克雷伯氏菌属(Klebsiella)细菌比较基因组学分析揭示多复制子抗性质粒介导广泛的抗性基因传播

目的] 研究克雷伯氏菌与多复制子抗性质粒间的关系，分析细菌携带多复制子质粒对抗生素环境的响应机制。方法] 以2018-2020年分离的56株不同来源克雷伯氏菌（Klebsiella sp.）分离株为研究对象，利用微量肉汤稀释法评估其多重耐药表型，对分离菌株进行全基因组测序（WGS），通过细菌全基因组关联分析（BGWAS）技术和比较基因组学方法深入解析多复制子抗性质粒形成的机制。结果] 耐药表型分析发现野生动物来源的菌株具有更广的耐药谱系，总体Klebsiella sp.对氨苄西林表现出很高的耐药率（80.36%），尤其是马来穿山甲来源菌株对头孢类抗生素高度耐受，同时对氯霉素、左氧氟沙星和复方新诺明等药物耐受，基因组分析发现这些菌株携带了抗性质粒和更多的抗生素抗性基因。进一步对69个质粒序列分析，发现有28个质粒为多复制子质粒，主要携带bla_CTX-M-15、bla_CTX-M-14、bla_CTX-M-55、bla_OXA-1和bla_TEM-1等β-内酰胺酶基因。细菌携带质粒类型分析认为Klebsiella pneumoniae可能是多复制子质粒的重要宿主，质粒骨架与结构分析发现多复制子质粒多由2个或2个以上单个质粒融合而成，携带此类质粒的菌株不仅获得了更广的耐药表型，而且在全球传播扩散分布逐年增加，因此产生对抗生素环境更强的适应性。结论] 多重耐药性细菌呈现的表型与携带的多复制子质粒有关，相比较下多复制子质粒比非多复制子质粒有更强的抗性基因携带能力，或许是细菌在强大的抗生素压力下产生的重要响应机制。本研究对于未来探索细菌抗性基因的传播扩散机制具有重要意义。

Comparative genomic analysis of Klebsiella reveals extensive transmission of resistance genes mediated by multireplicon resistance plasmids

Objective] To study the relationship between Klebsiella and multireplicon resistance plasmids, and to analyze the mechanism of multireplicon plasmid carrier in response to antibiotic pressure.Methods] We collected fifty-six isolates of Klebsiella from different wild animals and environments during 2018 to 2020. We analyzed the multidrug resistance (MDR) phenotype by microbroth dilution method, sequenced and analyzed the representative isolates by bacterial genome-wide association study (BGWAS).Results] Our results revealed that the isolates from non-human sources showed a more extensive drug resistance, and especially stronger resistance to ampicillin (reach 80.36%). In particular, the isolates from Malay pangolin were highly resistant to cephalosporins, chloramphenicol, levofloxacin and sulfamethoxazole. Genomic analysis showed that the resistance plasmids carried by these isolates were related to carry many antibiotic resistance genes. Further analysis of 69 plasmids demonstrated that 28 plasmids were multireplicon plasmids, mainly carrying beta-lactamase genes such as bla_CTX-M-15, bla_CTX-M-14, bla_CTX-M-55, bla_OXA-1 and bla_TEM-1. According to the analysis of plasmids carried by different isolates, Klebsiella pneumoniae may be an important host of multireplicon plasmid. Plasmid skeleton and structure analysis showed that multireplicon plasmid was formed by the fusion of two or more single plasmids, which made the isolates strong adaptability to the antibiotic environment, and increased the ability of drug-resistant isolates to spread around the world year by year.Conclusion] The phenotype of multidrug-resistant bacteria is related to the carrying multireplicon plasmid.Multireplicon plasmid has a stronger ability to carry resistance genes than non-multireplicon plasmid, which may be an important mechanism for bacteria response to adapt to stronger antibiotic pressure environment. This phenomenon is of great significance for exploring the transmission and diffusion mechanism of bacterial resistance genes in the future.