watery diarrhea (more than four episodes in 12 hours) in a patient aged ≥5 years, or 2) acute watery diarrhea in a patient aged ≥2 years in an area where there was an outbreak of cholera. An outbreak investigation was immediately initiated. A confirmed cholera case was defined as isolation of V. cholerae O1 or O139 from the stool of a patient with suspected cholera or a suspected cholera case that was epidemiologically linked to a confirmed case. By January 15, 2016, a total of 11,033 suspected or confirmed cases had been reported from 22 of Kenya's 47 counties (Table). The outbreak is ongoing.

Characterization of environmental Vibrio cholerae serogroups O1 and O139 in the Pearl River Estuary, China.

Toxigenic isolates of Vibrio cholerae serogroups O1 and O139 from aquatic reservoirs are a key source for recurrent epidemics of cholera in human populations. However, we do not have an optimal understanding of the microbiology of the strains within these reservoirs, particularly outside of the time periods when there are active cholera cases in the surrounding community. The main objective of the present study was to identify and characterize V. cholerae O1 and O139 in the Pearl River Estuary at a time when active disease was not being identified, despite prior occurrence of epidemic cholera in the region. Water samples were collected at 24 sites in the research area at monthly intervals between 2007 and 2010, and screened for the presence of V. cholerae O1 and O139. All isolates were screened for the presence of ctxAB, ompW, toxR, and tcpA genes. Multilocus variable number tandem repeat analysis (MLVA) was used to assess possible relationships among strains. The results show that Vibrio cholerae O1 or O139 was isolated, on average, from 6.7% of the sites screened at each time point. All V. cholerae O1 and O139 isolates were ctxAB negative, and 37% were positive for tcpA. Isolation was most common in the oldest, most urbanized district compared with other districts, and was associated with lower pH. Despite year-to-year variability in isolation rates, there was no evidence of seasonality. MLVA of 27 selected isolates showed evidence of high genetic diversity, with no evidence of clustering by year or geographic location. In this region where cholera has been epidemic in the past, there is evidence of environmental persistence of V. cholerae O1 and O139 strains. However, environmental strains were consistently nontoxigenic, with a high level of genetic diversity; their role as current or future agents of human disease remains uncertain.

Vibrio cholerae Serogroup O139: Isolation from Cholera Patients and Asymptomatic Household Family Members in Bangladesh between 2013 and 2014.

BACKGROUND: Cholera is endemic in Bangladesh, with outbreaks reported annually. Currently, the majority of epidemic cholera reported globally is El Tor biotype Vibrio cholerae isolates of the serogroup O1. However, in Bangladesh, outbreaks attributed to V. cholerae serogroup O139 isolates, which fall within the same phylogenetic lineage as the O1 serogroup isolates, were seen between 1992 and 1993 and in 2002 to 2005. Since then, V. cholerae serogroup O139 has only been sporadically isolated in Bangladesh and is now rarely isolated elsewhere. METHODS: Here, we present case histories of four cholera patients infected with V. cholerae serogroup O139 in 2013 and 2014 in Bangladesh. We comprehensively typed these isolates using conventional approaches, as well as by whole genome sequencing. Phenotypic typing and PCR confirmed all four isolates belonging to the O139 serogroup. FINDINGS: Whole genome sequencing revealed that three of the isolates were phylogenetically closely related to previously sequenced El Tor biotype, pandemic 7, toxigenic V. cholerae O139 isolates originating from Bangladesh and elsewhere. The fourth isolate was a non-toxigenic V. cholerae that, by conventional approaches, typed as O139 serogroup but was genetically divergent from previously sequenced pandemic 7 V. cholerae lineages belonging to the O139 or O1 serogroups. CONCLUSION: These results suggest that previously observed lineages of V. cholerae O139 persist in Bangladesh and can cause clinical disease and that a novel disease-causing non-toxigenic O139 isolate also occurs.

Toxigenic Vibrio cholerae identified in estuaries of Tanzania using PCR techniques.

The current study assessed the occurrence of the Vibrio cholerae serogroups O1 and O139 in environmental samples along salinity gradients in three selected estuaries of Tanzania both through culture independent methods and by cultured bacteria. Occurrence of V. cholerae was determined by PCR targeting the V. cholerae outer membrane protein gene ompW. Furthermore, the presence of toxigenic strains and serogroups O1 and O139 was determined using multiplex PCR with specific primers targeting the cholera toxin gene subunit A, ctxA, and serotype specific primers, O1-rfb and O139-rfb, respectively. Results showed that V. cholerae occurred in approximately 10% (n = 185) of both the environmental samples and isolated bacteria. Eight of the bacteria isolates (n = 43) were confirmed as serogroup O1 while one belonged to serogroup O139, the first reported identification of this epidemic strain in East African coastal waters. All samples identified as serogroup O1 or O139 and a number of non-O1/O139 strains were ctxA positive. This study provides in situ evidence of the presence of pathogenic V. cholerae O1 and O139 and a number of V. cholerae non-O1/O139 that carry the cholera toxin gene in estuaries along the coast of Tanzania.

IncA/C plasmids harboured in serious multidrug-resistant Vibrio cholerae serogroup O139 strains in China.

Vibrio cholerae serogroup O139 emerged in 1992 and is one of two major serogroups to have caused cholera epidemics. After 1998, serious multidrug-resistant (MDR) O139 strains quickly became common in China, showing a multidrug resistance profile to eight antibiotics. It is a great threat to public health, and elucidation of its mechanisms of resistance will provide a helpful guide for the clinical treatment and prevention of cholera. In this study, mega-plasmids from MDR V. cholerae O139 strains were identified by pulsed-field gel electrophoresis (PFGE) without enzyme digestion. One plasmid was isolated and sequenced, belonging to the IncA/C family. Ten antibiotic resistance genes were found in the MDR regions, including a blaTEM-20 gene, and these genes endowed the host with resistance to seven antibiotics. This kind of plasmid was positive in 71.2% (198/278) of toxigenic O139 strains, and the rate of plasmid positivity was consistent with the yearly change in MDR rates of these strains. This study reveals an important role of the IncA/C family plasmid in the spread of multiple antibiotic resistance of epidemic V. cholerae serogroup O139 strains, which has recombined with plasmids from different bacterial species and transferred among V. cholerae strains.

Detection of Vibrio cholerae O1 and O139 in environmental waters of rural Bangladesh: a flow-cytometry-based field trial.

Presence of Vibrio cholerae serogroups O1 and O139 in the waters of the rural area of Matlab, Bangladesh, was investigated with quantitative measurements performed with a portable flow cytometer. The relevance of this work relates to the testing of a field-adapted measurement protocol that might prove useful for cholera epidemic surveillance and for validation of mathematical models. Water samples were collected from different water bodies that constitute the hydrological system of the region, a well-known endemic area for cholera. Water was retrieved from ponds, river waters, and irrigation canals during an inter-epidemic time period. Each sample was filtered and analysed with a flow cytometer for a fast determination of V. cholerae cells contained in those environments. More specifically, samples were treated with O1- and O139-specific antibodies, which allowed precise flow-cytometry-based concentration measurements. Both serogroups were present in the environmental waters with a consistent dominance of V. cholerae O1. These results extend earlier studies where V. cholerae O1 and O139 were mostly detected during times of cholera epidemics using standard culturing techniques. Furthermore, our results confirm that an important fraction of the ponds' host populations of V. cholerae are able to self-sustain even when cholera cases are scarce. Those contaminated ponds may constitute a natural reservoir for cholera endemicity in the Matlab region. Correlations of V. cholerae concentrations with environmental factors and the spatial distribution of V. cholerae populations are also discussed.

Phylodynamic analysis of clinical and environmental Vibrio cholerae isolates from Haiti reveals diversification driven by positive selection.

UNLABELLED: Phylodynamic analysis of genome-wide single-nucleotide polymorphism (SNP) data is a powerful tool to investigate underlying evolutionary processes of bacterial epidemics. The method was applied to investigate a collection of 65 clinical and environmental isolates of Vibrio cholerae from Haiti collected between 2010 and 2012. Characterization of isolates recovered from environmental samples identified a total of four toxigenic V. cholerae O1 isolates, four non-O1/O139 isolates, and a novel nontoxigenic V. cholerae O1 isolate with the classical tcpA gene. Phylogenies of strains were inferred from genome-wide SNPs using coalescent-based demographic models within a Bayesian framework. A close phylogenetic relationship between clinical and environmental toxigenic V. cholerae O1 strains was observed. As cholera spread throughout Haiti between October 2010 and August 2012, the population size initially increased and then fluctuated over time. Selection analysis along internal branches of the phylogeny showed a steady accumulation of synonymous substitutions and a progressive increase of nonsynonymous substitutions over time, suggesting diversification likely was driven by positive selection. Short-term accumulation of nonsynonymous substitutions driven by selection may have significant implications for virulence, transmission dynamics, and even vaccine efficacy. IMPORTANCE: Cholera, a dehydrating diarrheal disease caused by toxigenic strains of the bacterium Vibrio cholerae, emerged in 2010 in Haiti, a country where there were no available records on cholera over the past 100 years. While devastating in terms of morbidity and mortality, the outbreak provided a unique opportunity to study the evolutionary dynamics of V. cholerae and its environmental presence. The present study expands on previous work and provides an in-depth phylodynamic analysis inferred from genome-wide single nucleotide polymorphisms of clinical and environmental strains from dispersed geographic settings in Haiti over a 2-year period. Our results indicate that even during such a short time scale, V. cholerae in Haiti has undergone evolution and diversification driven by positive selection, which may have implications for understanding the global clinical and epidemiological patterns of the disease. Furthermore, the continued presence of the epidemic strain in Haitian aquatic environments has implications for transmission.

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.

[Development of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae non-O1/O139 serogroups].

OBJECTIVE: To develop methodology of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae (V. cholerae) serogroups non-O1 and non-O139. METHODS: The outer membrane protein gene (ompW) specific for V. cholerae, as well as O antigen rfb genes specific for both O1 and O139, were used for the design of the PCR primers. Both multiple PCR and real-time SYBR green PCR systems were used to detect both O1 and O139. Specific rfb genes and ompW were developed to evaluate their specificity, limit of detection, reproducibility and consistency. RESULTS: We established multiple PCR and real-time SYBR green PCR methods. According to the specific electrophoretic bands (multiple PCR) and the specific melt curve temperature (real-time SYBR green PCR), both methods could specifically detect the non-O1, non-O139 V. cholerae, and to differentiate them from O1,O139 V. cholerae, other five Vibrios and 3 intestinal bacteria. The detection limits were 7 × 10(4) cfu/ml (multiple PCR) and 7 × 10² cfu/ml(real-time SYBR green PCR), with statistically significant difference seen (P < 0.05). For the reproducibility of real-time SYBR green PCR, the external coefficient variation ranging from 0.22% to 0.92% while the internal coefficient variation ranging from 0.27% to 1.41%. 370 strains of non-O1, non-O139 V. cholerae, were detected, with both consistency rates as 100% . CONCLUSION: Both multiple PCR and real-time SYBR green PCR could detect non-O1, non-O139 V. cholerae, rapidly, specifically, and reproducibly, that could all be used for the detection and identification of non-O1, non-O139 under different conditions.

A molecular surveillance reveals the prevalence of Vibrio cholerae O139 isolates in China from 1993 to 2012.

Vibrio cholerae serogroup O139 was first identified in 1992 in India and Bangladesh, in association with major epidemics of cholera in both countries; cases were noted shortly thereafter in China. We characterized 211 V. cholerae O139 isolates that were isolated at multiple sites in China between 1993 and 2012 from patients (n = 92) and the environment (n = 119). Among clinical isolates, 88 (95.7%) of 92 were toxigenic, compared with 47 (39.5%) of 119 environmental isolates. Toxigenic isolates carried the El Tor CTX prophage and toxin-coregulated pilus A gene (tcpA), as well as the Vibrio seventh pandemic island I (VSP-I) and VSP-II. Among a subset of 42 toxigenic isolates screened by multilocus sequence typing (MLST), all were in the same sequence type as a clinical isolate (MO45) from the original Indian outbreak. Nontoxigenic isolates, in contrast, generally lacked VSP-I and -II, and fell within 13 additional sequence types in two clonal complexes distinct from the toxigenic isolates. In further pulsed-field gel electrophoresis (PFGE) (with NotI digestion) studies, toxigenic isolates formed 60 pulsotypes clustered in one group, while the nontoxigenic isolates formed 43 pulsotypes which clustered into 3 different groups. Our data suggest that toxigenic O139 isolates from widely divergent geographic locations, while showing some diversity, have maintained a relatively tight clonal structure across a 20-year time span. Nontoxigenic isolates, in contrast, exhibited greater diversity, with multiple clonal lineages, than did their toxigenic counterparts.