江苏水产养殖区拟态弧菌种群结构及遗传多样性

Population structure and genetic diversity of Vibrio mimicus isolated from aquaculture area in Jiangsu, China

摘要:

【背景】拟态弧菌(Vibrio mimicus)是一种常见的革兰氏阴性病原菌，广泛分布于水环境和水生动物体内，可导致多种水产动物和人类感染。多位点序列分型(multilocus sequence typing, MLST)已被应用于多种病原菌的分子分型，其通过分析不同菌株之间的遗传关系，监测细菌传播的时间和地理分布，确定感染和传播途径，但目前未见有关拟态弧菌MLST的报道。【目的】开发一种基于MLST的拟态弧菌分型方法，并用于江苏水产养殖区拟态弧菌的种群结构和遗传进化分析，为拟态弧菌感染所引起的疾病防治提供理论基础。【方法】选择拟态弧菌的7个管家基因dnaE、gyrB、mdh、recA、rpoD、pntA和pyrH作为靶点，对江苏水产养殖区分离的155株拟态弧菌进行PCR扩增和测序。将测序结果分配等位基因，制作等位基因谱，分配不同的序列类型(sequence type, ST)，利用软件goeBURST-1.2.1和MEGA-X对分配的ST型进行克隆复合体和遗传进化树聚类分析；此外，利用Kirby-Bauer圆盘扩散法测试155株拟态弧菌的药敏特性。【结果】155株拟态弧菌被分为56个STs，其中ST11占比最高；在双位点变异(double locus variants, DLV)水平分析发现56个STs分为3个克隆复合体和3个单体；系统发育树显示，56个STs被分为3个集群(cluster I、cluster II、cluster III)。药敏结果显示，155株拟态弧菌对红霉素类抗生素的耐药性最高(88.39%, 137/155)，对氯霉素类抗生素敏感性最高(91.61%, 142/155)。【结论】本研究建立的MLST方法具有良好的分辨率，可作为拟态弧菌系统发育和未来流行病学调查有用的分子分型工具。根据抗生素耐药谱结果，提示在养殖过程中可选用氟苯尼考等国家批准使用的专用抗菌药对拟态弧菌进行防治。

Abstract:

[Background] Vibrio mimicus, a common Gram-negative pathogen, is widely distributed in the aquatic environment and often involved in infectious diseases of aquatic animals and humans. Multilocus sequence typing (MLST) has been applied to the molecular typing of many pathogens, which allows for investigations into genetic relatedness between different strains, therefore provides insights into bacterial evolution and epidemiology. However, no report is available on the MLST of V. mimicus. [Objective] To develop a powerful MLST typing method for V. mimicus and then apply it to analyze the population structure and genetic evolution of different V. mimicus strains, and eventually provide theoretical references for the prevention and control of diseases caused by V. mimicus infection. [Methods] Seven housekeeping genes (dnaE, gyrB, mdh, recA, rpoD, pntA, and pyrH) of V. mimicus, were selected as target genes for MLST, and their conserved fragments were obtained by polymerase chain reaction (PCR) from a total of 155 V. mimicus strains isolated from the aquaculture area in Jiangsu and sequenced. All unique DNA sequences were assigned alleles, made allelic profiles, and assigned different sequence type (ST). The clonal complex and genetic evolution tree clustering of the assigned ST types were analyzed using goeBURST-1.2.1 and MEGA-X. In addition, antibiotic sensitivity of the 155 V. mimicus strains was tested using the Kirby-Bauer disk diffusion method. [Results] The 155 V. mimicus strains were divided into 56 STs, among them ST11 had the highest proportion. At the level of double locus variants (DLV), 56 STs were further divided into three clonal complexes and three monomers. Phylogenetic tree showed that 56 STs fell into three clusters (cluster I, cluster II, and cluster III). The Kirby-Bauer disk diffusion assay showed that the 155 V. mimicus strains exhibited the highest resistance to erythromycin (88.39%, 137/155) and the highest sensitivity to chloramphenicol (91.61%, 142/155). [Conclusion] The MLST method established in this study displays good resolution and can be used as a molecular typing tool for phylogeny and future epidemiological investigation of V. mimicus. Antibiotic resistance profiles suggest that florfenicol, one of antibacterial agents that approved for application, can be used for the treatment of V. mimicus infections in the aquaculture process.