Resistance to ceftazidime-avibactam is due to transposition of KPC in a porin-deficient strain of Klebsiella pneumoniae with increased efflux activity

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 g/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.