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

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 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

OBJECTIVETo investigated the genetic polymorphism of the isolated strains using ribotyping method.

METHODSOne hundred twenty-two strains of V. cholerae O139 isolated from different areas of China from 1993 to 1999 were selected and characterized with ribotyping, including 16s rDNA and 23s rDNA probes.

RESULTSOne hundred twenty-two strains were differentiated into 10 different ribotypes (RT1-RT10) on the basis of rRNA gene probes hybridization (with Bgl I digestion), which consisted of 7 - 9 bands between 12 and 1.5 kb in size. RT1 and RT3, as two predominant ribotypes, comprised most number of the strains which spread to the extensive range. Nine strains, which are negative to ctxAB, zot and RS individually, belong to 4 special ribotypes. The dendrogram revealing genetic relationship among different clones of V. cholerae O139 showed that the clones belonging to RT1 and RT2 had genetic similarity on high degree, although they were isolated from different regions. The two predominant ribotypes (RT1 and RT3) were distant in genetic relationship.

CONCLUSIONResults showed the clonal diversity and the wide area distribution of V. cholerae O139 strains in China, suggesting the multiple origins of O139 epidemics.

Phylogeny ; Polymorphism, Genetic ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Ribotyping ; Species Specificity ; Vibrio cholerae O139 ; classification ; genetics

Animals ; Antibodies, Bacterial ; blood ; Cholera Toxin ; immunology ; Cholera Vaccines ; immunology ; Fimbriae, Bacterial ; immunology ; Mice ; Vibrio cholerae O139 ; immunology

In developing countries threat of cholera is a significant health concern whenever water purification and sewage disposal systems are inadequate. Vibrio cholerae is one of the responsible bacteria involved in cholera disease. The complete genome sequence of V. cholerae deciphers the presence of various genes and hypothetical proteins whose function are not yet understood. Hence analyzing and annotating the structure and function of hypothetical proteins is important for understanding the V. cholerae. V. cholerae O139 is the most common and pathogenic bacterial strain among various V. cholerae strains. In this study sequence of six hypothetical proteins of V. cholerae O139 has been annotated from NCBI. Various computational tools and databases have been used to determine domain family, protein-protein interaction, solubility of protein, ligand binding sites etc. The three dimensional structure of two proteins were modeled and their ligand binding sites were identified. We have found domains and families of only one protein. The analysis revealed that these proteins might have antibiotic resistance activity, DNA breaking-rejoining activity, integrase enzyme activity, restriction endonuclease, etc. Structural prediction of these proteins and detection of binding sites from this study would indicate a potential target aiding docking studies for therapeutic designing against cholera.
Bacteria ; Binding Sites ; Cholera ; Computer Simulation* ; Developing Countries ; DNA ; DNA Restriction Enzymes ; Drug Discovery ; Drug Resistance, Microbial ; Genome ; Humans ; Integrases ; Sewage ; Solubility ; Vibrio cholerae ; Vibrio cholerae O139* ; Water Purification

OBJECTIVETo study the survival and growth of Vibrio cholerae inside the Acanthamoeba polyphage.

METHODSSurvival and growth of Vibro cholerae O139, co-cultured with Acanthamoeba polyphaga, was observed inside the trophozoites and cysts, using Gram stain and electron microscope.

RESULTSViable O139 was observed inside the amoebal vacuoles in 24 hours. Vacuoles were filled with more bacteria along with the longer period of co-culture. The process of O139 infection with Amoebae would include uptake, formation of O139 vacuole, multiplication, trophozoites lysed and expel under electron microscopy. Some infected trophozoites could subsequently encyst and the surviving O139 could locate in the vesicles inside the cysts.

CONCLUSIONO139 might survive and multiply in the trophozoites and reside inside the cysts of Amoebae, suggesting that Acanthamoebae might serve as one of the environmental hosts of Vibro cholerae.

Acanthamoeba ; growth & development ; microbiology ; ultrastructure ; Animals ; Coculture Techniques ; Colony Count, Microbial ; Culture Media ; Vibrio cholerae O139 ; growth & development ; ultrastructure ; Water ; parasitology

OBJECTIVETo investigate the molecular epidemiological characteristics and antibiotic resistance profiles of Vibrio cholerae O139 in Shandong province.

METHODSA total of 13 strains of V. cholerae O139 (9 clinical strains and 4 environmental strains) isolated from cholera epidemics in Shandong province since 1997 were recovered and confirmed with serum agglutination and biochemical reaction. Pulsed-field gel electrophoresis (PFGE) was carried out for molecular subtyping. Virulence genes and drug resistance related genes were detected by PCR. Antibiotic susceptibility tests were performed using micro-broth dilution method.

RESULTSThirteen strains of V. cholerae O139 were differentiated into seven pulsetypes. One clinical strain and two environmental strains isolated from Jining in 2013 were clustered into the pulsetype namely KZGN11O139. CN0077, and an identical PFGE pattern of KZGN11O139. CN0002 was found among three clinical strains from Jinan in 2005, Jining in 2005 and Heze in 2009. Other pulsotypes were unique in China and found only in Shandong province. Because of deletion of ctxAB and tcpI, the PFGE patterns of two strains isolated from Yantai in 2000 and 2004 were different from other 11 strains which harbored ctxAB, tcpA, tcpI, rtxA, hlyA and toxR. All strains contained one or more drug resistance related genes such as intI 1, intI 4 and sxt, and were resistant to two kinds of antibiotics at least. Among the 12 kinds of antibiotics, the resistant ratioes to kamamycin, trimethoprim-sulfamethoxazole, ampicillin and gentamicin were 11/13, 9/13, 7/13 and 7/13, respectively.

CONCLUSIONMolecular subtyping indicates possible epidemiological links among V.cholerae O139 in Shandong province, and almost all strains were toxigenic and drug resistant.

China ; Cholera ; Cholera Toxin ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Epidemics ; Humans ; Molecular Epidemiology ; Polymerase Chain Reaction ; Vibrio cholerae O139 ; Virulence

OBJECTIVETo study the correlation between Vibrio cholerae strains isolated from natural enviroment and fishery products and the source of infection during V. cholerae outbreaks.

METHODSCholera toxin gene was detected by polymerase chain reaction (PCR) amplification. Pulsed-field gel electrophoresis (PFGE) was used to subtype the isolates. Results of PFGE were analyzed and clustered by BioNumerics software (Version 4.0).

RESULTSDuring the outbreaks, a total number of thirty O139 V. cholerae related serogroups were collected from patients, carriers, sewage and fishery products were identified and proved to be toxigenic. They could be clustered into four PFGE patterns when digested by Not I. These two V. cholerae outbreaks were caused by the same source of infection because of the following reasons: (1) PFGE patterns of the predominant strains isolated from two outbreaks were identical; (2) they were identical to the PFGE patterns of the strains isolated from the green turtle and rana catesbiana which were bought from the same wholesale store.

CONCLUSIONGreen turtle and rana catesbiana that were contaminated by toxigenic O139 V. cholerae strains seemed to be the source of infection causing the O139 V. cholerae outbreaks in Jiangxi province. Rapid laboratory surveillance and epidemiologic investigation were important in identifying the source of infection during the outbreaks of V. cholera.

Animals ; China ; epidemiology ; Cholera ; epidemiology ; Cluster Analysis ; Disease Outbreaks ; Disease Reservoirs ; microbiology ; Electrophoresis, Gel, Pulsed-Field ; methods ; Fisheries ; Humans ; Ranidae ; microbiology ; Sewage ; microbiology ; Turtles ; microbiology ; Vibrio cholerae O139 ; isolation & purification