鳖源铜绿假单胞菌的分离鉴定及多位点序列与全基因组分析

Identification, multilocus sequence typing and whole-genome analysis of Pseudomonas aeruginosa strains isolated from diseased Trionyx sinensis

摘要:

【目的】查明引起湖北仙桃某中华鳖养殖场中华鳖发病死亡的病原及其特征。【方法】本研究分离患病中华鳖的病原，并结合形态特征、生理生化试验、16S rRNA基因鉴定，鉴定分离菌株；通过人工回归感染试验、药敏试验、全基因组测序与分析对分离菌株的致病性和耐药性进行研究；通过多位点序列分型(multilocus sequence type，MLST)分析，对分离菌株的流行情况进行探究。【结果】从患病中华鳖肝脏等部分分离到3株优势菌株HX8、FG10和GC20，16S rRNA基因同源性和生化特征分析鉴定为铜绿假单胞菌(Pseudomonasaeruginosa)。回归感染试验证实该菌株是引起本次中华鳖患病的病原菌。3株分离株的药敏实验结果基本一致，均对诺氟沙星、恩诺沙星等8种抗生素敏感，对氟苯尼考、多西环素、磺胺异恶唑等6种抗生素耐药。MLST鉴定表明， 3株分离株均属于序列型(sequence type，ST)252型，eBURST分析进一步显示ST252型与一些ST共同构成了克隆复合体(clonal complexes，CC) CC252，且ST252是CC252的原始序列型(founder ST)。全基因组测序结果显示，FG10的全基因组大小为5.65 Mb，GC含量为65.3%，共预测到5 956个编码序列(CDS)，GenBank登录号为JAJGXC000000000。通过与毒力因子数据库(the virulence factor database，VFDB)比对，预测到873个毒力相关因子，主要与黏附、分泌系统以及毒素等相关。通过与耐药基因数据库(the comprehensive antibiotic resistance database，CARD)比对，结果显示FG10中含有喹诺酮类、碳青霉烯类、肽类等抗生素的耐药相关基因。【结论】本研究分离获得了中华鳖的病原细菌铜绿假单胞菌，MLST分析推测了其流行情况，基因组数据揭示了其毒力及耐药基因，为防控水产养殖中铜绿假单胞菌感染提供了一定的参考依据。

关键词:中华鳖;铜绿假单胞菌;分离鉴定;多位点序列分型;全基因组;毒力及耐药

Abstract:

[Objective] This study aims to determine and characterize the pathogen of the diseased Trionyx sinensis in a farm in Xiantao, Hubei Province. [Methods] The suspicious pathogenic bacteria were isolated from the diseased T.sinensis and identified based on the morphological, physiological, and biochemical characteristics and the phylogenetic tree constructed with the 16S rRNA gene sequence. The artificial infection tests, drug sensitivity tests, multilocus sequence typing (MLST), eBURST analysis, and whole-genome sequencing were then carried out for the isolates. [Results] Three predominant strains, HX8, FG10, and GC20, were isolated from the diseased T.sinensis and all identified as Pseudomonas aeruginosa. Artificial infection tests confirmed that the isolates were the pathogen causing the disease of T.sinensis. Drug sensitivity tests demonstrated that the three isolates were sensitive to eight antibiotics (such as enrofloxacin and norfloxacin) and resistant to six antibiotics (such as florfenicol, doxycycline, and sulfagan). According to the results of MLST, the three isolates all belonged to sequence type 252 (ST252). The further eBURST analysis showed that ST252 formed a clonal complex CC252, of which ST252 was the founder ST. The whole genome of strain FG10 had a size of 5.65 Mb, the average G+C content of 65.3%, and 5 956 coding sequences. The whole genome has been deposited at GenBank under the accession number JAJGXC000000000. In comparison with the virulence factor database (VFDB), 873 virulence-related genes were predicted, which were mainly associated with adhesion, secretion systems, and toxin. In comparison with the comprehensive antibiotic resistance database (CARD), some drug (such as fluoroquinolone, carbapenem, and peptide) resistance related-genes were predicted.[Conclusion] We isolated and identified the pathogens of the diseased T.sinensis and preliminarily analyzed the prevalence and predicted the genes related to the virulence and drug resistance of P.aeruginosa, which provided a basis for the prevention and control of infections caused by P.aeruginosa in aquaculture.

Key words:Trionyx sinensis;Pseudomonas aeruginosa;isolation and identification;multilocus sequence typing;whole genome;virulence and drug resistance