湖北省2012年流行株O139霍乱弧菌脉冲场凝胶电泳分型分析

目的分析3起霍乱疫情病原O139霍乱弧菌分子分型特征和遗传相关性,探讨O139霍乱疫情流行特征。方法对2012年湖北省3起霍乱疫情分离鉴定的35株O139霍乱弧菌菌株用水煮法提取DNA,利用聚合酶链反应检测霍乱肠毒素CT基因;取新鲜培养的菌株,制备约4.3个麦氏单位的细菌悬浮液,经裂解、洗涤及限制性内切酶NotI和SfiI酶切后进行脉冲场凝胶电泳(PFGE)分子分型,用凝胶成像仪获取电泳图像,分析DNA片段并用BioNumerics V4.6软件UPGMA方法(复选Dice相关系数)进行聚类分析。结果 35株O139霍乱弧菌ctxA基因PCR扩增产物约为308bp,分离自聚餐食用的凉菜、病人、带菌者及厕所标本(对应病人家)O139霍乱弧菌均为产毒株,经NotI酶切分为9种PFGE带型,SfiI酶切分为6种PFGE带型;A市2株病人分离菌和4株带菌者分离菌PFGE带型为KZGN11O139.CN0077+KZGS12O139.CN0054,B市1株病人分离菌和1株厕所分离菌PFGE带型为KZGN11O139.CN0302+KZGS12O139.CN0058,C市和D市分离菌株优势型为KZGN11O139.CN0276+KZGS12O139.CN0007,包括3株食品分离菌、2株厕所分离菌及16株病人和带菌者分离菌,另有6株PFGE带型呈现多样性。结论 2012年湖北省霍乱疫情特点为散发以及聚餐导致的局限暴发,3起疫情的分离菌株带型各不相同,呈现多样性,其中2起为单一菌型感染,1起为混合菌型感染,传染来源复杂且不明确,提示应加强霍乱的病原学监测。

Molecular typing of Vibrio cholerae O139 strains isolated in Hubei Province in 2012 using pulsed-field gel electrophoresis （PFGE）

Objectives To analyze the molecular typing characteristics and genetic correlation among 35 strains of Vibrio cholerae O139 samples from three cholera epidemics in Hubei Province, and to explore the correlation between the charac-teristics of V. cholerae O139 and cholera outbreaks. Methods The genomic DNA of 35 strains of V. cholerae O139 was extracted. These samples came from three cholera outbreaks that occurred in Hubei Province. The cholera enterotox in （CT） gene was then characterized using a polymerase chain reaction （PCR）. A fresh cultured bacterial suspension of 35 strains of V. cholerae O139 was prepared with a turbidity of about 4.3 McFarland units. After cell lysis, washing, and digestion with the restriction enzymes NotI and Sill, molecular typing was performed using pulsed-field gel electrophoresis （PFGE） . PFGE profiles were subjected to cluster analysis using UPGMA with BioNumerics software V4.6 and the Dice correlation coefficient. Results The 35 V. cholerae O139 strains were samples isolated from the cold dishes in communal meals, patients, carriers, and the toilet （used by the patient）. All 35 were toxin-producing strains. PCR amplification of the ctxA gene yielded a product of about 308 bp. In total, 9 different patterns of PFGE bands were obtained using NotI digestion and 6 were obtained using SfiI digestion. Strains isolated from 2 patients and 4 carriers in City A had PFGE bands in the form of KZGN110139. CN0077 ＋ KZGS120139. CN0054. Strains isolated from 1 patient and 1 toilet sample in City I3 had PFGE bands in the form of KZGN11O139. CN0302 ＋ KZGS120139. CN0058. The predominant strains isolated in the cities of C and D had PFGE bands in the form of KZGN110139. CN0276 ＋ KZGS120139. CN0007. These i solates included 3 isolates from food, 2 from toilets, and 16 from patients and carriers. Moreover, 6 other isolates had di- verse PFGE bands. Conclusion The main epidemiological characteristics of cholera in Hubei Province are sporadic and limited outbreaks as a result of c