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

【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、16SrRNA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GC20,16SrRNA基因同源性和生化特征分析鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。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(sequencetype,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)...

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

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.