Molecular Characteristics Of Non-O1/O139 Vibrio Cholerae Isolated From 1985 To 2013 In Guangdong Province

BackgroundCholera is a potent intestinal infectious diseases caused by Vibrio cholerae, which spread fast with acute onset and wide spread range. It can lead to painless watery diarrhea, as patients often did not receive timely treatment,lead to severe dehydration,even poisoning to death. Cholera is a disease of international inspection in the country and is classified as a category "A" infectious diseases by the Law of Infectious Diseases in China,which is still a major public health problem in developing country threatening the lives and health .So far, there are more than 200 found Vibrio cholerae serogroups, only 01 and O139 can cause outbreaks and epidemic cholera.Non-O1/O139 Vibrio cholerae (V. cholerae) refers to serogroups other than O1 and O139. In the past that non-O1/ O139 Vibrio cholerae was thought non-pathogenic or caused only mild scattered diarrhea. In recent years, numerous outbreaks of diarrhoea caused by non-O1/O139 Vibrio cholerae had been reported, thus gradually attracted widespread attention of bacteriologist and epidemiologist. Non-O1/non-O139 Vibrio cholerae strains are frequently isolated from the environment, especially from seafood and aquatic sources, and is one of the main pathogens causing acute diarrhea, which lead to similar clinical symptoms of cholera elicited by the 01 toxigenic strains, mainly watery, slightly abdominal pain, fever or no fever, clinicians often have difficults to identify. Meanwhile reports of food poisoning caused by this kind of bacteria gradually increased. In view of the risks in food safety, non-O1/0139 Vibrio cholerae is officially included in the scope of monitoring of China’s food safety risk assessment program in 2015.Since 1817 to 1923,there were six cholera worldwide pandemics caused by classic Vibrio cholerae 01. The El Tor Vibrio cholerae 01 seroproup is the pathogen of the 7th pandemic of cholera, which originated from the island of Sulawesi in Indonesia in 1961,spreading to Guangdong,China in July the same year, and has continued over 40 years, spreading to more than 140 countries on five continents and regions. Vibrio cholera epidemic in Guangdong Province, was mainly caused by group O1.Since 1993, group 0139 appeared,mainly caused dissemination and/or small outbreaks. From 1961 to 2000, the cholera epidemic in Guangdong Province was serious, spread to the whole province with high mortality.After 2000, Cholera was under control, mainly caused limited outbreak and distribution, and gradually into the cholera epidemic intermittent period.During the interim period,we often found non-O1/0139 V. cholerae specimens in some cases of infectious diarrhea monitoring.These strains could obtain pathogenic virulence factors like cholera toxin genes to further strengthen its toxicity, and even possiblely cause a new epidemic due to a new 0139-similar serogroups.Therefore,during the interim period, in order to reduce new epidemic and the burden of infectious diarrhea disease caused by such pathogens, it is necessary to make a close monitoring of non-01/0139 Vibrio cholerae and analyze their molecular characteristics.Objective1.To know the distributions and characteristics of the virulence genes of 219 non-01/0139 Vibrio cholerae strains isolated from different areas of Guangdong province during the past decades.2.To illustrate the process and mechanism of strain evolution of non-01/0139 Vibrio cholerae, and to explore the epidemiological patterns of cholera at molecular level.3.To illustrate the changes and trends of those non-01/0139 Vibrio cholerae strains from different sources and different ages in Guangdong Province. And provide scienfitic evidences for policy making on control and prevention of the disease in Guangdong province.MethodsMaking a epidemiological description of the collected 219 non-01/0139 Vibrio cholerae strains from different sources in Guangdong Province, from 1985 to2013, by using the basic theory of epidemiology. Conduct automatic identification by using traditional biochemical tests and serotyping tests. Apply PCR methods to detect CTXΦ genetic elements (ctxB, rstR4, rstR6, rstR232, rstR-18, rstR264, rstR, rstRclac, rstRclass, rstRElTor), VPI pathogenicity island (tcpl, tcpA-El Tor, tcpR), VSP-I (0175,0178, O180,0183,0185), VSP-Ⅱ (O490,0493,0498, O502, O504,0512, 0514,0516) and other potential virulence genes (chxA, VCS (N2), SXT, ST, rtxA, IS 1004, hlyA, mshA). Apply pulsed-field gel electrophoresis (PFGE) method for molecular typing, analyzed by electrophoresis pattern BioNumerics software.Contents1. Epidemiological information:Making a three-dimension distribution of the collected 219 non-O1/0139 Vibrio cholerae strains from different years, different regions and different sources in Guangdong Province, to illustrate the changes and trends of those strains.2. Strain identification and recovery training serotype:Conduct recovery activation, biochemical identification and serotyping tests after pure culture of the strains. Making a comparative analysis of serotype distribution of 219 non-O1/0139 Vibrio cholerae strains during different pandemic in Guangdong.3.Virulence gene detection:Apply PCR methods to detect CTXΦ genetic elements (ctxB, rstR4, rstR6, rstR232, rstR-18, rstR264, rstR, rstRclac, rstRclass, rstRElTor), VPI pathogenicity island (tcpI, tcpA-El Tor, tcpR), VSP-I (0175,0178,0180,0183,0185), VSP-II (O490,0493,0498, O502, O504,0512,0514,0516) and other potential virulence genes (chxA, VCS (N2), SXT, ST, rtxA, IS1004, hlyA, mshA). Calculate the positive rates and virulence gene types to provide the basic data for the further study of non-01/0139 Vibrio cholerae pathogenic mechanism. And illustrate the distribution characteristics and changes,further explore the popular rule among the219 non-01/0139 Vibrio cholerae strains collected from different sources in Guangdong Province from 1985 to 2013 at the molecular level.4. Molecular subtyping:PFGEBy pulsed-field gel electrophoresis (PFGE) method for molecular typing of isolates,compare the PFGE patterns with the Pulse Net China central database, calculate the similarity, drawing PFGE clustering patterns. To investigate the association of different PFGE clone type and virulence genes, tracing the source of the strains and changes.ResultsThe majority of those non-01/0139 Vibrio cholerae strains were isolated from1985 to 1989,mainly from diarrhea patients(110 strains) and environmental water sources(21 strains), and mainly in Zhanjiang(67 strains), Guangzhou(20 strains), Shantou(16 strains) and Haikou(14 strains) (a separate province of Hainan, after 1988) regions. From 1990 to 1999, the number of non-O1/O139 Vibrio cholerae strains was significantly reduced to 31 strains, patients(17 strains) and external environmental 11 strains) water aquatic origins(3 strains) were detected, and mainly distributed in Yangjiang(13 strains) and Guangzhou(7 strains) regions. Since 2001, the number of non-01/0139 Vibrio cholerae strains has declined for four consecutive years,2007 appears modest recovery,in 2012 there was a certain rise, scattered in various regions.Serotyping results show that 219 non-O1/O139 Vibrio cholerae strains were into VBO2,4,6,7,8,10,11,12,13,14,18,22,23,25,28,29,30,32,38,39,45,46,51,71,76,84,140,1 41 and 155, total 29 serotypes. The serotypes were dispersed and complex, typing rate was 27.40%t, far below serotype range of more than 200 non-01/0139 Vibrio cholerae serotypes.Virulence gene analysis showed that only 13 non-01/0139 Vibrio cholerae strains contain ctxB gene, which accounted for 76.92% of patients sources (10/13), 23.08%of the external environment water sources (3/13), after further sequencing showed all were EL Tor type.7 strains of which were from patients from 1985 to 1987,2 of which were from patients in 1996 and 1998,3 of which were from environment water sources in 1999, and after 2000,no non-O1/O139 Vibrio cholerae contain ctxB gene was detected.33 non-O1/O139 Vibrio cholerae strains carried different types of rstR virulence genes,15 non-O/O139 Vibrio cholerae detected tcpA positive, as well as tcpl.CTXΦ phage and TCP virulence gene distribution was:ctxB +rstR+tcpA+tcpl+(n=13), ctxB-rstR+tcpA-tcpI+(n=18), ctxB-rstR+tcpA+ tcpl+(n= 2).48.32%(72/149)patient-derived strains carried different types of VSP Island virulence genes, only 3 of which carried complete VSP pathogenicity island genes, other strains carried parts of VSP-I or VSP-II genes.46.43%(26/56) external environmental water sourced strains carried different types of VSP island of virulence genes, only 1 of which carried complete VSP pathogenicity island genes, other strains carried parts of VSP-Ⅰ or VSP-Ⅱ virulence genes.64.29%(9/14) aquatic origined strains carried different types of VSP Island virulence genes, none of which carried a complete VSP strain pathogenicity island genes,all carried parts of VSP-I or VSP-II virulence genes. The results of 8 potential virulence genes showed that35.57% (53/149) patient-derived strains contained chxA virulence genes,62.42%(93/149) contained TTSS (vcsC2) gene,14.10%(21/149) contained SXT virulence genes, 2.01%(3/149) contained ST virulence genes,95.97%(143/149) contained rtxA virulence genes,96.64%(144/149) contained mshA virulence genes,89.26% (133/149) contained ISI004 virulence genes,93.29%(139/149) contained hlyA virulence genes.55.36%(31/56) external environmental water sourceed strains contained chxA virulence genes,33.93%(19/56) contained TTSS (vcsC2) virulence genes,14.29%(8/56) contained SXT virulence genes,1.79%(1/56) contained ST virulence genes,78.57%(44/56) contained rtxA virulence genes,87.5%(49/56) contained mshA virulence genes,64.29%(36/56) contained ISI004 virulence genes, 85.71%(48/56) contained hlyA genes.71.43%(10/14) aquatic origined strains contained chxAgenes,57.14%(8/14) contained TTSS (vcsC2) gene,7.14%(1/14) contained SXT genes,7.14%(1/14) contained ST genes,85.71%(12/14) contained rtxA enes,92.86%(13/14) contained mshA genes,85.71%(12/14) contained ISI004 virulence genes,100%(14/14) contained hlyA genes.Pulsed-field gel electrophoresis (PFGE) molecular typing results showed that 149 cases of sources of non-O1/0139 Vibrio cholerae could be divided into 143 PFGE band patterns, the similarity between the spectral shape of 62.4% to 100%,while did not form distinct clusters.70 strains from environmental waters and aquatic sources could be divided into 67 PFGE band patterns, fingerprint similarity between 62.4% to 100%, no significant clustering among strains isolated from different years,areas and sources, indicating that there is a significant genetic diversity in non-O1/0139 strains in Guangdong Province.