Centre of Laboratory Medicine, Zhejiang Provincial People' s Hospital, People' s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, China.
Department of Biophysics and Department of Pathology, Sir Run Run Shaw Hospital, Zhejiang Universitygrid.13402.34 School of Medicine, Hangzhou, China.
mSphere. 2021 Dec 22;6(6):e0085921. doi: 10.1128/mSphere.00859-21.
Here, we characterized the mechanisms resulting in the development of KPC-71-mediated resistance to ceftazidime-avibactam (CZA) during treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. CZA-susceptible and CZA-resistant K. pneumoniae strains, namely, KP357 and KP697, were isolated from the same patient. Whole-genome sequencing revealed that KP357 and KP697 belonged to the ST11 type and KP697 strain possessed a mutation in the plasmid-borne gene. Compared to KPC-2, this gene () showed a mutated nucleotide and an insertion of 3 nucleotides at positions 542 to 545, which resulted in a variant with the subsequent insertion of a serine between the Ambler positions 182 and 183. This plasmid, carrying , successfully transformed its CZA-resistant phenotype to Escherichia coli DH5α. Cloning and expression of in E. coli DH5α demonstrated that KPC-71 resulted in a 16-fold increase in the MIC value for CZA. Kinetic parameters showed that KPC-71, compared to wild-type KPC-2, exhibited a lower (∼13-fold) with ceftazidime and a higher (∼14-fold) 50% inhibitory concentration with avibactam. In addition, both and gene expression have a negative impact on fitness. In conclusion, we detected a novel KPC variant, KPC-71, in a clinical ST11 CRKP strain resulting in CZA resistance development during treatment. The KPC-71 enzyme was associated with a higher affinity toward ceftazidime and a reduced sensitivity to avibactam, conferring resistance to CZA. Considering the wide application of CZA, clinicians should pay attention to the risk of the development of CZA resistance in CRKP strains under treatment pressure. In this study, we report an ST11-type clinical CRKP isolate that produces KPC-71, a novel plasmid backbone KPC variant that confers the development of CZA resistance during treatment. Furthermore, we reveal that resistance to CZA is mediated by the 182S insertion mutation in the KPC enzyme, which increases ceftazidime affinity and decreases avibactam inhibition. In addition, KPC-71 has reduced hydrolysis activity, which leads to susceptibility to carbapenems. To the best of our knowledge, this is a novel KPC-2 variant conferring resistance to CZA and the first report of its emergence. Considering the widespread presence of the ST11 CRKP strain in China, clinicians should pay attention to the risk of the development of CZA resistance in CRKP strains under treatment pressure.
在这里，我们研究了在治疗碳青霉烯类耐药肺炎克雷伯菌（CRKP）感染期间，导致产 KPC-71 对头孢他啶-阿维巴坦（CZA）产生耐药性的机制。CZA 敏感和 CZA 耐药的肺炎克雷伯菌菌株，即 KP357 和 KP697，是从同一位患者中分离出来的。全基因组测序显示，KP357 和 KP697 属于 ST11 型，而 KP697 菌株在质粒携带的 基因中发生了突变。与 KPC-2 相比，该 基因（）发生了一个突变核苷酸和三个核苷酸的插入，位置在 542 到 545 之间，导致在位置 182 和 183 之间插入丝氨酸的变体。这个携带 的质粒，成功地将其 CZA 耐药表型转化为大肠杆菌 DH5α。在大肠杆菌 DH5α 中克隆和表达 表明，KPC-71 使 CZA 的 MIC 值增加了 16 倍。动力学参数表明，与野生型 KPC-2 相比，KPC-71 对头孢他啶的亲和力降低（约 13 倍），对阿维巴坦的 50%抑制浓度升高（约 14 倍）。此外， 和 基因的表达都对适应性有负面影响。总之，我们在临床 ST11 型 CRKP 菌株中检测到一种新型的 KPC 变体 KPC-71，该变体导致 CZA 在治疗过程中耐药性的发展。KPC-71 酶与头孢他啶的亲和力更高，对阿维巴坦的敏感性降低，从而对 CZA 产生耐药性。考虑到 CZA 的广泛应用，临床医生应该注意在治疗压力下 CRKP 菌株产生 CZA 耐药性的风险。在本研究中，我们报告了一种 ST11 型临床 CRKP 分离株，该分离株产生 KPC-71，这是一种新型的质粒骨架 KPC 变体，可导致 CZA 在治疗过程中产生耐药性。此外，我们揭示了 CZA 耐药性是由 KPC 酶中的 182S 插入突变介导的，该突变增加了头孢他啶的亲和力，降低了阿维巴坦的抑制作用。此外，KPC-71 的水解活性降低，导致对碳青霉烯类药物的敏感性增加。据我们所知，这是一种新型的对 CZA 耐药的 KPC-2 变体，也是首次报道其出现。考虑到中国 ST11 型 CRKP 菌株的广泛存在，临床医生应该注意在治疗压力下 CRKP 菌株产生 CZA 耐药性的风险。
