The blaKPC-249-mediated mechanism drives the transition of ST463 Pseudomonas aeruginosa from Ceftazidime-Avibactam sensitivity to resistance during clinical treatment

TL;DR

This study identifies how a gene variant, blaKPC-249, causes Pseudomonas aeruginosa to become resistant to a key antibiotic during treatment.

Contribution

The discovery of blaKPC-249 as a novel resistance mechanism in P. aeruginosa during ceftazidime-avibactam treatment.

Findings

The blaKPC-249 gene is located on a 37 kb plasmid and can be transferred between bacteria.

The gene encodes a protein with two additional amino acids, Thr at position 182 and Ser at position 183.

The gene is flanked by insertion sequences ISKpn6 and ISKpn27, aiding its spread.

Abstract

Ceftazidime-avibactam represents a crucial therapeutic option for managing infections attributable to carbapenem-resistant P. aeruginosa. Nonetheless, the emergence of resistance to ceftazidime-avibactam in P. aeruginosa presents a significant challenge for clinical anti-infective therapy. This study primarily elucidates the mechanisms by which P. aeruginosa transitions from drug sensitivity to resistance during ceftazidime-avibactam treatment, ultimately resulting in therapeutic failure.

The susceptibility testing was performed using the broth microdilution method, conjugation experiment was performed via the filter mating method, the genetic surroundings of blaKPC-249 and comparison of plasmids structures was performed using short/long-read genome sequencing and analysis method, the resistance of transconjugant carried the blaKPC-249 to ceftazidime-avibactam was performed via molecular cloning method.

For P. aeruginosa isolates from a patient, the minimum inhibitory concentrations (MICs) of ceftazidime-avibactam (CAZ-AVI) were determined as follows: isolates from sputum and bronchoalveolar lavage fluid both exhibited an MIC of 2 mg/L without blaKPC. In comparison, the blood-isolated strain P. aeruginosa PAE045 showed a significantly elevated MIC of >128 mg/L against CAZ-AVI. Plasmid conjugation experiments results demonstrated that the plasmid harboring the blaKPC-249 gene could be successfully transferred to the recipient strain PAO1rifR (rifampicin-resistant P. aeruginosa PAO1). Third-generation sequencing results revealed that the blaKPC-249 gene was located on a plasmid with an approximate size of 37 kb. Compared with the wild-type e blaKPC-2 gene, the blaKPC-249 gene had two additional amino acid residues in its encoded protein: threonine (Thr, T) at position 182 and serine (Ser, S) at position 183. Furthermore, the upstream and downstream regions of the blaKPC-249 gene were flanked by the insertion sequences ISKpn6 and ISKpn27, respectively.

These mobile genetic elements may play a role in the capture and dissemination of the blaKPC-249 gene. The blaKPC-249 gene is identified as a novel mutant variant of the blaKPC gene family, which mediates the resistance of P. aeruginosa to the antimicrobial agent ceftazidime-avibactam.

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