Inhibition of biofilm formation and preformed biofilm in Acinetobacter baumannii by resveratrol, chlorhexidine and benzalkonium: modulation of efflux pump activity

These results demonstrate that: (i) CHX and BZK alone or in the presence of RV inhibit biofilm growth and preformed biofilm in A. baumannii; (ii) tolerance to CHX and BZK during biofilm growth is dependent on the activation of AdeB and AdeJ EPs; and (iii) the inhibitory effect of RV on biofilm growth is mediated by the inhibition of EP genes expression in A. baumannii.

Biofilm formation and preformed biofilm were tested by Crystal violet and tetrazolium salt reduction assay, respectively. Analysis of efflux pump (EP) expression during biofilm growth was performed by Real-time RT-PCR assays.

CHX and BZK at ¼ and ½ MICs alone or in combination inhibited biofilm growth of A. baumannii ATCC 19606, 4190, and 3909 strains. RV at 32 mg/L and CHX and BZK at ¼ or ½ MICs showed a synergistic effect and completely inhibited biofilm formation in all A. baumannii strains. Similarly, RV at 32 mg/L and CHX and BZK at ½ MIC significantly inhibited air-liquid biofilm formation of A. baumannii ATCC 19606, 4190 and 3909 strains. The inactivation of AdeB and AdeJ RND EPs in A. baumannii ATCC19606 increased the susceptibility to CHX and BZK alone or in the presence of 32 mg/L RV. Concordantly, carbonyl cyanide m-chlorophenylhydrazine (CCCP) increased the susceptibility to CHX, BZK and RV and dose-dependently inhibited biofilm formation in A. baumannii ATCC 19606, 4190 and 3909 strains. RV at 32 mg/L inhibited basal and CHX-induced EP genes expression, while increased EP gene expression in the presence of BZK during A. baumannii ATCC19606 biofilm growth. In addition, CHX and BZK alone or in combination dose-dependently reduced preformed biofilm of all A. baumannii strains. The combination of RV with CHX and BZK additively decreased minimal biofilm eradicating concentrations in A. baumannii strains.