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 TnAs1-orf-orf-orf-orf-orf-orf-ISKpn6-bla_KPC-ISKpn27 structure. The bla_KPC-like gene was flanked by various insertion sequences and transposon elements, including the Tn3 family transposon, such as TnAs1, TnAs3, IS26, and IS481-ISKpn27.

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.

Keywords:

• KPC
• antibiotic resistance
• whole genome sequencing
• ceftazidime–avibactam
• susceptibility testing

Ethics Approval

The Qingdao Municipal Hospital’s Ethics Committee approved the whole plan for this study’s research. The authors of this paper have not performed any studies on humans or animals. The need for informed consent was waived because this study was about bacteria and did not involve patients.

Acknowledgments

The authors would like to thank the Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China, for its technical help, including provision of instruments, equipment and relevant experimental material.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

In this work, the authors state that they have no conflicts of interest.

Additional information

Funding

This work was funded by Qingdao Municipal Clinical Laboratory key specialty.