Synergistic activities of antibiotic combinations against carbapenem-resistant Klebsiella pneumoniae and mechanisms of carbapenem resistance in Klebsiella pneumoniae clinical isolates.

Abstract

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has been increasing reported worldwide. The aims of this study were to investigate the mechanisms of carbapenem resistance in K. pneumoniae Thai isolates and study the synergistic activities of antibiotic combinations against CRKP isolates. Two-hundred and forty CRKP clinical isolates were obtained from patients at King Chulalongkorn Memorial Hospital between September 2016 and April 2020. The antibiotic susceptibility testing was determined by agar dilution to imipenem, meropenem, amikacin, fosfomycin, ciprofloxacin and by broth microdilution to colistin. The prevalence rates of resistance to imipenem, meropenem, ciprofloxacin, fosfomycin, amikacin, and colistin were 90.42%, 91.67%, 95.84%, 41.25%, 20%, and 12.08%, respectively. The majority of CRKP isolates (62.92%) were multidrug-resistant (MDR). Of the 240 CRKP isolates, 236 (98.33%) harbored at least one carbapenemase gene. The blaNDM-like plus blaOXA-48-like were the most common carbapenemase genes (43.75%), followed by blaNDM-like (27.50%), blaOXA-48-like (25.42%), blaIMP-like (0.83%), and blaOXA-48-like plus blaIMP-like (0.83%). Neither blaKPC-like nor blaVIM-like were detected. All isolates harboring carbapenemase genes were positive for carbapenemase activity by modified carbapenem inactivation method (mCIM). The overexpression of efflux pump was detected in 13.33% of CRKP isolates by using carbonyl cyanide m-cholorophenyl hydrazone (CCCP) inhibitor. The loss of OmpK35 and/or OmpK36 was found in 98.33% of CRKP isolates by SDS-PAGE. The combination of carbapenemase production and loss of OmpK was the most common mechanism of carbapenem resistance (83.33%). High level of carbapenem MIC (≥64 mg/L) was found in isolates harboring blaNDM-like plus blaOXA-48-like together with loss of both OmpK35 and OmpK36, and overexpression of efflux pump.

Antibiotic combinations including colistin plus imipenem, colistin plus meropenem, fosfomycin plus amikacin, and fosfomycin plus imipenem were screened for the synergistic activity by checkerboard assay in 31 representative isolates. The most effective combination was fosfomycin plus imipenem which showed synergism in 22.58%, followed by 14.81% in fosfomycin plus amikacin combination. Three CRKP isolates that had additive effect in colistin-based combination showed synergistic effect in fosfomycin plus imipenem combination. Time-kill assay was used to confirm the synergism against 7 CRKP isolates. The results from time-kill assay were consistent with the checkerboard assay. The bactericidal activity was found in 6 isolates (85.71%). In conclusion, this study showed that the carbapenemase production and loss of OmpK were the major mechanisms of carbapenem resistance in CRKP Thai isolates. The combination of fosfomycin plus imipenem may be one of the alternative antibiotic combination for treatment of CRKP infections.