Zhou Peiyao, Gao Haojin, Li Meilan, Wu Chunyang, Han Weihua, Wan Cailing, Shen Li, Yuan Xinru, Shi Junhong, Huang Yu, Lv Jianbo, Zhou Ying, Yu Fangyou
Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
Department of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
Int J Antimicrob Agents. 2025 Mar;65(3):107411. doi: 10.1016/j.ijantimicag.2024.107411. Epub 2024 Dec 19.
With the widespread clinical use of ceftazidime-avibactam (CZA), reports of resistance have increased continuously, posing immense threats to public health worldwide. In this study, we explored the underlying mechanisms leading to the development of CZA resistance in an ST11-KL64 hypervirulent Klebsiella pneumoniae CRE146 that harbored the bla gene.
Twelve carbapenem-resistant Klebsiella pneumoniae (CRKP) strains were isolated from the same patient, including K. pneumoniae CRE146. Whole-genome sequencing (WGS), phylogenetic analysis, bla gene cloning and pACYC-KPC construction assays were conducted to further explore the molecular mechanisms of CZA resistance. Quantitative siderophore production assay, string test, capsule quantification and Galleria mellonella in vivo infection model were applied to verify the level of pathogenicity of K. pneumoniae CRE146.
This strain carried key virulence factors, iutA-iucABCD operon and rmpA gene. Compared to the wild-type KPC-2 carbapenemase, the novel KPC-228 enzyme exhibited a deletion of four amino acids in the Ω-loop (del_167-170_ELNS). In addition, the emergence of CZA resistance appeared to be associated with drug exposure, and we observed the in vivo evolution of wild-type KPC-2 to KPC-228 and then the reversion to its original wild-type KPC-2. The bla gene was located within the double IS26 flanking the ISKpn6-bla-ISKpn27 core structure and carried on an IncFII/IncR-type plasmid. Notably, CRE146 exhibited high-level resistance to CZA (64/4 mg/L) but increased susceptibility to meropenem (1 mg/L) and imipenem (0.5 mg/L) respectively. PACYC-KPC plasmids were constructed and expressed in K. pneumoniae ATCC13883. Compared to K. pneumoniae ATCC13883 harboring bla, K. pneumoniae ATCC13883 harboring bla exhibited a high-level resistance to CZA (32/4 mg/L) and increased susceptibility to meropenem (1 mg/L) and imipenem (0.5 mg/L). Interestingly, K. pneumoniae ATCC13883 harboring bla showed a significant decrease in their resistance to all β-lactamases tested except CZA and ceftazidime.
In conclusion, we reported a novel KPC variant, KPC-228, in a clinical ST11-KL64 hypervirulent K. pneumoniae strain, which conferred CZA resistance, possibly through enhancing ceftazidime affinity and reducing avibactam binding. The bla can mutate back to bla under carbapenem pressure, which was very detrimental to clinical treatment. This strain carried both resistance and virulence genes, posing a major challenge in clinical management.
随着头孢他啶-阿维巴坦（CZA）在临床上的广泛应用，耐药报告不断增加，对全球公共卫生构成巨大威胁。在本研究中，我们探究了一株携带bla基因的ST11-KL64高毒力肺炎克雷伯菌CRE146中导致CZA耐药的潜在机制。
从同一患者分离出12株耐碳青霉烯类肺炎克雷伯菌（CRKP）菌株，包括肺炎克雷伯菌CRE146。进行全基因组测序（WGS）、系统发育分析、bla基因克隆及pACYC-KPC构建试验，以进一步探究CZA耐药的分子机制。应用定量铁载体产生试验、拉丝试验、荚膜定量及黄粉虫体内感染模型，验证肺炎克雷伯菌CRE146的致病水平。
该菌株携带关键毒力因子iutA-iucABCD操纵子和rmpA基因。与野生型KPC-2碳青霉烯酶相比，新型KPC-228酶在Ω环中出现四个氨基酸缺失（del_167 - 170_ELNS）。此外，CZA耐药的出现似乎与药物暴露有关，我们观察到野生型KPC-2在体内演变为KPC-228，随后又恢复为原始野生型KPC-2。bla基因位于双IS26侧翼的ISKpn6-bla-ISKpn27核心结构内，并携带在IncFII/IncR型质粒上。值得注意的是，CRE146对CZA表现出高水平耐药（64/4 mg/L），但对美罗培南（1 mg/L）和亚胺培南（0.5 mg/L）的敏感性分别增加。构建了PACYC-KPC质粒并在肺炎克雷伯菌ATCC13883中表达。与携带bla的肺炎克雷伯菌ATCC13883相比，携带bla的肺炎克雷伯菌ATCC13883对CZA表现出高水平耐药（32/4 mg/L），对美罗培南（1 mg/L）和亚胺培南（0.5 mg/L）的敏感性增加。有趣的是，携带bla的肺炎克雷伯菌ATCC13883对除CZA和头孢他啶外的所有测试β-内酰胺酶的耐药性显著降低。
总之，我们在一株临床ST11-KL64高毒力肺炎克雷伯菌菌株中报道了一种新型KPC变体KPC-228，其赋予了CZA耐药性，可能是通过增强头孢他啶亲和力和减少阿维巴坦结合来实现的。在碳青霉烯压力下，bla可突变为bla，这对临床治疗非常不利。该菌株同时携带耐药和毒力基因，给临床管理带来了重大挑战。
