Search

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.

To evaluated the fundamental role of stage control technology (SCT) on the detectability for Salmonella networking laboratories.Appropriate Salmonella detection methods after key point control being evaluated, were establishment and optimized. Our training and evaluation networking laboratories participated in the World Health Organization-Global Salmonella Surveillance Project (WHO-GSS) and China-U.S. Collaborative Program on Emerging and Re-emerging infectious diseases Project (GFN) in Shanghai. Staff members from the Yunnan Yuxi city Center for Disease Control and Prevention were trained on Salmonella isolation from diarrhea specimens. Data on annual Salmonella positive rates was collected from the provincial-level monitoring sites to be part of the GSS and GFN projects from 2006 to 2012.The methodology was designed based on the conventional detection procedure of Salmonella which involved the processes as enrichment, isolation, species identification and sero-typing. These methods were simultaneously used to satisfy the sensitivity requirements on non-typhoid Salmonella detection for networking laboratories. Public Health Laboratories in Shanghai had developed from 5 in 2006 to 9 in 2011, and Clinical laboratories from 8 to 22. Number of clinical isolates, including typhoid and non-typhoid Salmonella increased from 196 in 2006 to 1442 in 2011. The positive rate of Salmonella isolated from the clinical diarrhea cases was 2.4% in Yuxi county, in 2012. At present, three other provincial monitoring sites were using the SBG technique as selectivity enrichment broth for Salmonella isolation, with Shanghai having the most stable positive baseline.The method of SCT was proved the premise of the network laboratory construction. Based on this, the improvement of precise phenotypic identification and molecular typing capabilities could reach the level equivalent to the national networking laboratory.

According to results from the two-month consecutive surveillance program in Maanshan, six suspected cases of non-O1 non-O139 Vibrio (V.) cholerae infection, were found that called for identification of pathogens as well as molecular-epidemiological analysis to determine the aggregation of the epidemic situation.Biochemical and serotype identification, hemolysis test, and drug sensitive test were used to detect the drug resistance spectrum. Real-time PCR and conventional PCR were used to detect the presence of V. cholerae specific genes, virulent genes and its related genes, including ompW, ctx, tcpA, toxR, hlyA, zot, ace, rstR and gIII(CTX). Pulsed-field gel electrophoresis (PFGE) was used to analyze the molecular type of strains.All the six isolates of non-O1 non-O139 V. cholerae were identified by biochemical and serologic tests, and appeared to be β hemolytic. Twelve out of the 14 kinds of drugs showed 100% sensitive. All isolates were positive of ompW gene by real-time PCR, but negative for ctx, tcpA, zot, ace, rstR and gIII(CTX). Five of the six isolates were positive for toxR and hlyA, except for strain 1001434446. All strains had different PFGE types, but two strains had similar types. All strains had a low similarity compared to the toxigenic V. cholerae.Six cases of non-O1 and non-O139 nontoxigenic V. cholerae infection appeared in the same period. Along with epidemiological information, we noticed that these cases had a sporadic nature, but frequently appeared in the same area. We got the impression that public health measurements should be strengthened, with special attention paid to those diarrhea outbreaks caused by non-O1/non-O139 strains since V. cholerae had appeared in low incidence.

To build the Geographical Information System (GIS) database for prevention and control of cholera programs as well as using management analysis and function demonstration to show the spatial attribute of cholera. Data from case reporting system regarding diarrhoea, vibrio cholerae, serotypes of vibrio cholerae at the surveillance spots and seafoods, as well as surveillance data on ambient environment and climate were collected. All the data were imported to system database to show the incidence of vibrio cholerae in different provinces, regions and counties to support the spatial analysis through the spatial analysis of GIS. The epidemic trends of cholera, seasonal characteristics of the cholera and the variation of the vibrio cholerae with times were better understood. Information on hotspots, regions and time of epidemics was collected, and helpful in providing risk prediction on the incidence of vibrio cholerae. The exploitation of the software can predict and simulate the spatio-temporal risks, so as to provide guidance for the prevention and control of the disease.

To understand the distribution, molecular characteristics and virulence genes of the O1 and O139 Vibrio cholerae isolates from the Pearl River Estuary water.Vibrio cholerae isolates collected from the Pearl River estuary waters from January 2009 to December 2010, were tested by PCR for eight virulence-related genes, including cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and the regulatory protein genes (tcpI, toxR). Genetic relation was assessed by pulsed-field gel electrophoresis (PFGE) and the patterns were clustered by BioNumerics.From 1152 aquatic samples, 69 isolates were identified, including 41 Inaba, 18 Ogawa and 10 O139. All the isolates showed ctxA negative, while the hlyA and toxR genes were positive in all the isolates. 34.15% (14/41) of the Inaba strains were hlyA(+) toxR(+) ompU(+) ace(+) zot(+) tcpI(+), while 66.67% (12/18) belonged to Ogawa strains and 70% (7/10) of the O139 strains were hlyA(+) toxR(+). Through PFGE analysis, the O1 isolates formed three clusters in this study. The patterns of O1 isolates differed widely, with the similarity as 72.8% - 100.0%, while the patterns of O139 isolates having the similarity of 69.9% - 95.5%.The non-toxigenic O1 and O139 V. cholerae had a wide distribution in the environment of Pearl River estuary water during the non-epidemic period of cholera. All the aquatic isolates presented diversities on the related virulent genes.

To characterize the spatial distribution of typhoid and paratyphoid fever (TPF) in Yunnan province, China and to determine the effectiveness of meteorological factors on the epidemics of TPF.Data of reported TPF cases in Yunnan province (2001 - 2007) from the China Information System for Diseases Control and Prevention was applied to GIS-based spatial analyses to detect their spatial distribution and clustering of TPF incidence at the county level. Panel data analysis was used to identify the relationships between the TPF incidence and meteorological factors including monthly average temperature, monthly cumulative precipitation and monthly average relative humidity.During the study period, the average incidence of TPF in Yunnan province was 23.11/100 000, with majority of the TPF cases emerged in summer and autumn. Although widely distributed, two TPF clusters were detected in Yunnan province based on the spatial analysis: one area around Yuxi city with the average annual incidence as 207.45/100 000 and another at the junctions of Yunnan province with Burma and Laos. Based on results from panel data analysis, the incidence of TFP was shown to be associated with meteorological factors such as temperature, precipitation, relative humidity and one month lag of temperature increase [10°C increase in the monthly average temperature: IRR = 1.30 (95%CI: 1.24 - 1.36); 10% increase in monthly average relative humidity: IRR = 1.07 (95%CI: 1.05 - 1.09); 100 mm rise in monthly cumulative precipitation: IRR = 1.02 (95%CI: 1.00 - 1.03); and 10°C average temperature increase, the last month: IRR = 1.73 (95%CI: 1.64 - 1.82)].Areas with high TPF incidence were detected in this study, which indicated the key areas for TPF control in Yunnan province. Meteorological factors such as temperature, precipitation and humidity played a role in the incidence of TPF.

To analyze the molecular characteristics and genetic correlations of Vibrio cholerae isolates in Hainan in 2008, so as to provide pathogenic proof to diagnose the plague.Seventy six cholera strains were isolated from this cholera epidemic.69 strains were obtained from patients, 7 were isolated from external environment, among which, one was from patient's toilet, one from water sample, three were isolated from fish pond near patient's home, one came from swab of the patient vomit on the ground of health center and one from swab of kitchen knife from Hainan University canteen respectively. With conventional aetiological methods, pulse-field gel electrophoresis was conducted and the patterns of the 76 isolates were analyzed. The PFGE image was analyzed using BioNumerics (Version4.0, Applied Maths BVBA, Belium). Image bands were identified and similarity coefficient was automatically generated.Seventy six strains were isolated from Vibrio cholerae outbreaks in Hainan in 2008.5 PFGE patterns of patient's isolates in June were the same, sharing a similarity coefficient of 100%. 70 PFGE patterns of patients and water in October and November were completely same, the similarity coefficient being 100%. But they were not same as that of June. 1 PFGE pattern of isolate from the sample in Hainan University was different, only sharing a similarity coefficient of 79.7%, which showed no correlation with the outbreak.Different outbreaks of Vibrio cholera occurred in Hainan in 2008. The epidemic in October and November at different counties was one outbreak. The pollution of water in environment was an important factor for outbreak.

To identify the isolates of Shewanella spp. from specimens of food poisoning based on biological and biochemical analysis.Strains were obtained from the investigation on two food poisoning episodes in September and October, 2007 in Ma'anshan city, Anhui province. In accordance with the national standard protocol (GB/T 4789), all specimens were enriched and isolated on selective medium, and the suspected strains were identified by the VITEK-32 and API20E systems. For Shewanella spp. identified by the biochemical system, more characteristics were analyzed using auxiliary biochemical, growth, hemolytic and drug-resistance tests. DNAs of Shewanella spp. were extracted, 16S rDNA was PCR amplified and sequenced with universal 16S rDNA primers. Phylogenetic tree was constructed with MEGA 4.0.After enrichment, all specimens were inoculated to selective medium and Shewanella spp. strains were isolated from 8 samples with single colony on both TCBS and BP media. The characteristics of growth in the Triple Sugar Iron (TSI) agar appeared to have had hydrogen sulfide production but no gas production or positive oxidase. No Shewanella spp. strain was detected in WS, SS and EMB media. The 8 strains were identified as Shewanella algae (S. algae) or Shewanella putrefaciens (S. putrefaciens) by VITEK-32, as S. putrefaciens by API20E system. No other enteropathogenic bacteria, including Vibrio cholerae, Salmonella, Vibrio parahaemolyticus, Proteus vulgaris or Staphylococcus aureus, were detected from those 8 samples. From 16S rDNA phylogenetic trees, 7 out of 8 Shewanella spp. were identified as S. algae, 1 as S. putrefaciens.Strains of Shewanella spp. were isolated from samples of the food poisoning episodes, providing a possible clue to investigate the role of Shewanella spp. on food poisoning.

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

To develop a TaqMan real-time PCR for the detection of Aeromonas hydrophila.The conserved region of major adhesion gene of Aeromonas hydrophila (aha) was used to design primers and TaqMan probe. A total of six concentration gradients for forward and reverse primers ranging from 200 -700 nmol/L were chosen, and four concentration gradients for probe ranging from 100 - 400 nmol/L were chosen. And then the concentration of primers and probe were optimized by ANOVA of completely randomized design respectively. The specificity of the established method was evaluated by using bacteria as contrast, including 45 strains Vibrio cholerae, 20 strains Vibrio parahaemolyticus, 10 strains Vibrio fluvialis, 4 strains Vibrio mimicus, 5 strains Vibrio vulnificus, 1 strain Vibrio alginolyticus, 1 strain Vibrio furnissii, 5 strains Salmonella, 10 strains Shigella and 2 strains Plesiomonas shigelloides. The sensitivity, bacterial sensitivity and DNA sensitivity included,were evaluated. The stool of healthy people was contaminated by Aeromonas hydrophila artificially, and the ability of the established TaqMan real-time PCR system for detection of Aeromonas hydrophila was also evaluated.The cycle threshold (Ct) value deserved from 6 groups of primers concentration gradient was (x +/- s):20.69 +/- 0.33, 20.72 +/- 0.21, 20.81 +/- 0.12, 20.74 +/- 0.12, 20.51 +/- 0.16 and 20.69 +/- 0.11, respectively, and the concentration of forward primer and reverse primer was determined to be 200 nmol/L (F = 1.33, P = 0.28). The Ct value deserved from 4 groups of probe concentration gradient was (x +/- s):20.56 +/- 0.08, 20.82 +/- 0.05, 20.82 +/- 0.11 and 20.93 +/- 0.09, respectively, and the concentration of probe was determined to be 100 nmol/L (F = 5.26, P = 0.01). The bacterial sensitivity and DNA sensitivity were 80 CFU/ml and 100 fg/microl respectively, and the sensitivity to detect Aeromonas hydrophila from stool was 8 x 10(3) CFU/ml, which might be 8 CFU/ml after 8 hours' enrichment. No amplification was observed in the templates of other bacterial.The TaqMan real-time PCR method targeting the aha gene of Aeromonas hydrophila had a high sensitivity and specificity and might be used to detect Aeromonas hydrophila from pure bacterial and stool rapidly.

To understand the biochemical characteristics and 16S rDNA genetic sequence evolution of strains isolated from diarrhea specimens so as to provide basis for classification and identification of Klebsiella pneumoniae.Specimens were cultured using MacConkey and SS medium. All isolates were identified as K. pneumoniae by automated biochemical tests. DNA was extracted, 1500 bp fragments of the 16S rDNA gene were by amplified PCR and sequenced with K. pneumoniae 16S rDNA primer, after being cut. Fragments of 1000 bp overlapping sequences were analyzed by Blast to confirm the identity of the isolates. A phylogenetic tree was constructed by PHYLIP process to analyze the 16S rDNA sequence of the isolated strain with other relative bacteria species in the GenBank databases.Among 113 specimens of infectious diarrhea, 25 K. pneumoniae strains were identified by biochemical tests, of which 21 subsp. pneumoniae and 4 subsp. ozaenae, no subsp. of rhinoscleroma were isolated. Strains of subsp. pneumoniae were found having nature of resistance. All isolates were resistant to penicillin G and susceptible to polymyxin with some strains were resistant to Nitrofurantoin, Cephalothin, Kanamycin, Tobramycin. After searching in GenBank of 16S rDNA, strains biochemical identified as subsp. ozaenae shared high similarity with Salmonella strains and other intestinal bacteria. 16S rDNA phylogenetic analysis could be used to confirm subsp. pneumoniae, but could not separate other subspecies of K. pneumoniae completely.16S rDNA phylogenetic analysis useful in identifying and classifying K. pneumoniae.

To analyze the etiologic characteristics of Vibrio cholerae in Guangdong province in 2007. Genetic relationship was observed including among predominated biotype isolates from different areas within the province and among same biotypes isolates from cholera cases and regular surveillance.Isolates from cholera cases and through environmental surveillance were typed by sero- and phage- typings. Similarity of molecular fingerprinting was analyzed through comparing the pulsed field gel electrophoresis(PFGE) pattern of predominated biotype isolates, and those of the same biotype isolates from cholera cases a nd environment surveillance, respectively. In addition, genetic relationship was determined by clustering analysis, using bionumerics software.In total, 31 isolates from cholera cases were collected and subtyped for 3 serogroups. V. cholerae O1 El Tor Inaba phage 1d was the predominant biotype which causing most of the cases in Guangdong province in 2007. Data from cluster analysis showed that the similarity among Inaba phage 1d strains from different areas were from 94.5% to 100%. However, 16 isolates were collected from environment surveillance programs and the predominated biotype could not be found. Additionally, the biotype distribution of cases isolates was not consistent with those isolates through surveillance. High phylogenetic diversity was observed for the same biotypes isolates from cases and surveillance samples.Our data showed that V. cholerae O1 El Tor Inaba phage 1d was the predominated biotype with multi-clone coexisting and circulating in Guangdong province in 2007. It also appeared to be the characteristics of cholera in the non-epidemic period, suggesting that it was necessary to enhance the alert surveillance programs for cholera epidemic based on the molecular typing techniques.

Through 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.Twenty-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.862 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%.V. 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.

To 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.O 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.The 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.The 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.

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

To understand the pollution rates of vibrio cholera (V. cholera) in different seafood, aquatic products and their circulatory processes, so as to help making measures for cholera control and prevention.Different seafood, aquatic products and breed water specimen collected from 12 provinces of China were tested from July to September in 2005.A total of 12 104 samples of seafood and aquatic products were tested and the average pollution rate of vibrio cholera was 0.52%. The positive isolate rate of turtle sample (1.72%) was the highest among all samples. The second higher isolated rate was 1.14% in water specimen of turtle breed pool. The positive rate of bullfrog was 0.50%. The percentage of toxin strains was 47.54% and 79.31% of them were isolated from turtle and water samples of turtle breed pool. The important sector of the pollution of vibrio cholera was in turtle breed pool (2.38%).The average pollution rate of vibrio cholera in seafood and aquatic products in 12 provinces of China was low. It should be very necessary to supervise the sanitation in turtle breed for controlling and preventing the vibrio cholera.

DNA sequence and the genome of phage VP3 (a typing phage of V. cholera) were analyzed. A random library of VP3 DNA was constructed by shot-gun library method. The VP3 genome sequence was assembled with contigs sequences, the gaps between different contigs were filled with sequencing data from primer walking. ORFs were predicted; Phylogeny of DNA polymerase sequences was analyzed to determine the class of VP3; The activity of putative promoter genes were analyzed using lacZ report system. VP3 genome is a 39504bp of circular double-stranded DNA. Twenty-seven out of forty-nine putative ORFs were annotated; twenty gene products were homologous with T7-like phages, including DNAP, DNA replicative protein, capsid, tail tubular, tail fiber protein, and DNA packaged protein. The activity of the putative promoter regions was confirmed through cloning those regions to LacZ-fuse plasmid pRS1274 and analysis of the expression of beta galactosidase. The complete genomic sequence of VP3 and phylogenetic tree analysis suggests VP3 is a member of T7 phage family.

Phage VP1 infects and lyses Vibrio cholerae. The VP1 genome is a circular double-strand DNA and its size is 32176 base pairs. Analysis of the sequence of the VP1 genome revealed the presence of 15 putative promoter sequence. The activities of these putative promoters in V. cholerae were assayed by transformation of reporter gene plasmid and phage infection together. Promoter regions were ligated into pRS1274/BamH I/EcoR I. Then transformed into E. coli JM109 and all of clone display blue. The recombinant plasmids were transformed into V. cholerae 7743 deltaZ by electroporation, then bacteriophage VP1 infect transformant. The time-course expressing lacZ gene and detecting change of beta-galactosidase enzyme activity in V. cholerae transformants at latent period, indicated P17 probably is a early promoter; P2 and P3 and P9 etc are medium-term promoters; P18 is a late promoter.