2019-2020年枣庄市副溶血性弧菌病人分离株的耐药性、毒力基因及分子分型分析

Analysis of the drug resistance,virulence gene and molecular typing of Vibrio parahaemolyticus identified from patients in Zaozhuang City,2019-2020

目的了解枣庄市2019—2020年副溶血性弧菌(VP)病人分离株的耐药性、毒力基因携带情况和分子分型特征。方法参照GB4789.7—2013《食品安全国家标准食品微生物学检验副溶血性弧菌检验》进行VP菌株的分离和鉴定,采用微量肉汤稀释法测定VP分离株对12种抗生素的耐药性,利用荧光定量聚合酶链式反应(PCR)技术检测VP分离株的毒力基因(tlh、tdh、trh),使用脉冲场凝胶电泳(PFGE)对分离株进行分子分型检测,并进行聚类分析。采用χ2检验对结果进行统计分析。结果678份病人粪便标本中VP的检出率为4.57%,31株VP分离株对头孢唑啉耐药率为58.06%,多重耐药率为12.90%,其毒力基因tlh、tdh、trh的携带率分别为100%、96.78%、3.22%。31株VP分为17个带型,2个带型簇,优势带型簇涵盖了93.55%的菌株。结论枣庄市VP病人分离株对头孢唑啉有较高耐药率,出现了多重耐药株。携带的主要毒力基因为tdh和tlh。PFGE带型集中、同源性高,提示应加强该地区VP的流行病学调查和溯源工作。

Objective The study aims to analyze the drug resistance,virulence genes and molecular typing of Vibrio parahaemolyticus(VP)identified in Zaozhuang from 2019 to 2020.Methods The VP strains were isolated and identified,following the Vibrio parahaemolyticus test protocol in the National Food Safety Standard(GB 4789.7—2013).The broth microdilution method was performed to determine the resistance of VP to 12 antibiotics.Their virulence genes(tlh,tdh,trh)were detected by real-time quantitative polymerase chain reaction(PCR).Pulsed field gel electrophoresis(PFGE)was applied for molecular typing,and the strain clustering was analyzed by BN software.The chi-square test was used to analyze the statistical result(α=0.05).Results The detection rate of VP was 4.57%out of 678 patients’stool samples.Among the 31 identified VP strains,the resistance rate to cefazolin was 58.06%,and the multi-drug resistance rate was 12.90%.The positive rates of virulence genes tlh,tdh,trh were 100%,96.78%,3.22%respectively.The 31 VP strains were further divided into 17 band types and 2 band clusters.The dominant band type cluster covered 93.55%of the strains.Conclusion The VP strains in Zaozhuang from 2019 to 2020 were highly resistant to cefazolin.Some strains showed multiple drug resistance.They mainly carry two virulence genes,tdh and tlh.The PFGE bands were well-clustered,suggesting high homology among these VP strains.Epidemiological investigation and tracing of VP strains should be strengthened in this region.