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

INTRODUCTION

The persistence of in the contaminated environment is sustained by tolerance to biocides and ability to growth as biofilm. The aim of the study was to analyze the susceptibility of biofilms to chlorhexidine (CHX) and benzalkonium (BZK) biocides and the ability of natural monomeric stilbenoid resveratrol (RV) to modulate the phenomenon.

METHODS

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.

RESULTS

CHX and BZK at ¼ and ½ MICs alone or in combination inhibited biofilm growth of 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 strains. Similarly, RV at 32 mg/L and CHX and BZK at ½ MIC significantly inhibited air-liquid biofilm formation of ATCC 19606, 4190 and 3909 strains. The inactivation of AdeB and AdeJ RND EPs in 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 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 ATCC19606 biofilm growth. In addition, CHX and BZK alone or in combination dose-dependently reduced preformed biofilm of all strains. The combination of RV with CHX and BZK additively decreased minimal biofilm eradicating concentrations in strains.

CONCLUSION

These results demonstrate that: (i) CHX and BZK alone or in the presence of RV inhibit biofilm growth and preformed biofilm in ; (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 .