Emergence of a KPC-90 Variant that Confers Resistance to Ceftazidime-Avibactam in an ST463 Carbapenem-Resistant Pseudomonas aeruginosa Strain

Abstract

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has become a serious challenge in the clinic. Recently, the prevalence of CRPA isolates carrying the bla_KPC-2 gene has been increasing in China. Ceftazidime-avibactam (CZA) has shown good efficacy against large portions of KPC-2-producing CRPA strains. However, with the increasing usage of this drug, CZA resistance in CRPA strains has been reported. Here, we reported for the first time that resistance of the ST463 CRPA strain to CZA was caused by a novel variant in the KPC gene that arose after CZA exposure. The CRPA strain PA2207 is a carbapenem- and CZA-resistant strain that harbors a mutated bla_KPC gene, named bla_KPC-90. Cloning and expression of bla_KPC-90 in Escherichia coli DH5α revealed that KPC-90 led to a 64-fold increase in the MIC value of CZA. Conjugation experiments further confirmed that bla_KPC-90 was located on a conjugative plasmid. Whole-genome sequencing analysis showed that this plasmid had high sequence similarity to a previously reported novel bla_KPC-2-harboring plasmid in a clinical P. aeruginosa strain isolated in China. In addition, overexpression of an efflux pump (MexXY-OprM) might be associated with the CZA resistance phenotype, as determined by reverse transcription-quantitative PCR and efflux pump inhibition experiments. For the first time, we reported a KPC variant, KPC-90, in a clinical ST463 CRPA strain with CZA resistance that was mediated by a 2 amino acid insertion outside the KPC omega-loop region. Our study further highlights that diverse KPC variants that mediate CZA resistance have emerged in the CRPA strain. Furthermore, KPC-90 mutation combined with efflux pump overexpression resulted in a high level of resistance to CZA in the PA2207 isolate. Effective surveillance should be conducted to prevent CZA resistance from spreading in the CRPA strain. IMPORTANCE For the first time, we reported a KPC variant, KPC-90, in a clinical ST463 CRPA strain with CZA resistance. CZA resistance was mediated by a 2 amino acid insertion outside the KPC omega-loop region in CRPA. Our study further emphasized that CZA resistance caused by bla_KPC gene mutation could be selected in CRPA after CZA therapy. Considering the widespread presence of the ST463 CRPA strain in China, clinicians should pay attention to the risk of the development of CZA resistance in CRPA strains under treatment pressure.

Keywords: CRPA; KPC-90; ST463; ceftazidime-avibactam; resistance.

FIG 1

Amplicon alignments between bla_KPC-90 and bla_KPC-2 in nucleotide and amino acid (aa) sequences surrounding the mutation. Six nucleotide deletions were identified at the bla_KPC-90 gene compared to bla_KPC-2, which result in amino acid insertions at the 180 and 181 amino acid positions of the KPC-2 protein. The red letters represent mutant amino acids. Dotted line, common sequence; broken line, deletion of six nucleotides; *, common amino acid; bold font, deletion of two amino acids; A, Ala; D, Asp; E, Glu; G, Gly; I, Ile; P, Pro; R, Arg; S, Ser; T, Thr; V, Val; Y, Tyr.

FIG 2

Plasmid analysis of pPA2207_2. Schematic map of plasmid pPA2207_2, this plasmid sequence was compared with plasmids pSRRSH1002-KPC (accession number CP0643988) and pP23 (CP065418).

FIG 3

Linear characterization between the plasmid pPA2207_2 (CP080290) and pP23 (CP065418). The gray regions between plasmids indicate nucleotide identity (90 to 100%) by BLASTn. Arrows indicate predicted ORFs. Colored arrows represent open reading frames, with red, yellow, blue, and green representing antibiotic resistance genes, common function genes, replication genes, and mobile elements, respectively.

FIG 4

Relative ratio of gene expression of AmpC and efflux pumps. Gene expression was normalized versus the rpsL housekeeping gene and expression levels were indicated as a ratio to the expression level in P. aeruginosa strain PA01.