Abstract：
Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses immense threats to the health of infected patients worldwide, especially children. This study reports the infection caused by CRKP in a paediatric intensive care unit (PICU) child and its drug-resistant mutation during the treatment. Twelve Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains were isolated from the child. Broth microdilution method, plasmid transformation assay, and whole genome sequencing (WGS) were performed to investigate the antimicrobial susceptibility, resistance mechanisms, and genetic structural features of CRKPs. The results showed that 12 strains were highly resistant to most available antimicrobial agents. Among them, K. pneumoniae FD11 and K. pneumoniae FD12 were resistant to ceftazidime-avibactam (CZA, MIC >64 mg/L) and restored the carbapenem susceptibility (Imipenem, MIC =0.25 mg/L; Meropenem, MIC =2 mg/L). The patient improved after treatment with CZA in combination with aztreonam. Plasmid transformation assay demonstrated that the blaKPC-33-positive transformant increased MICs of CZA by at least 33-fold and 8-fold compared with the recipient Escherichia coli DH5α and blaKPC-2-positive transformants. WGS analysis revealed that all strains belonged to the ST11-KL64 type and showed highly homologous (3-26 single nucleotide polymorphisms [SNPs]). A single base mutation (G532T) of blaKPC-2 resulted in a tyrosine to aspartic acid substitution at Ambler amino acid position 179 (D179Y), which conferred CZA resistance in K. pneumoniae. This is the first report of a drug-resistant mutation evolving into blaKPC-33 during the treatment of blaKPC-2-positive CRKP in paediatric-infected patients. It advises clinicians that routine sequential antimicrobial susceptibility testing and KPC genotyping are critical during CZA therapy in children infected with CRKP.
摘要：
耐碳青霉烯类肺炎克雷伯菌（CRKP）对全球感染患者的健康构成巨大威胁，尤其是儿童。这项研究报告了儿科重症监护病房（PICU）儿童中CRKP引起的感染及其在治疗过程中的耐药突变。从该儿童中分离出12种产生肺炎克雷伯菌碳青霉烯酶（KPC）的肺炎克雷伯菌菌株。肉汤微量稀释法,质粒转化试验,和全基因组测序（WGS）进行，以调查抗菌药物的敏感性，抵抗机制，和CRKPs的遗传结构特征。结果表明，12株菌株对大多数可用的抗菌剂具有高度抗性。其中,肺炎克雷伯菌FD11和肺炎克雷伯菌FD12对头孢他啶-阿维巴坦(CZA，MIC>64mg/L)并恢复碳青霉烯敏感性(IMP，MIC=0.25mg/L;MEM,MIC=2mg/L）。CZA联合氨曲南治疗后，患者病情好转。质粒转化测定表明，与受体大肠杆菌DH5α和blaKPC-2阳性转化体相比，blaKPC-33阳性转化体使CZA的MIC增加了至少33倍和8倍。WGS分析显示，所有菌株均属于ST11-KL64型，并显示高度同源（3至26个单核苷酸多态性（SNP））。blaKPC-2的单碱基突变（G532T）导致在Ambler氨基酸位置179（D179Y）的酪氨酸到天冬氨酸取代，这在肺炎克雷伯菌中赋予了CZA耐药性。这是关于在儿科感染患者中治疗blaKPC-2阳性CRKP期间，耐药性突变演变成blaKPC-33的第一份报告。它建议临床医生，在感染CRKP的儿童中，在CZA治疗期间，常规的序贯抗菌药物敏感性测试和KPC基因分型至关重要。
