Resistance to Ceftazidime-Avibactam Is Due to Transposition of KPC in a Porin-Deficient Strain of Klebsiella pneumoniae with Increased Efflux Activity

Abstract

Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of bla_KPC-3 and bla_SHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring bla_KPC-3 into a second plasmid, pIncX3, which also harbored bla_SHV-12, ultimately resulting in a higher copy number of bla_KPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 μg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.

Keywords: KPC; Klebsiella pneumoniae; ceftazidime-avibactam; porin mutation; resistance.

FIG 1

IncX3 plasmids found in KP1244 and KP1245. pUCLAKPC-2 and pIncX-KP1245 are 66.6- and 55.0-kb plasmids, respectively, with homology to pIncX-SHV (GenBank accession no. JN247852). pULCAKPC-2, identified in isolate KP1244, features a Tn4401d insertion in replication initiation protein repX at amino acid 7 and a unique IS1294 insertion in the intergenic region between repX and dnaJ. pUCLAKPC-2 and pIncX-KP1245 share a 9.9-kb region encoding aminoglycoside (aph3′Ia) and macrolide mph(A) resistance determinants homologous to two regions of pKPN-a68 (CP009777) inserted between the genes umuD and bla_SHV-12, as well as an intergenic IS1294 insertion between the gene dnaJ and a conserved hypothetical protein, and an SNP, resulting in an early stop for the transcriptional activator gene actX. Orange shading marks the region of homology between the Tn4401d from pUCLAKPC-2 and pNJST258N2. Red arrows mark beta-lactamase genes. Dark blue arrows mark the genes of Tn4401. Dark gray arrows and shading mark regions of homology to pKPN-a68. Light blue arrows and shading mark regions of homology to pIncX-SHV. Yellow arrows mark the IS26 and IS1294 transposons.

FIG 2

Plasmid map of pUCLAKPC-1, a 51.2-kb plasmid with large regions of homology to published plasmids pNJST258N2 (GenBank accession no. CP006926) and pNJST258N3 (GenBank accession no. CP006925). Orange shading marks regions of homology between pUCLAKPC-1 and pNJST258N2. Blue arrows mark the genes of Tn4401. Red arrows mark bla_KPC-3. Gray arrows mark nickel, cobalt, and chromium resistance genes. Orange arrows mark genes from the end of Tn4401 to aac6′-Ib. Green arrows and shading mark regions of homology between pUCLAKPC-1 and pNJST258N3. White arrows mark genes in pNJST258N2 and pNJST258N2 without homology in pUCLAKPC-1. Yellow arrows mark genes in pUCLAKPC-1 without homology in pNJST258N2 or pNJST258N2.

FIG 3

(A) Relative KPC-3 expression determined by Western blotting, using anti-RNA polymerase B as a loading control, and corresponding expression levels calculated by densitometry analysis from three experiments. (B) Beta-lactamase activities of KP1244 and KP1245. The relative rates of ceftazidime and meropenem hydrolysis by lysates were normalized to that of the lysate prepared from KP1244 (ceftazidime-avibactam-resistant isolate).

FIG 4

bla_KPC-3 gene copy number in strains KP1245 and KP1074 relative to strain KP1244 in log- and stationary-phase cells.

FIG 5

Uptake of meropenem into the cellular periplasm, normalized to that of KP1245, as an assay for the activity of OmpK36.