High-level ertapenem resistance in Klebsiella pneumoniae is due to RamA downregulation of ompK35 through micF

• A Klebsiella pneumoniae clinical strain resistant to ertapenem but susceptible to meropenem and imipenem was isolated. • The isolate was non-carbapenemase-producing and efflux pumps were not involved in resistance. • This study reveals the mechanism of ertapenem resistance in K. pneumoniae . • Termination mutation in ramR resulted in overexpression of RamA, causing loss of OmpK35 through upregulation of micF. 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 KP20Δ ramA by SDS-PAGE. Complementation of ramA in KP20Δ ramA 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 .