Effect Of Forsythoside B On Efflux Pump Activity Of Klebsiella Pneumoniae

Objectives Efflux pump is one of the major mechanisms of antibiotic resistance inKlebsiella pneumoniae. The research is to determine whether the use of low efficacy antibiotics hasany effect on the activity of efflux pumps and induces drug resistance in Klebsiella pneumoniae, toclarify antibacterial effective ingredients of forsythia, to interpret the effect of forsythia effectiveingredients on efflux pump activity of Klebsiella pneumoniae, in order to provide detailedinformation and scientific evidence for the exploitation and application of forsythia in antibacterialtreatment.Methods Twenty susceptible Klebsiella pneumoniae strains were isolated from patients andextended-spectrum β-lactamases (ESBLs) were negative. Their minimal inhibitoryconcentrations (MICs) of ciprofloxacin were measured in the absence and presence of the pumpinhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The strains were then induced witha gradient of ciprofloxacin until the MICs of the strains showed no further increase, to obtaininduced resistant strains. ESBLs-negative Klebsiella pneumoniae strains after induction wereselected. The efflux pump activities of the strains before and after induction were compared usingefflux pump inhibition and EtBr accumulation assays. The bacteriostatic effect of three monomersof forsythia was compared. Ten strains after induction were co-cultured with subinhibitoryconcentration of forsythoside B. EtBr accumulation assay was applied to evaluate efflux pumpactivity of Klebsiella pneumoniae with or without forsythoside B culture.Results Sixteen ciprofloxacin-induced strains were identified as Klebsiella pneumoniaeand ESBLs were negative. The MIC values of the strains were16-256times higher after inductionthan before induction. CCCP had only a2-fold inhibition effect on the activity of the efflux pumpin four strains before induction, while CCCP inhibited efflux pump activity by2or4–fold in halfof the strains after induction. CCCP had no effect on seven strains before induction, but had a2-or4-fold inhibition effect on their induced strains. CCCP showed a2-fold inhibition effect on threestrains before induction, but had no effect on their induced strains. The EtBr accumulation assayshowed that accumulation of EtBr inside the bacterial cells increased in the presence of CCCP. Thefluorescence of EtBr in the induced cells was lower than that in the cells before induction, eitherwith or without CCCP. The bacteriostasis effect of forsythoside A and forsythoside B is better than forsythin. The combined use of antibiotics and forsythoside A or forsythoside B can producesynergistic effect in antimicrobial activity. The subinhibitory concentrations of forsythoside B was512mg/L. For induced strains with or without forsythoside B, their fluorescence value in thepresence of CCCP was higher than that of the one in the absence of CCCP. The fluorescencevalues of two strains with forsythoside B culture were higher than those of them co-culturedwithout forsythoside B, even higher than those without forsythoside B culture in the presence ofCCCP. The fluorescence values of six strains with forsythoside B culture were higher than those ofthem co-cultured without forsythoside B, but lower than those without forsythoside B culture in thepresence of CCCP. Efflux pump activity of two strains was unchanged before and afterforsythoside B culture.Conclusion Efflux pumps are widespread in susceptible and drug resistant Klebsiellapneumoniae strains. Induction with ciprofloxacin can increase efflux pump activity in Klebsiellapneumoniae. Efflux pump inhibition assay was not the best method for evaluating the activity levelof the efflux pump in Klebsiella pneumoniae and EtBr accumulation assay is more sensitive. Thebacteriostasis effect of forsythoside A and forsythoside B is better than forsythin. Antibiotics andforsythoside A or forsythoside B have synergistic effect in antimicrobial activity. Forsythoside Bcould inhibit partially efflux pump activity of Klebsiella pneumoniae.