辽宁省溶藻弧菌REP-PCR和PFGE分子分型、耐药谱的研究

REP-PCR, PFGE molecular typing and drug resistance spectrum research for Vibrio alginolyticus

目的利用PFGE和REP-PCR两种分型方法对溶藻弧菌进行分子分型和亲缘关系的研究;并将两种分型方法进行对比,并结合对抗生素耐药试验结果对其关联性进行初步探讨。方法应用脉冲场凝胶电泳(PFGE)方法对辽宁省2017年食品中分离的39株溶藻弧菌进行分型,应用BN(7.6版本)软件分析同源性。以基因外重复回文序列(REP)为引物,对40株溶藻弧菌DNA进行扩增,得到DNA指纹图谱,并利用SPSS 13.0统计软件对DNA扩增图谱进行聚类分析,同时将两种聚类分析结果进行比较,并用肉汤稀释法测定对15种抗生素的耐药性。结果 PFGE和REP-PCR两种方法的聚类分析结果均可分出两种优势带型A型和B型,且包含的菌株一致。PFGE分型方法在一些菌株中出现的DNA条带更多更复杂一些,分辨力指数(DI)为0.974,REP-PCR分型方法的DI为0.936。溶藻弧菌对头孢唑啉耐药率最高达57.5%,其次是氨苄西林(20.0%)和红霉素(12.5%),三重耐药菌比例达12.5%。结论 PFGE和REP-PCR两种方法均可以对溶藻弧菌进行分型,且均具有较好的分型能力,分辨力和再现性都很好,但PFGE分型方法的分辨力更优于REP-PCR分子分型。耐药菌株间带型具有高度同源性,优势带型菌株易耐药。综上所述,PFGE和REP-PCR两种分子分型方法对于评估溶藻弧菌流行及分布规律均是有用的,并且分型结果是一致的,都可以对菌株间亲缘关系进行有效溯源,因此在溶藻弧菌所致疾病爆发流行时,在普遍缺乏昂贵的PFGE专业设备和昂贵的配套BN软件以及专业操作人员的实验室,可以应用REP-PCR方法和SPSS统计软件代替PFGE方法和BN软件对菌株进行快速有效溯源。

Objective To compare PFGE and REP-PCR methods for molecular typing of Vibrio alginolyticus,and explore their correlation.Drug sensitivity were detected and discussed.Methods Thirty-nine strains of Vibrio alginolyticus detected in Liaoning in 2017 were molecularly typed by using PFGE method.BioNumerics software Version 7.6 was used to analyze the homology.REP sequences were used as the PCR primers to amplify the DNAs of the Vibrio alginolyticus strains.Clustering analysis was performed with SPSS 13.0 software.The dendrograms of the two methods were compared,and the relevance between the two methods were assessed.Drug sensitivity to 15 antibiotics were determined by broth dilution test.Results Both PFGE and REP-PCR fingerprints grouped the Vibrio alginolyticus strains into predominant patterns A and B which contained the same strains.PFGE resulted in more bands.The DI of PFGE was0.974 while that of REP-PCR was 0.936.The resistance rates to CFZ were the highest（57.5%）,followed by AMP（20.0%）and ERY（12.5%）,three-drug-fast bacteria was 12.5%.Conclusion Vibrio alginolyticus can be typed by both PFGE and REP-PCR methods with good discriminability and reproducibility,although the discriminability of PFGE is better.The drug resistant strains are high degree of homology.Strains of predominant patterns resist to drug easily.Since both the two methods can be used to assess the prevalence and distribution of Vibrio alginolyticus and effectively trace the genetic relationship between Vibrio alginolyticus strains,REP-PCR combined with SPSS 13.0 software is an alternative method when Vibrio alginolyticus-associated disease breaks out and there are no sufficient PFGE equipments and professional personnel.