sodium-dodecyl-sulfate and Klebsiella-Infections

An ertapenem-resistant Klebsiella pneumoniae clinical isolate (KP20) without carbapenemase and negative for the efflux pump inhibition test was resistant to ertapenem at a high level [minimum inhibitory concentration (MIC) = 64 mg/L] but susceptible to meropenem and imipenem. Second-generation sequencing was performed and a termination mutation was found in ramR. Complementation of ramR in KP20 reduced the ertapenem MIC by 128 times (from 64 mg/L to 0.5 mg/L). Overexpression of ramA and loss of OmpK35 were discovered in strain KP20 by quantitative reverse transcription PCR (RT-qPCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Furthermore, ramA deletion in strain KP20 resulted in a 128-fold decrease in the MIC of ertapenem (from 64 mg/L to 0.5 mg/L), and expression of OmpK35 was observed in KP20ΔramA by SDS-PAGE. Complementation of ramA in KP20ΔramA led to a 45.45-fold downregulation of ompK35. Complementation of ompK35 in KP20 could restore susceptibility to ertapenem (MIC reduced from 64 mg/L to 0.25 mg/L). Furthermore, results of the electrophoretic mobility shift assay showed that RamA could bind to the promoter of micF. These results showed that the termination mutation in ramR resulted in overexpression of ramA causing loss of OmpK35 expression through upregulation of micF, revealing the mechanism of ertapenem resistance only in K. pneumoniae.

Topics: Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Down-Regulation; Ertapenem; Humans; Imipenem; Klebsiella Infections; Klebsiella pneumoniae; Meropenem; Microbial Sensitivity Tests; Sodium Dodecyl Sulfate