Molecular Mechanisms Driving the In Vivo Development of KPC-71-Mediated Resistance to Ceftazidime-Avibactam during Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections

Abstract

ABSTRACT 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 blaKPC-2 gene. Compared to KPC-2, this blaKPC gene (blaKPC-71) 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 blaKPC-71, successfully transformed its CZA-resistant phenotype to Escherichia coli DH5α. Cloning and expression of blaKPC-71 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) Km with ceftazidime and a higher (∼14-fold) 50% inhibitory concentration with avibactam. In addition, both blaKPC-2 and blaKPC-71 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. IMPORTANCE 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.

Keywords

• CRKP
• KPC-71
• CZA resistance
• fitness