Phenotypic and Genetic Analysis of KPC-49, a KPC-2 Variant Conferring Resistance to Ceftazidime–Avibactam and Maintaining Resistance to Imipenem and Meropenem

Abstract

Purpose

Klebsiella pneumoniae, a gram-negative bacterium, poses a severe hazard to public health, with many bacterial hosts having developed resistance to most antibiotics in clinical use. The goal of this study was to look into the development of resistance to both ceftazidime–avibactam and carbapenems, including imipenem and meropenem, in a K. pneumonia strain expressing a novel K. pneumoniae carbapenemase-2 (KPC-2) variant, referred to as KPC-49.

Methods

After 1 day of incubation of K1 on agar containing ceftazidime–avibactam (MIC = 16/4 mg/L), a second KPC-producing K. pneumoniae strain (K2) was recovered. Antimicrobial susceptibility assays, cloning assays, and whole genome sequencing were performed to analyse and evaluate antibiotic resistance phenotypes and genotypes.

Results

K. pneumoniae strain (K1), that produced KPC-2, was susceptible to ceftazidime–avibactam but resistant to carbapenems. The K2 isolate harboured a novel bla _KPC-49 variant, which differs from bla _KPC-2 by a single nucleotide (C487A), and results in an arginine-serine substitution at amino acid position 163 (R163S). The mutant K2 strain was resistant to both ceftazidime–avibactam and carbapenems. We demonstrated the ability of KPC-49 to hydrolyse carbapenems, which may be attributed to high KPC-49 expression or presence of an efflux pump and/or absence of membrane pore proteins in K2. Furthermore, bla _KPC-like was carried on an IncFII (pHN7A8)/IncR-type plasmid within a Tn As1-orf-orf-orf-orf-orf-orf-IS Kpn6-bla _KPC-IS Kpn27 structure. The bla _KPC-like gene was flanked by various insertion sequences and transposon elements, including the Tn3 family transposon, such as Tn As1, Tn As3, IS 26, and IS 481-IS Kpn27.

Conclusion

New KPC variants are emerging owing to sustained exposure to antimicrobials and modifications in their amino acid sequences. We demonstrated the drug resistance mechanisms of the new mutant strains through experimental whole genome sequencing combined with bioinformatics analysis. Enhanced understanding of laboratory and clinical features of infections due to K. pneumoniae of the new KPC subtype is key to early and accurate anti-infective therapy.