OBJECTIVETo understand the phenotypic diversity of toxigenic Vibrio cholerae O1 El Tor strains isolated from different provinces in China during the last 50 years.

METHODSTraditional biotyping testings including susceptibility to polymyxin B, sensitivity to group IV phage, Voges-Proskauer test and haemolysis of sheep erythrocytes were conducted.

RESULTSData from Biotype-specific phenotype analysis revealed that only 133 isolates carried the typical El Tor phenotypes while the other 251 isolates displayed atypical El Tor phenotypes. Combined with ctxB, rstR genotypes and phenotypic characteristics, 64 isolates were identified as typical El Tor biotype, 21 were El Tor variants that showing the typical El Tor biotype-specific phenotype but with ctxB(class). 280 isolates were defined as the hybrid groups with traits of both classical and El Tor biotypes that could be further classified into 45 groups, based on the combination of genotypes of ctxB, rstR and phenotypic characteristics.

CONCLUSIONToxigenic Vibrio cholerae O1 El Tor strains that isolated from different provinces in China displayed high phenotypic diversity. The traditional biotype traits could not be used to correctly distinguish the two different biotypes.

China ; Genotype ; Phenotype ; Polymorphism, Genetic ; Vibrio cholerae O1 ; genetics

OBJECTIVETo understand the ctxB and rstR variations of toxigenic Vibrio cholerae (V.cholerae) O1 El Tor strains isolated from different provinces in China from 1961 to 2010.

METHODSAll 385 toxigenic V.cholerae O1 El Tor strains were selected, which were isolated in China between year 1961 and 2010. ctxB gene was amplified by PCR method and sequenced for further analysis. rstR was detected with PCR by using the genotype specific primers.

RESULTSctxB sequence analysis revealed that 52.5% (202/385) isolates carried ctxB(ET) and 47.5% (183/385) carried ctxB(class), namely Y(39) to H and I(68) to T substitutions which were specific to the classical biotype CT-B sequence. From 1961 to 1992, strains carrying ctxB(ET) were predominant and the proportion was as high as 98.4% (182/185). After 1993, strains carrying ctxB(class) were sharply increased. Especially during year 1993 to year 2005, 97.2% (174/179) of the isolated strains carried ctxB(class). Since 2006, resurgence of dominant strains carrying ctxB(ET) or co-existing of strains with ctxB(ET) or ctxB(class) was noticed. rstR genotype detection showed that 62.9% (242/385)of the tested strains carried the rstR(ET), while 6.8% (26/385) with rstR(class), and the remainings contained at least two types of rstR in different combination forms, among which rstR(ET)+rstR(class) combination were the most, accounting for 75.7% (75/99) . Similar to the ctxB, the distribution of rstR genotypes showed time specificity. From 1961 to 1992, strains carrying rstR(ET) predominated (87.0%, 161/185). After 1993, the diversity of rstR genotypes was observed accompanying by a sharp increase of strains containing other rstR genotype, such as rstR(class), rstR(env) and different combinations. There were separately 96% (25/26), 84% (63/75) and 18/18 strains containing rstR(class), rstR(ET)+rstR(class) and rstR(ET)+rstR(class)+rstR(env) isolated after 1993.

CONCLUSIONThe distribution of different genotypes of ctxB and rstR showed obvious time-specificity, and there were various combining forms of rstR, reflecting the diversity of the genetic and evolutionary characteristics of Chinese V.cholerae isolates.

China ; Cholera ; Genotype ; Molecular Epidemiology ; Polymerase Chain Reaction ; Vibrio cholerae ; Vibrio cholerae O1

Vibrio cholerae non-O1/O139 strains are the organisms that are biochemically indistinguishable from V. cholerae but do not agglutinate in Vibrio cholerae O1 and O139 antisera. V. cholerae non-O1/O139 strains are associated with gastroenteritis and extraintestinal infections such as bacteremia, peritonitis and wound infections. Gastroenteritis by V. cholerae non-O1/O139 is uncommon in Korea. We isolated V. cholerae non-O1/O139 from a stool specimen of a one year-old female with diarrhea and high fever.
Bacteremia ; Cholera ; Diarrhea ; Female ; Fever ; Gastroenteritis* ; Humans ; Immune Sera ; Korea ; Peritonitis ; Vibrio cholerae O1 ; Vibrio cholerae* ; Wound Infection

OBJECTIVETo investigate the serologic type, phage-biotype and toxic factor of Vibrio cholerae isolated from different sea products, analyze the relation between the Vibrio cholerae in sea products and cholera epidemiology, and provide references for forecasting cholera epidemic situation and drawing out a preventing plan.

METHODThe biotype of strains isolated was analyzed by using type and phage-biotype serological methods. The toxic gene was detected by PCR.

RESULTSThe constituent ratio of V. cholerae O139, Ogawa and Inaba were, respectively, 48.44%, 20.31% and 31.25% in 64 strains of V. cholerae. The result of phage-biotype showed that the 26 strains of V. cholerae O1 were all non-epidemic strains. The result of toxic gene detecting showed that positive rate of V. cholerae O139 was higher than those of Ogawa and Inaba.

CONCLUSIONThe positive rate of toxic gene in V. cholerae O139 was high and the V. cholerae O139 was mainly in turtle, breed aquatics water and crustacean, so these sea products were the important sectors in cholera prevention and control.

Animals ; Bacteriophage Typing ; DNA, Bacterial ; genetics ; Seafood ; microbiology ; Serotyping ; Vibrio cholerae ; classification ; genetics ; isolation & purification ; Vibrio cholerae O1 ; isolation & purification ; Vibrio cholerae O139 ; isolation & purification

BACKGROUND: Cholera is a representative water-borne disease that is caused by V. cholera ctx (+). V. cholera El Tor was previously the primary pathogen, but after the seventh pandemic outbreak, it was replaced by a V. cholera El Tor variant with a classical phenotype and genotype. In this study, we investigated the genotypic and phenotypic characteristics of imported V. cholerae El Tor in Korea. METHODS: Forty-nine V. cholerae O1 El Tor strains isolated from 2004 to 2011 were used in this study. Polymerase chain reaction amplification of the ctxB and rstR genes was used for biotype determination. An antimicrobial susceptibility test was performed for phenotypic analysis, and pulse field gel electrophoresis (PFGE) was used for analysis of genetic relatedness. RESULTS: Classical ctxB genes were found in all of the isolates, while classical, El Tor, and combined rstR genes were found. Twenty strains showed antimicrobial resistance against streptomycin, sulfamethoxazole/trimethoprim, nalidixic acid, and ciprofloxacin. Based on PFGE, all isolates were grouped as cluster B. The country of origin and resistance pattern were highly related, although the time of influx and serogroup were not. CONCLUSION: Isolates of V. cholera El Tor imported since 2004 were hybrids of V. cholera El Tor, which has the classical ctxB gene and is considered to be a CTX prophage. The SXT element plays an important role in antimicrobial resistance. PFGE patterns, which can be used for analysis of imported V. cholera, revealed the relatedness of the resistant isolates.
Chimera ; Cholera ; Ciprofloxacin ; Electrophoresis ; Genotype ; Korea ; Nalidixic Acid ; Pandemics ; Phenotype ; Polymerase Chain Reaction ; Prophages ; Streptomycin ; Vibrio ; Vibrio cholerae ; Vibrio cholerae O1

Vibrio cholerae non-O1 mainly causes gastroenteritis and rarely causes extraintestinal infections, such as bacteremia. Skin and soft tissue infections are also possible, but the incidence rate is very low. Although the most common cause of pyomyositis is Staphylococcus aureus, Gram-negative organisms such as Vibrio species may also cause pyomyositis in patients with chronic liver disease. Pyomyositis caused by Vibrio cholerae non-O1 has not been reported in Korea. Here, we report a case of pyomyositis caused by V. cholerae non-O1 bacteremia in a patient with liver cirrhosis following seafood exposure. This case study suggests that V. cholerae, as well as V. vulnificus, should be considered when soft tissue infections occur in patients with liver cirrhosis after seafood exposure. In addition, physicians should consider imaging studies for a prompt diagnosis if the patient complains of severe pain disproportionate to the skin manifestation.
Bacteremia ; Cholera ; Gastroenteritis ; Humans ; Incidence ; Korea ; Liver ; Liver Cirrhosis ; Liver Diseases ; Pyomyositis ; Seafood ; Skin ; Skin Manifestations ; Soft Tissue Infections ; Staphylococcus aureus ; Vibrio ; Vibrio cholerae ; Vibrio cholerae non-O1

OBJECTIVETo develop a real-time SYBR Green polymerase chain reaction (PCR) for detection of Vibrio cholerae serogroups O1 and O139, and to evaluate its reliability through detection of estuary water samples.

METHODSO antigen rfb genes specific for O1 and O139 were used for the design of PCR primers. The real-time SYBR Green PCR system in detecting O1 and O139 specific rfb genes in one tube was developed, and its sensitivity, specificity and reproducibility were evaluated. The ability of the real-time PCR in detection of estuary water samples was compared with the routine PCR and bacteria isolation.

RESULTSThe amplification of O1 or O139 specific target gene could be detected according to the melt curve temperature of amplicons. No amplification was observed in the templates of other 10 non-cholerae vibrios. When comparing to the real-time PCR to bacteria isolation in detection of 524 estuary water samples, it showed high sensitivity, plus also positive in real-time PCR detection among all the samples in which bacteria of O1 or O139 were isolated.

CONCLUSIONThe real-time SYBR Green PCR could be used as the first step of rapid environment screen of V. cholerae in water samples thus might enhance the efficiency of isolation in screening of large amount of water samples.

Environmental Monitoring ; methods ; Genes, Bacterial ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Rivers ; microbiology ; Sensitivity and Specificity ; Vibrio cholerae O1 ; isolation & purification ; Vibrio cholerae O139 ; isolation & purification

OBJECTIVETo assess the antibiotic resistance and molecular characterization of cholera strains and to provide basis for clinical treatment and prevention of cholera.

METHODS4 stains isolated from an outbreak of cholera epidemic in Huai'an City in Jiangsu province in September 2010 were characterized using antibiotic susceptibility, biotype analysis, virluence genes detection, ctxB gene sequencing, and PFGE analysis.

RESULTSThe 4 strains were all resistant to sulphamethoxazole/trimethoprim, erythromycin, streptomycin. High drug susceptibility of the samples was found to 6 kinds of antibiotics such as amikacin, norfloxacin, ciprofloxacin, gentamicin, chloramphenicol, ampicillin. The isolates expressed phenotypic traits of both serogroup O1 ogawa and El Tor and carried 9 kinds of virulence genes, ctxA, ace, zot, toxR, tcpI, ompU, rtxC, tcpA, and hlyA gene. They were also identified as harboring the classical ctxB genotype based on amino acid residue substitutions. The PFGE profiles of NotI showed a single banding pattern, while SfiI's was 2 banding patterns.

CONCLUSIONThe bacterium type of Vibrio cholerae causing the epidemic outbreak of cholera belonged to the atypical EL Tor variant which was also identified as toxicogenic strain. The mapping of the strains prompted that there should be the common contamination source. Drug sensitivity test can guide the clinical drug use, in order to reduce the emergence of resistant strains.

Anti-Bacterial Agents ; Cholera ; Cholera Toxin ; Disease Outbreaks ; Drug Resistance, Bacterial ; Drug Resistance, Microbial ; Epidemics ; Genotype ; Humans ; Vibrio cholerae ; Vibrio cholerae O1 ; Virulence

OBJECTIVETo establish a duplex nested PCR assay system which is capable for detecting O1 and O139 groups of Vibrio cholerae simultaneously, and is applicable to environmental specimens from routine cholera surveillance.

METHODSBased on nucleic acid sequences available in GenBank, six sets of primers were designed by PrimerSelect program of DNAStar, targeting the rfb gene that encodes the O antigens of O1 and O139 V. cholerae, respectively. The specificity of several primer combinations was tested. A duplex nested PCR assay system for simultaneously detecting O1 and O139 V. cholerae was established, subsequently, its sensitivity, specificity, reproducibility and field evaluation were tested. The sensitivity of this assay was evaluated by comparing detection limits of nested PCR and conventional PCR. Its reproducibility was tested by 32 positive samples (11 samples positive for O1, 21 samples positive for O139) from environmental surveillance. In addition, the selected amplicons from positive samples were sequenced and analyzed with relevant sequences.

RESULTSThis newly-established duplex nested PCR assay might distinguish O1 V. cholerae from O139 V. cholerae, based on fragment lengths of amplicons, with reliable reproducibility, and no specific amplification was observed as compared with other vibrio species. The sensitivity of this nested PCR was (15 000) higher than conventional PCR, and there was no interference observed with multiple primers and complicated templates in the same vial. In its field evaluation, 32 positive DNA samples were detected and be further confirmed with double or triple tests, implying reliable reproducibility and consistency of this system. These results indicated that this assay had reliable reproducibility. No amplification was observed in all negative specimens and also suggested the acceptable specificity of this assay. Sequence analysis of the selected amplification products revealed 100% homogeneous with relevant genes from V.cholerae, indicating that these amplicons were originated from V. cholerae.

CONCLUSIONThis duplex nested PCR assay system should be rapid, sensitive and especially applicable to small laboratories, and be suitable for dynamic environmental surveillance.

DNA, Bacterial ; Environmental Monitoring ; methods ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; Vibrio cholerae O1 ; genetics ; isolation & purification ; Vibrio cholerae O139 ; genetics ; isolation & purification

OBJECTIVEThrough systematic monitoring of the number and strain types of O1 and O139 Vibrio cholerae in the Pearl River estuary waters to analyze it's relevance with the temperature of environment, and the relevance between strains in water and isolates during outbreaks and epidemics as well as to estimate the methods used for environmental water detection and the potential role in cholera surveillance program.

METHODSTwenty-four stations along the Pearl River were selected and the water samples were collected monthly from March 2006 to February 2007. V. cholerae O1 and O139 strains were isolated from the samples. Real-time PCR established in our laboratory was used to detect V. cholerae O1 and O139. Air temperature and water temperature were collected during sampling. Pulsed field gel electrophoresis (PFGE) was applied in molecular typing of the isolates.

RESULTS862 water samples were collected during the study period. A total number of 77 O1 and O139 V. cholerae were isolated in 67 water samples and the positive rates were 7.77% for isolation and 26.33% for real-time PCR. Seasonal trend of positive rates by month were approximately coincident with the change of water temperature. The positive rates in the stations in urban area were higher than those in other areas. Toxigenic O139 strains were found in one station located in downstream of a marine market. Most of the O1 and O139 isolates were non-toxigenic. No trend of seasonal variation of the strains was noticed. Within these 75 isolates, 49 PFGE patterns were identified and the patterns differed widely with the similarity of 57.4% - 100%.

CONCLUSIONV. cholerae existed as the natural habitat in estuary water of the Pearl River and showed obvious genetic diversity. Data from monitoring waters might show the separation of strains with certain seasonal association. But the crowd did not show the relationship between the infections. Results from water surveillance program might provide indicators on the appearance of cholera pathogen which might be used in assessing the environmental risk of cholera epidemics as well as the alert of cholera.

Electrophoresis, Gel, Pulsed-Field ; Environmental Monitoring ; Phylogeny ; Polymerase Chain Reaction ; Seasons ; Temperature ; Vibrio cholerae O1 ; classification ; genetics ; isolation & purification ; Vibrio cholerae O139 ; classification ; genetics ; isolation & purification