南美白对虾养殖水体中霍乱弧菌的毒力、耐药及重金属抗性研究

Virulence genes and susceptibility to antimicrobial agents and heavy metals of Vibrio cholerae isolated from aquaculture water farming Litopenaeus vannamei

摘要:

旨在分析南美白对虾养殖水体中霍乱弧菌(Vibrio cholerae)的毒力、抗菌素耐药性以及重金属抗性特性,为致病性霍乱弧菌流行预警的研究提供数据支撑。利用聚合酶链反应技术检测了84株霍乱弧菌分离菌株的毒力以及整合接合元件(ICEs)相关基因;采用美国临床与实验室标准研究所(CLSI)标准Kirby-Bauer纸片扩散法以及最小抑菌浓度法测定了受试菌株的抗菌素和重金属抗性;运用脉冲场凝胶电泳(PFGE)技术对部分受试菌株进行了分子分型。结果显示:受试霍乱弧菌菌株均不携带致病性肠毒素基因ctxAB以及毒素相关基因sto、tcpA、ace、zot;含有毒素表达调控蛋白基因toxR,其中19.05%菌株还含有RTX家族毒素基因rtxA;携带SXT-R391家族ICEs保守功能模块部分相关基因(int、traI、traC、setR、attR)。受试霍乱弧菌菌株对六大类十种抗菌素的耐药性存在显著差异,其中对氨苄西林的耐药率最高为13.10%,其次是庆大霉素(2.38%);利福平、链霉素、氨苄西林、卡那霉素、壮观霉素的中介耐药率为25.00%～1.19%;对四环素、氯霉素、甲氧胺苄嘧啶、复方新诺明均敏感。此外,30%的受试霍乱弧菌菌株对重金属铅、锌具有高度抗性,而对铬、镉、铜、镍、汞、钡敏感;PFGE分子分型可分为5种NotI-PFGE型13种亚型,相似性值为56%～100%。本研究结果为进一步探讨致病性霍乱弧菌的进化起源奠定了基础。

Abstract:

The aim of this study was to investigate virulence genes and susceptibility to antimicrobial agents and heavy metals of Vibrio cholerae strains isolated from aquaculture water farming Litopenaeus vannamei, in order to provide scientific data support for early warning of food-borne and life-threatening cholera disease caused by V. cholerae. The genes involved in pathogenicity and integrative conjugative elements (ICEs) were detected by polymerase chain reaction (PCR). The virulence-related genes were absent in all V. cholerae isolates, including ctxAB, sto, tcpA, ace and zot. However, all the isolates were tested positive for a toxR gene, 19.05% of which also contained a rtxA gene. Some of the functional genes (int, traI, traC, setR, attR) involved in conserved module structures of SXT-R391 family ICEs were detected positive, suggesting no possible transfer activity of the ICEs detected in the isolates. Antimicrobial susceptibility of V. cholerae strains were examined by using standard Kirby Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute (USA,2010 Edition),showing distinct susceptibility to ten antimicrobial agents belonging to six drug classes tested.Total 13.10% and 2.38% of the V. cholerae strains were resistant to ampicillin and gentamycin, respectively. About 25.00%-1.19% of the isolates also displayed intermediate resistance to rifampicin, streptomycin, ampicillin, kanamycin and spectinomycin. In addition, a narrow heavy metal resistance profile was detected in the isolates tested, showing strong resistance to Pb and Zn. By pulsed-field gel electrophoresis (PFGE) genetyping, these V. cholerae isolates were clustered into five NotI-PFGE types and thirteen subtypes with 56%-100% identity. The data in this study constituted the first evidence for molecular and resistance phenotype characterization of the V. cholerae isolates of aquaculture environmental origin.