Characterization of a Novel KPC-2 Variant, KPC-228, Conferring Resistance to Ceftazidime-Avibactam in an ST11-KL64 Hypervirulent Klebsiella pneumoniae

Abstract

With the widespread clinical use of ceftazidime-avibactam (CZA), reports of resistance have increased continuously, posing immense threats to public health worldwide. In this study, we explored the underlying mechanisms leading to the development of CZA resistance in an ST11-KL64 hypervirulent Klebsiella pneumoniae CRE146 that harbored the bla_KPC-228 gene. Twelve carbapenem-resistant Klebsiella pneumoniae (CRKP) strains were isolated from the same patient, including K. pneumoniae CRE146. Whole genome sequencing (WGS), phylogenetic analysis, bla_KPC gene cloning and pACYC-KPC construction assays were conducted to further explore the molecular mechanisms of CZA resistance. Quantitative siderophore production assay, string test, capsule quantification and Galleria mellonella in vivo infection model were applied to verify the level of pathogenicity of K. pneumoniae CRE146. This strain carried key virulence factors, iutA-iucABCD operon and rmpA gene. Compared to the wild-type KPC-2 carbapenemase, the novel KPC-228 enzyme exhibited a deletion of four amino acids in the Ω-loop (del_167-170_ELNS). In addition, the emergence of CZA resistance appeared to be associated with drug exposure, and we observed the in vivo evolution of wild-type KPC-2 to KPC-228 and then the reversion to its original wild-type KPC-2. The bla_KPC-228 gene was located within the double IS26 flanking the ISKpn6-bla_KPC-228-ISKpn27 core structure and carried on an IncFII/IncR-type plasmid. Notably, CRE146 exhibited high-level resistance to CZA (64/4 mg/L) but increased susceptibility to meropenem (1 mg/L) and imipenem (0.5 mg/L) respectively. PACYC-KPC plasmids were constructed and expressed in K. pneumoniae ATCC13883. Compared to K. pneumoniae ATCC13883 harboring bla_KPC-2, K. pneumoniae ATCC13883 harboring bla_KPC-228 exhibited a high-level resistance to CZA (32/4 mg/L) and increased susceptibility to meropenem (1 mg/L) and imipenem (0.5 mg/L). Interestingly, K. pneumoniae ATCC13883 harboring bla_KPC-228 showed a significant decrease in their resistance to all β-lactamases tested except CZA and ceftazidime. In conclusion, we reported a novel KPC variant, KPC-228, in a clinical ST11-KL64 hypervirulent K. pneumoniae strain, which conferred CZA resistance, possibly through enhancing ceftazidime affinity and reducing avibactam binding. The bla_KPC-228 can mutate back to bla_KPC-2 under carbapenem pressure, which was very detrimental to clinical treatment. This strain carried both resistance and virulence genes, posing a major challenge in clinical management.

Keywords: Carbapenem-resistant Klebsiella pneumoniae; ST11-KL64; bla(KPC-228); ceftazidime-avibactam; hypervirulent.