深圳地区2002-2008年副溶血弧菌分子特征研究

Molecular characterization of Vibrio parahaemolyticus collected from human infections in Shenzhen, between 2002 and 2008

目的 了解深圳地区副溶血弧菌主要血清型和分子分型以及毒力因子分布,分析新O3：K6型克隆群与国际流行株的进化关系.方法 对2002-2008年深圳地区1005株副溶血弧菌临床分离株进行血清型分型；采用荧光PCR方法对68种不同血清型的28 1株副溶血弧菌进行tlh、toxR、tdh和trh毒力基因检测；对281株菌株进行orf8检测；采用脉冲场凝胶电泳（PFGE）对不同分离时间的5种主要血清型菌株进行分子分型,根据有代表性的分子分型图谱选取82株副溶血弧菌菌株进行GS-PCR检测,并选取60株副溶血弧菌菌株进行toxRS基因测序；进而对41株O3∶K6和O1∶K25副溶血弧菌菌株进行多位点序列分型（MLST）分析,采用eBURST软件分析进化关系.结果 1005株菌共得到79种不同的血清型,主要血清型为O3∶K6、O4∶K8、O1∶K25、O1∶KUT、O4∶K68、O1∶K56和O9∶K44,所占比例分别为57.9％、8.16％、5.27％、5.87％、1.39％、1.39％和0.99％.243株为tlh＋、toxR＋、tdh＋、trh-,37株为tlh＋、toxR＋、tdh-和trh-,1株tlh＋、toxR＋、tdh＋和trh＋.35株O3∶K6型菌株的MLST序列类型为ST3,是同源复合体CC3,O4∶K8型副溶血弧菌的序列类型为ST189,无同源复合体.结论 深圳地区主要流行的副溶血弧菌为tdh＋、trh-的O3∶K6、O4∶K8和O1∶K25菌株,2006年后出现O1∶K56、O9∶K44、O3∶K29、O4∶K9新的血清型；其中O3∶K6血清型属于新的O3∶K6克隆群,与其他国家流行的副溶血弧菌属于同一克隆群,同时也存在新、旧克隆群.血清的多样性和新旧克隆群的存在是造成深圳地区副溶血弧菌腹泻病高发态势的主要原因之一.

Objective To determine the occurrence and distribution of specific clones of pathogenic Vibrio parahaemolyticas （VP） isolated in Shenzhen and to assess the relationship between serotype O3 ∶K6 and the globally distributed pandemic clone.Methods A total of 1005 VPs isolated from diarrhea patients in 2002-2008 were sero-typed.Real-time PCR was used to detect the virulence genes tlh,toxR,tdh,trh and orf8 in 281 isolates from 68 different serotypes.The main serotypes were typed by pulsed field gel electrophoresis （PFGE）.Strains with dominant serotypes and PFGE pattems were assayed by GS-PCR and toxRS sequencing for the identification of pandemic clone.Multilocus sequence typing （MLST） analysis was reserved for exemplary 41 O3 ∶ K6 and O1 ∶ K25 isolates.Results Seventy-nine serotypes were observed among the 1005 isolates,including O3 ∶ K6 （57.9%）,O4 ∶ K8 （8.16%）,O1 ∶ KUT （5.87%）,O1 ∶ K25 （5.27%）,O4 ∶ K68（1.39%）,O1 ∶ K56（1.39%） and O9 ∶ K44 （0.99%）.Most of the strains （99.36%） showed PCR positive to tlh,toxR,and tdh but eleven strains were tdh negative.MLST showed that all the 36 O3 ∶ K6 isolates belonged to ST3 and all the 5 O4∶K8 strains were ST189.These results matched the description of the pandemic VP clone.Conclusion A recognizable burden of diarrheal illness caused by VP had been seen in Shenzhen.Results from serotyping indicated that although there exsiting a large variety of diversities,the dominant serotype appeared to be O3 ∶ K6.VP isolates identified in Shenzhen mainly showed as tdh positive but trh negative,in consistent with the current pandemic O3 ∶ K6 clone.The pandemic O3 ∶ K6 clone did appear to co-exist with other clones of O3 ∶ K6,as well as O4 ∶ K8,O1 ∶ K25.Potential outbreak of VP could be monitored through the laboratory-based surveillance programs,suggesting that the strategies related to prevention and control of VP should be prioritized in Shenzhen.