Phenotypic, molecular, and in silico characterization of coumarin as carbapenemase inhibitor to fight carbapenem-resistant Klebsiella pneumoniae.

Background: Carbapenems represent the first line treatment of serious infections caused by drug-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) is one of the urgent threats to human health worldwide. The current study aims to evaluate the carbapenemase inhibitory potential of coumarin and to test its ability to restore meropenem activity against CRKP. Disk diffusion method was used to test the antimicrobial susceptibility of K. pneumoniae clinical isolates to various antibiotics. Carbapenemase genes (NDM-1, VIM-2, and OXA-9) were detected using PCR. The effect of sub-MIC of coumarin on CRKP isolates was performed using combined disk assay, enzyme inhibition assay, and checkerboard assay. In addition, qRT-PCR was used to estimate the coumarin effect on expression of carbapenemase genes. Molecular docking was used to confirm the interaction between coumarin and binding sites within three carbapenemases.

Results: K. pneumoniae clinical isolates were found to be multi-drug resistant and showed high resistance to meropenem. All bacterial isolates harbor at least one carbapenemase-encoding gene. Coumarin significantly inhibited carbapenemases in the crude periplasmic extract of CRKP. The checkerboard assay indicated that coumarin-meropenem combination was synergistic exhibiting a fractional inhibitory concentration index ≤ 0.5. In addition, qRT-PCR results revealed that coumarin significantly decreased carbapenemase-genes expression. Molecular docking revealed that the binding energies of coumarin to NDM1, VIM-2, OXA-48 and OXA-9 showed a free binding energy of -7.8757, -7.1532, -6.2064 and - 7.4331 Kcal/mol, respectively.

Conclusion: Coumarin rendered CRKP sensitive to meropenem as evidenced by its inhibitory action on hydrolytic activity and expression of carbapenemases. The current findings suggest that coumarin could be a possible solution to overcome carbapenems resistance in CRKP.