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 <i>Klebsiella pneumoniae</i> 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 <i>Klebsiella pneumoniae</i> 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 <i>bla</i>_KPC-3 and <i>bla</i>_SHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn<i>4401</i> transposon harboring <i>bla</i>_KPC-3 into a second plasmid, pIncX3, which also harbored <i>bla</i>_SHV-12, ultimately resulting in a higher copy number of <i>bla_KPC</i>_-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient <i>K. pneumoniae</i> background that harbored a mutation to the <i>ramR</i> regulator of the <i>acrAB</i> efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 μg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.