Li Xi, Quan Jingjing, Ke Huanhuan, Wu Wenhao, Feng Yu, Yu Yunsong, Jiang Yan
Centre of Laboratory Medicine, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, China.
Department of Infectious Diseases, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Front Microbiol. 2021 Aug 16;12:724272. doi: 10.3389/fmicb.2021.724272. eCollection 2021.
Carbapenem-resistant (CRKP) infection poses a great threat to public health worldwide, and KPC-2-producing strains are the main factors responsible for resistance to carbapenems in China. Ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor combination with good activity against KPC-2 carbapenemase and is becoming the most important option for treating KPC-producing CRKP infection. Here, we report the emergence of a novel KPC-2 variant, designated KPC-74, produced by strain KP55, that conferred CZA resistance in a patient after CZA exposure. The novel variant showed a deletion of 6 nucleotides at positions 712-717 compared with , and this deletion resulted in the consequent deletion of glycine and valine at positions 239 and 240. Antimicrobial susceptibility testing showed that KP55 presents multidrug resistance, including resistance to CZA and ertapenem, but is susceptible to imipenem, meropenem, and colistin. The gene was located on a plasmid, as determined by S1-nuclease pulsed-field gel electrophoresis followed by southern blotting, and confirmed to be 133,766 bp in length by whole-genome sequencing on both the Illumina and MinION platforms. The CZA resistance phenotype of the novel KPC variant was confirmed by both transformation of the -harboring plasmid and a gene cloning assay, showing a 64-fold higher CZA minimum inhibitory concentration (MIC) than the recipient strains. The G239_V240del observed in KPC-74 was outside the omega-loop region but was still close to the active site Ser70 and omega-loop in the protein tertiary structure. The enzyme kinetic parameters and IC values further indicated that the hydrolytic activity of the KPC-74 enzyme against ceftazidime was potentiated twofold and that the affinity between KPC-74 and avibactam was alleviated 17-fold compared with that of the KPC-2 allele. This CZA resistance mediated by KPC-74 could be selected after CZA therapy and evolved to be more diverse and heterogeneous. Surveillance of CZA resistance is urgently needed in clinical settings.
耐碳青霉烯类肺炎克雷伯菌（CRKP）感染对全球公共卫生构成巨大威胁，产KPC-2菌株是中国碳青霉烯类耐药的主要因素。头孢他啶/阿维巴坦（CZA）是一种新型β-内酰胺/β-内酰胺酶抑制剂组合，对KPC-2碳青霉烯酶具有良好活性，正成为治疗产KPC的CRKP感染的最重要选择。在此，我们报告了由菌株KP55产生的一种新型KPC-2变体，命名为KPC-74，该变体在患者接受CZA治疗后导致对CZA耐药。与KPC-2相比，该新型变体在712-717位缺失6个核苷酸，这种缺失导致239和240位的甘氨酸和缬氨酸缺失。药敏试验表明，KP55呈现多重耐药，包括对CZA和厄他培南耐药，但对亚胺培南、美罗培南和黏菌素敏感。通过S1核酸酶脉冲场凝胶电泳和Southern印迹分析确定，KPC基因位于质粒上，通过Illumina和MinION平台的全基因组测序证实其长度为133,766 bp。通过携带KPC-74质粒的转化和KPC基因克隆试验证实了新型KPC变体的CZA耐药表型，其CZA最低抑菌浓度（MIC）比受体菌株高64倍。在KPC-74中观察到的G239_V240del位于ω-环区域之外，但仍靠近蛋白质三级结构中的活性位点Ser70和ω-环。酶动力学参数和IC值进一步表明，与KPC-2等位基因相比，KPC-74酶对头孢他啶的水解活性增强了两倍，而KPC-74与阿维巴坦之间的亲和力降低了17倍。这种由KPC-74介导的CZA耐药可在CZA治疗后被选择出来，并演变得更加多样和异质。临床环境中迫切需要对CZA耐药进行监测。
