二甲双胍与替加环素协同抑制TMexCD1-TOprJ1阳性肺炎克雷伯菌的机制OA北大核心

This study aims to investigate the antibacterial effect and mechanism of metformin combined with tigecycline on TMexCD1-TOprJ1 efflux pump-mediated tigecycline-resistant Klebsiella pneumoniae.The checkerboard broth microdilution method and time-kill curve assay were used to evaluate the in vitro antibacterial effect of metformin combined with tigecycline against Klebsiella pneumoniae.The mechanism of combined bactericidal action was explored by measuring efflux pump function(Rhodamine B efflux assay and tigecycline accumulation assay),proton motive force(PMF),outer membrane permeability,and biofilm formation ability.The results showed that the fractional inhibitory concentration index(FICI)of metformin combined with tigecycline against TMexCD1-TOprJ1 positive strains ranged from 0.187 5 to 0.5,indicating a synergistic antibacterial effect.The time-kill curve results showed that the concentration of the original bacterial solution in the combined metformin and tigecycline group was reduced by 2.38 log10 CFU/mL compared to the tigecycline monotherapy group.The Rhodamine B efflux assay results showed that the fluorescence intensity of Rhodamine B in the 20 mg/mL and 40 mg/mL metformin groups was significantly reduced compared to the control group(P＜0.001).The tigecycline accumulation assay results indicated that metformin significantly increased the accumulation of tigecycline in TMexCD1-TOprJ1 positive strains(P＜0.01).PMF measurement results showed that the fluorescence intensity of DiSC3(5)in the metformin group was significantly increased compared to the tigecycline group(P＜0.001),indicating that metformin could disrupt the membrane potential difference(Δψ)in PMF,affecting the energy source of the efflux pump.The outer membrane permeability assay results showed that the fluorescence intensity of 1-N-phenyl-naphthylamine(NPN)in TMexCD1-TOprJ1 positive strains treated with sub-inhibitory concentrations of metformin was significantly increased compared to the control group(P＜0.000 1),indicating a significant increase in outer membrane permeability.The biofilm formation ability assay results showed that the biofilm formation rate of TMexCD1-TOprJ1 positive strains treated with the combination of metformin and tigecycline(22％)was significantly reduced compared to the control group(P＜0.000 1).The results indicate that metformin combined with tigecycline has a good inhibitory effect on TMexCD1-TOprJ1 positive Klebsiella pneumoniae,and metformin can exert a synergistic effect with tigecycline through multiple mechanisms.This study provides a strategy for the clinical treatment of infections caused by TMexCD1-TOprJ1 positive tigecycline-resistant Klebsiella pneumoniae.