Reduced imipenem susceptibility in Klebsiella pneumoniae clinical isolates with plasmid-mediated CMY-2 and DHA-1 beta-lactamases co-mediated by porin loss

We investigated the resistance mechanisms and clonality among 42 imipenem-non-susceptible Klebsiella pneumoniae isolated at a tertiary care hospital in Korea. Two isolates had bla(VIM-2) alleles, whereas bla(CMY-2)- and bla(DHA-1)-like alleles were detected in 24 and 16 isolates, respectively, with these enzymes confirmed by sequencing for representative isolates. Transfer of bla(CMY-2) and bla(DHA-1) was achieved by conjugation. Addition of 300 mg/L 3-aminophenylboronic acid (APB) reduced the minimum inhibitory concentration for 90% of the organisms (MIC(90)) of imipenem and meropenem eight- and four-fold, respectively, for the bla(CMY-2)- and bla(DHA-1)-positive isolates, confirming the role of these enzymes in resistance. SDS-PAGE of outer membrane proteins for representative isolates showed lack or greatly diminished expression of OmpK35 and OmpK36 porins. Pulsed-field gel electrophoresis of XbaI-restricted genomic DNA revealed two closely related clusters among 23 bla(CMY-2)-positive isolates, whereas those with bla(DHA-1) were more heterogeneous. In conclusion, reduced imipenem susceptibility among K. pneumoniae at this Korean hospital was largely co-mediated by production of plasmid-mediated AmpC beta-lactamases along with lack or greatly diminished expression of OmpK35 and OmpK36 porins.