Department of Molecular Medicine, Sapienza University of Romegrid.7841.a, Rome, Italy.
Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy.
Antimicrob Agents Chemother. 2021 Sep 17;65(10):e0057421. doi: 10.1128/AAC.00574-21. Epub 2021 Aug 2.
From January 2019 to April 2020, 32 KPC-producing, ceftazidime-avibactam (CZA)-resistant Klebsiella pneumoniae strains were isolated in a university hospital in Rome, Italy. These strains belonged to the sequence type 512 (ST512), ST101, and ST307 high-risk clones. Nine different CZA-resistant KPC-3 protein variants were identified, five of them never previously reported (KPC-66 to KPC-70). Among the nine, KPC-31, KPC-39, KPC-49, KPC-66, KP-68, KPC-69, and KPC-70 showed amino acid substitutions, insertions, and deletions in the Ω loop of the protein. KPC-29 has a duplication, while the novel KPC-67 has a triplication, of the KDD triplet in the 270-loop, a secondary loop of the KPC-3 protein. Genomics performed on contemporary resistant and susceptible clones underlined that these novel mutations emerged in genes located on conserved plasmids: in ST512, all mutant genes were located in pKpQIL plasmids, while the three novel mutants identified in ST101 were on FIIk-FIA(HI1)-R plasmids. Selection also promoted multiplication of the carbapenemase gene copy number by transposition, recombination, and fusion of resident plasmids. When expressed in Escherichia coli recipient cells cloned in the high-copy-number pTOPO vector, the Ω loop mutated variants showed the CZA-resistant phenotype associated with susceptibility to carbapenems, while KPC variants with insertions in the 270-loop showed residual activity on carbapenems. The investigation of CZA resistance mechanisms offered the unique opportunity to study vertical, horizontal, and oblique evolutionary trajectories of K. pneumoniae high-risk clones.
从 2019 年 1 月到 2020 年 4 月，意大利罗马一家大学医院分离出 32 株产 KPC、头孢他啶-阿维巴坦（CZA）耐药肺炎克雷伯菌。这些菌株属于序列型 512（ST512）、ST101 和 ST307 高风险克隆。鉴定出 9 种不同的 CZA 耐药 KPC-3 蛋白变体，其中 5 种从未报道过（KPC-66 至 KPC-70）。在这 9 种变体中，KPC-31、KPC-39、KPC-49、KPC-66、KP-68、KPC-69 和 KPC-70 蛋白的 Ω 环中存在氨基酸取代、插入和缺失。KPC-29 有一个 KDD 三联体的重复，而新型 KPC-67 则有一个 270 环内 KPC-3 蛋白的二级环的三倍重复。对当代耐药和敏感克隆进行基因组分析表明，这些新的突变出现在位于保守质粒上的 基因中：在 ST512 中，所有 突变基因都位于 pKpQIL 质粒上，而在 ST101 中鉴定的 3 种新型 突变基因位于 FIIk-FIA(HI1)-R 质粒上。选择还通过转座、重组和驻留质粒的融合促进了碳青霉烯酶基因拷贝数的倍增。当在高拷贝数 pTOPO 载体中克隆的大肠杆菌受体细胞中表达时，Ω 环突变变体表现出与对碳青霉烯类药物敏感相关的 CZA 耐药表型，而 270 环插入的 KPC 变体对碳青霉烯类药物仍有残留活性。对 CZA 耐药机制的研究提供了一个独特的机会，可以研究肺炎克雷伯菌高危克隆的垂直、水平和斜向进化轨迹。
