Preliminary Study on the Inhibiting Mechanism of Gallic Acid for Biofilm Formation
Salmonella pullorum and Staphylococcus aureus are important pathogenic bacteria that endanger poultry industry and public health and the formation of biofilm is an important factor of their persistent infection and repeated occurrence.In order to investigate the effect of gallic acid on the biofilm formation,the minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),minimum biofilm eradication concertation(MBEC)and minimum biofilm inhibitory concentration(MBIC)for S.pullorum CVCC519 and S.aureus ATCC 25923 were determined by microdilution method.Crystal violet staining and phenol-sulfuric acid method were used to determine the inhibition rate of the bacteria on the biofilm and exopolysaccharide(EPS).Moreover,the morphology of the biofilm was observed by environmental scanning electron microscopy.Finally,qRT-PCR was used to detect the expression of genes related to biofilm formation and gallic acid inhibition.The results showed that the MIC,MBC,MBIC and MBEC of gallic acid were 4 mg/mL,8 mg/mL,8 mg/mL and 16 mg/mL for CVCC519,and 8 mg/mL,16 mg/mL,8 mg/mL and 16 mg/mL for ATCC 25923.In addition,gallic acid significantly inhibited biofilm formation and EPS production at a concentration that did not significantly affect growth of test bacteria.Environmental scanning electron microscopy showed that the biofilm formation was significantly reduced by gallic acid treatment and the structure of biofilm became loose and thin.The results of qRT-PCR showed that gallic acid decreased the expression levels of curli fimbriae genes csgA and csgD,cellulose genes bcsA and adrA,and quorum sensing system related gene luxS in a dose-dependent manner in CVCC519 at a non-inhibitory concentration while the ica operon and luxS of ATCC 25923 were not significantly regulated.These results suggested that gallic acid produced an anti-biofilm effect to S.pullorum via down-regulating the expression of genes related to biofilm formation and reducing the synthesis of corresponding extracellular matrix.Gallic acid also effectively inhibited the secretion of EPS and biofilm formation of S.aureus through ica independent pathways.Taken together,this study provided a theoretical basis for gallic acid in the prevention and control of S.pullorum and S.aureus biofilm formation.
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