浙江省海、水产品霍乱弧菌污染状况调查及分子生物学研究

Investigation on polluting situation and study on molecular biology of Vibrio cholerae in aquatic products of Zhejiang Province

目的：了解浙江省海、水产品霍乱弧菌污染情况及海、水产品中霍乱弧菌的毒力特征，并对其进行耐药性和同源性分析，研究其分子特征及流行病学意义。方法：2005年7～9月采集浙江省温州、宁波、舟山的海、水产品及杭州某批发市场的甲鱼，对检出的霍乱弧菌菌株进行耐药性分析，采用聚合酶链反应（PCR）方法进行霍乱弧菌肠毒素基因（Ctx）、小带联结毒素基因（Zot）、辅助霍乱肠毒素（Ace）的检测，并参照美国CDC PulseNet的统一方法进行脉冲场凝胶电泳（PF—GE）分型并作同源性分析。结果：从采集的990份海、水产品标本中检出14株霍乱弧菌，其中分离自青蛙1株、甲鱼9株、贝类1株、甲壳类1株、鱼类1株、其它1株。经耐药性分析，菌株对诺氟沙星、头孢噻肟100％敏感；对复方新诺明、萘啶酸耐药率最高，达64．3％，其次为庆大霉素、利福平，耐药率为35．7％。毒力基因检测表明，14株霍乱弧菌中3种毒力基因均阳性的有5株，占35．7％；携带Ctx的有7株，占50．0％；只携带Zot的有4株，占28．6％；另有3株毒力基因均阴性，占21．4％。脉冲场凝胶电泳分型显示，2株稻叶1f同源；2株小川1k同源；1株小川19k降解；9株0139群霍乱弧菌除1株死亡未作分析外，其余8株分为3个型。结论：此次海、水产品检测的霍乱弧菌血清型较多，但以0139群霍乱弧菌为主，其脉冲场凝胶电泳分型较多，这与浙江省近几年霍乱时有散发和多菌型并存的特点一致。耐药率及毒力基因携带情况与广东省有较大的差别。

Objective：The polluting situation and the vindence characteristics of Vibrio cholerae in aquatic products of Zhejiang Province were investigated to study the molecular characteristics and its epidemiological significance by analysis of drug resistance and homology. Methods：Aquatic products from Wenzhou, Ningbo, Zhoushan and soft shell turtle from a wholesale market in Hangzhou were collected during July to September in 2005. The drug sensitivity test were used to analyze V. cholerae isolates resistant to drugs. The virulence gene Ctx, Zot and Ace were detected by PCR. The molecular types were classified by pulsed - field gel electrophoresis（PFGE）. Results： Fourteen V. cholerae strains were isolated from 990 aquatic products samples,9 from soft shell turtle and the left 5 from frog, seashell, crustacea, fish and other aquatic products respectively. Drug resistance analysis showed that the sensitivity of isolates to norfloxacin and cefotaxime was 100%. The resistant rate to trimethoprim and sulphame - thoxazole and nalidixic acid was highest, it was 64. 3%. The resistant rate to gentamycin and rifampicin was 35. 7%. Virulence gene analysis showed that 35.7% （5/14） of V. cholerae isolates carried all of 3 virulence genes. 50.0% （7/14） isolates carried vindence gene Ctx. 28. 6% （4/14） isolates carried vindence gene Zot and there are 3 isolates which can＇t be detected any of 3 virulence genes. Classification of molecular types with PFGE showed 2 Inaba If strains and 20gawa lk strains were homologous respectively, and 10gawa 19k strain degraded. Except 1 isolate which was not be analyzed because of death, 8 of 9 V. cholerae O139 isolates can be separated into 3 genotypes. Conclusion：This investigation shows that there are more serotypes in Zhejiang and E cholerae 0139, which shows more genotypes is still in predominance. It is in accordance with the characteristics that ema- nation and muhibacillary type of V. cholerae strains in Zhejiang often coexist in these years. There are great differences in the resistance rate and virulence gene between V. cholerae isolates separated from aquatic products in Zhejiang and those of Guangdong.