Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaIMP-4 induce resistance transmission and fitness variation

To investigate the epidemiology of ST20 carbapenem-resistant Klebsiella pneumoniae (CRKP) in China, and further explore the genomic characteristics of blaIMP-4 and blaNDM-1 coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-β-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that blaIMP-4 was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of blaIMP-4 in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that blaIMP-4 on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS26 and ltrA, respectively, and the circular transposon unit was related to cointegration, however, blaIMP-4 in different locations did not affect the gene stability. The blaNDM-1-harbouring plasmid contributed to the increased resistance to β-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the K. pneumoniae ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.

为探究中国境内ST20型碳青霉烯类耐药肺炎克雷伯菌（carbapenem-resistant Klebsiella pneumoniae, CRKP）的流行现状，并进一步解析同时携带blaIMP-4与blaNDM-1菌株的基因组特征，以及质粒在抗菌耐药性与适应性形成中的作用。本研究共收集全国范围内的7株ST20型CRKP，开展抗菌药物敏感性试验；通过全基因组测序鉴定抗菌耐药基因、毒力基因及质粒复制子，并采用核心基因组多位点序列分型评估菌株的克隆相关性。此外，本研究共检出4株携带双重金属β-内酰胺酶（metallo-β-lactamases, MBL）的菌株，通过Southern印迹杂交检测耐药基因的定位，质粒定位分析显示，blaIMP-4可分别存在于独立质粒、自主接合型融合质粒或细菌染色体上。对上述菌株开展长读长测序，通过基因组比对明确blaIMP-4的不同定位形式，并采用反向PCR检测转座子单元。后续研究发现，融合质粒与细菌染色体上的blaIMP-4分别由IS26与ltrA介导的完整质粒重组形成，环状转座子单元与共整合事件相关；但不同定位的blaIMP-4均不会影响该基因的稳定性。通过质粒消除试验证实，携带blaNDM-1的质粒可提升菌株对β-内酰胺类抗菌药物的耐药性，并缩短菌株的生长迟滞期。综上，肺炎克雷伯菌ST20克隆株属于高风险耐药克隆株。临床在使用头孢他啶/阿维巴坦时，需重点关注金属β-内酰胺酶阳性菌株，尤其是携带双重金属β-内酰胺酶的菌株。