KPC variants conferring resistance to ceftazidime-avibactam in Pseudomonas aeruginosa strains

Abstract

Background

This study aimed to characterize three KPC variants (KPC-33, KPC-100, and KPC-201) obtained from a clinical isolate of Pseudomonas aeruginosa (#700), along with two induced strains C109 and C108.

Methods

Genomic DNAs of #700 (ST235), C109 (ST463), and C108 (ST1076) were sequenced using Illumina and Oxford Nanopore technologies. The transferability and stability of the plasmid was assessed through conjugation experiments and plasmid stability experiments, respectively. Minimum inhibitory concentrations of bacterial strains were determined using broth microdilution methods. In vitro induction was performed using ceftazidime-avibactam (CZA) at concentrations of 6/4 µg/ml. Linear genomic alignments were visualized using Easyfig, and protein structure modeling of the novel KPC variant (KPC-201) was conducted using PyMol.

Results

The plasmids carrying the KPC variants in the three CZA-resistant strains (C109, C108, and #700) had sizes of 39,251 bp (KPC-100), 394,978 bp (KPC-201), and 48,994 bp (KPC-33). All three plasmids belonged to the IncP-like incompatibility (Inc) groups, and the plasmid exhibited relatively high plasmid stability, KPC-33 and KPC-201-harboring plasmids were successfully transferred to the recipient strain P. aeruginosa PAO1^rifR. The genetic environments of the three bla_KPC genes differed from each other. The mobile elements of the three bla_KPC genes were as follows, TnAS1-IS26-ΔISKpn27-bla_KPC-33-ISKpn6-IS26, IS6-ΔISKpn27-bla_KPC-100-ISKpn6-IS26-Tn3-IS26, and IS6100-ISKpn27-bla_KPC-201-ISKpn6-TnAS1. Notably, the length of ΔISKpn27 upstream of the bla_KPC-33 and bla_KPC-100 genes were remarkably short, measuring 114 bp and 56 bp, respectively, deviating significantly from typical lengths associated with ISKpn27 elements. Moreover, the novel KPC variant, KPC-201, featured a deletion of amino acids LDR at positions 161–163 in KPC-3, resulting in a looser pocket structure contributing to its avibactam resistance.

Conclusions

KPC-201, identified as a novel KPC variant, exhibits resistance to CZA. The presence of multiple mobile elements surrounding the bla_KPC-variant genes on stable plasmids is concerning. Urgent preventive measures are crucial to curb its dissemination in clinical settings.