| Cholera has caused seven globle pandemic since1817, the first six pandemic were caused by01classical Vibrio cholerae. Since1961, the group01El Tor biotype of V. cholerae replaced the classical biotype and caused the7th pandemic in the world. In India and Bangladesh in1992, a new serogroup0139of V. cholerae emerged, causing the cholera’s outbreak and prevalent and then spreading in Southeast Asia (including China). All previous cholera pandemic has spread to China, whereas the molecular characters and evolution of the strains of the7th pandemic in China has not yet been fully elucidated. In this study, we selected865strains of the seventh pandemic from different years and different regions (provinces), including650strains of01serotype (between1961and2010) and215strains of0139serotype (between1993and2010). We analyzed these strains to elucidate the molecular characters of the epidemic cholera strains in China.The causative agent of cholera is the cholera toxin (CT), encoding by ctxAB gene. PCR detection of all865strains selected,56.3%(487/865) of the strains were toxigenic strains (ctxAB+), among them,85.5%(359/420) of the patient isolates (n=420) and28.8%(128/445) of445environmental isolates were toxigenic strains, respectively. According to the serogroup, of O1strains (n=650), the toxigenic strains of the patient and environmental strains were278(84.2%) and83(25.9%), respectively; of the O139strains (n=215), the toxigenic strains of the patient and environmental strains were81(90.0%) and45(36.0%), respectively. CtxB gene sequencing showed that, of O1toxigenic strains(n=361), the ctxB type of El Tor (ctxBET) and Classical (ctxBCL) accounted for67.6%and29.6%, respectively. It is interesting that, ctxBCL of strains were emerged in1992, in the subsequent period appeared the long-term and coexistence with ctxBET of strains. Whereas all O139strains were ctxBET type. Meanwhile, we also found three novel types of ctxB, which have not been reported previously.We detected the marker genes of7th pandemic Ⅴ. cholerae-rtxC and VSP-Ⅰ/Ⅱ gene cluster, the positive rate of rtxC was95.4%(825/865). Meanwhile,87.0%(423/486) of the toxigenic strains (n=486) and16.6%of non-toxigenic strains (n=379) carried VSP-Ⅰ/Ⅱ gene cluster. According to the serotype, of all650O1isolates,82.8%and20.1%of toxigenic and non-toxigenic strains were positive for VSP-Ⅰ/Ⅱ, respectively; Of215O139strains,99.2%and5.6%of toxigenic and non-toxigenic strains were positive for VSP-Ⅰ/Ⅱ, respectively, this results showed that in either O1or O139strains, positive of VSP-Ⅰ/Ⅱ in toxigenic strains was significantly higher than the non-toxigenic strains (P＜0.0001), the results also indicated the majority strains after1961were El Tor type,in addition, the VSP-Ⅰ/Ⅱ clusters were mainly existed in the pathogenicity of Ⅴ. cholerae. The role of the VSP-Ⅰ/Ⅱ cluster in the pathogenic of Ⅴ. cholerae need further study.Previous study has analyzed the O1Ⅴ. cholerae using muti-locus sequence typing(MLST) based on seven housekeeping genes. In the present study, we focused on O139Ⅴ. cholerae from1993to2010, also using MLST of seven housekeeping genes.72O139strains were divided into16sequence types (STs), toxigenic strains have two STs, remaining14STs belonged to non-toxigenic strains, suggested different lineages origins of toxigenic and non-toxigenic strains, the toxigenic O139strains predominant in China were origined from limited lineages. PFGE (Not Ⅰ digestion) results showed that197strains can be divided into five groups, toxigenic strains were clustered into one group, remaining four groups were non-toxigenic strains, while within each group, there is more than one PT, the similarity of toxigenic strains (84%) was higher than that of non-toxigenic strains (66%), suggested the0139strains predominant in China were evoluted to variant strains within settled lineages. In addition, the nontoxigenic0139strains exhibited greater diversity than that of toxigenic strains,.CTXΦ carries the gene of ctxAB, which can transfer between toxigenic strains and non-toxigenic strain, is an important mechanism for the appearance of the new type V. cholerae, this need toxin-coregulated pilus (major subunit was encoded by tcpA) as the receptor, but tcpA gene is polymorphism in different source of strains(the majority were non-toxigenic strains). From the results of the first part of research, we screened different types the advantages cloning group of of non-toxigenic strains (ctxAB-but tcpA+), using chloromycetin gene markered CTXΦ carrying ctxAB to infect the non-toxigenic strains, the results showed that the El Tor type (tcpAET) of strains, but not other types (such as tcpAVCE232, tcpA153-94, and tcpA06-18) of strains, can obtained CTXΦ effectively, the results suggested that tcpAE1of strains could be the potential receptors and then obtained the virulence genes, to cause cholera epidemic. |
PDF Full Text Request