野生鸟类分离的多重耐药肺炎克雷伯氏菌(Klebsiella pneumoniae)耐药基因分子进化特征研究

Molecular evolutionary characterization of multidrug-resistant Klebsiella pneumoniae genes isolated from wild birds

摘要:

【目的】调查野生鸟类携带菌的耐药状况，探索其在细菌耐药性传播过程中的作用。【方法】从野生鸟类石鸡、绯胸鹦鹉、太阳锥尾鹦鹉和黑领椋鸟的新鲜粪便分离4株Klebsiella pneumoniae，采用微量肉汤稀释法评估其多重耐药表型，并利用全基因组测序技术和细菌全因组关联分析、比较基因组学方法对分离株进行分子溯源，系统解析其携带的多重耐药质粒或基因与其宿主、同源质粒间的关联。【结果】4株肺炎克雷伯菌的耐药谱各不相同，来自石鸡样本的分离株S90-2对9种药物耐受，绯胸鹦鹉样本分离株S141对3种药物耐受，太阳锥尾鹦鹉分离株M911-1仅耐受氨苄西林，黑领椋鸟的样本分离株S130-1对所使用的14种药物完全敏感。S90-2属于ST629型，携带bla_CTX-M-14、fosA6、aac(3)-Iid和bla_SHV-11为主的30个耐药基因和携带1个耐药性质粒pS90-2.3 (IncR型)。S141属于ST1662型，携带fosA5、bla_SHV-217等27个耐药基因，1个质粒pS141.1 [IncFIB(K)(pCAV1099-114)/repB型]仅携带耐药基因adeF。M911-1为新ST类型，携带bla_SHV-1、fosA6等共计27个耐药基因，其质粒pM911-1.1携带了3个耐药基因。S130-1属于ST3753型，携带bla_SHV-11、fosA6等27个耐药基因，pS130-1 [IncFIB(K)型]则仅携带一个耐药基因tet(A)。质粒比对表明，质粒pS90-2.3携带的耐药基因片段源自不同的肠杆菌科菌株染色体或质粒。pS90-2.3的同源质粒主要来自人类宿主菌，且主要在中国分布，这些质粒主要细菌宿主为K. pneumoniae和Escherichia coli，且ST11型K. pneumoniae分离株为重要宿主菌。【结论】本研究中来自野生鸟类的多重耐药K. pneumoniae，其耐药基因主要来自质粒，质粒耐药基因主要由转座子、插入序列、整合子和前噬菌体等可移动元件介导，这些多重耐药质粒与人类的宿主菌密切相关。

Abstract:

[Objective] To investigate the drug resistance status of wild birds carrying bacteria and to explore their role in the transmission of bacterial drug resistance. [Methods] Four Klebsiella pneumoniae were isolated from fresh feces of captured Alectoris chukar, Psittacula alexandri, Aratinga solstitialis and Sturnus nigricollis, and were assessed for multidrug resistance phenotypes by micro-broth dilution method. Bacterial genome-wide association analysis and comparative genomics were used to trace the isolates and systematically analyze the association between the multidrug resistance plasmids/genes and their hosts/homologous plasmids. [Results] Four strains of K. pneumoniae showed different drug resistance phenotypes. Specifically, S90-2 from A. chukar was resistant to nine drugs including ampicillin, cefuroxime, cefazolin, ceftriaxone and cefepime; S141 from P. alexandri was resistant to ampicillin, cefuroxime and cefazolin; M911-1 from A. solstitialis was resistant to ampicillin only; S130-1 from S. nigricollis was sensitive to all of the 14 drugs. S90-2 belonged to ST629 type and carried 30 resistance genes including bla_CTX-M-14, fosA6, aac(3)-Iid and bla_SHV-11, and its plasmid pS90-2.3 (IncR) carried resistance genes of mphA, dfrA12, aadA2, qacEdelta1, sul1, tet(A), aph(3')-Ia, sul2 and aph(3')-Ib. S141 belonged to ST1662 type and carried 27 resistance genes including fosA5 and bla_SHV-217, and only plasmid pS141.1 [IncFIB(K)(pCAV1099-114)/repB] carried one resistance gene adeF. M911-1 was a new ST type, carrying 27 resistance genes such as bla_SHV-1 and fosA6, and its plasmid pM911-1.1 (novel) carried three resistance genes qnrS1, bla_LAP-2 and tet(A). S130-1 belonged to ST3753 type, carrying 27 resistance genes such as bla_SHV-11 and fosA6, and its plasmid pS130-1 [IncFIB(K)] carried only one resistance gene tet(A). The plasmids pM911-1.1 and pS90-2.3 failed to perform conjugative transfer, but their resistance gene fragments were derived from multiple homologous chromosomes or plasmids of Enterobacteriaceae strains, and the formation of resistance gene fragments (MDR region) involved interactions between multiple mobile element genes, resulting in a complex and diverse structure of resistance plasmid. The homologous plasmids related to pM911-1.1 and pS90-2.3 were mainly from human bacteria hosts in China, such as K. pneumoniae and Escherichia coli, and the K. pneumoniae ST11 was a major host for the above drug-resistant homologous plasmids. [Conclusion] The multidrug-resistant K. pneumoniae from wild birds in this study had resistance genes mainly from plasmids, which were mediated by transposons, insertion sequences, integrons and prophage and other mobile elements, and these multidrug-resistant plasmids were closely related to the human host bacteria.