Abstract

The present study investigated the mechanisms involved in decreased imipenem susceptibility in Klebsiella pneumoniae. Pulsed field gel electrophoresis was used for homology analysis, and a modified Hodge test was used to detect carbapenemase expression. An ethylenediaminetetraacetic acid (EDTA) synergy test was used to screen for metallo-β-lactamase. An E-test and a conjugation experiment were used to detect drug susceptibility, whereas specific polymerase chain reaction (PCR) amplification and sequencing, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and carbonyl cyanide-m-chlorophenylhydrazone (CCCP) inhibition tests were used to analyze mechanisms underlying the decrease in imipenem susceptibility in K. pneumoniae. Kp1, Kp2, and Kp3 did not belong to the same clone strain and their minimum inhibitory concentrations for imipenem and meropenem were 32, 4, and 24 µg/ml and 4, 8, and 4 µg/ml, respectively. Kp1 and Kp3 were resistant to piperacillin/tazobactam, whereas Kp2 was not. Kp1, Kp2, and Kp3 were highly resistant to cephalosporins and aztreonam. Kp2 and Kp3 were resistant to gentamicin. All produced β-lactamase DHA-1, CTX-M-14, SHV-12, and KPC-2. Kp3 produced CMY-2 and cephalosporinase. Gene sequencing analysis and outer membrane porin SDS-PAGE showed that the Ompk36 gene of Kp1 and Kp3 was inserted by IS5 and IS903, which resulted in the loss of the outer membrane porin Ompk36. CCCP did not increase the sensitivity of Kp1, Kp2, and Kp3 to imipenem. Therefore, the loss of the outer membrane porin Ompk36 in β-lactamase KPC-2, DHA-1, CTX-M-14 and SHV-12 producing K. pneumoniae induces the development of high-level imipenem resistance.

Key words: Klebsiella pneumoniae, β-lactamase, imipenem, drug sensitivity, outer membrane porin.