Influences Of Chlorogenic Acid And Amphotericin B On Aspergillus Fumigatus Biofilm And Its Interference Mechanism In Vitro

PARTI IN VITRO INTERACTION BETWEEN CHLOROGENIC ACID AND AMPHOTERICIN B AGAINST ASPERGILLUS FUMIGATUS BIOFILMObjective:To explore the effect of the main active component of Honeysuckle(Chlorogenic acid,CRA)alone and in combined with amphotericin B(AMB)on Aspergillus fumigatus biofilm(BF)in vitro.Methods:The A.f004 strain isolated from clinical patient and exhibited strongest and stable biofilm formation ability,was used in this study.A.fumigatus biofilm models were established in vitro after 24h and 48h.Each model divided into the control group,the AMB group,the CRA group and the CRA-AMB combination group;the double dilution method was used to measure the minimum inhibitory concentration(MIC)and the minimum fungicidal concentration(MFC)of testing drugs.Crystal violet assay(CV)was used for the quantification of biofilm;the XTT reduction assay was used for the metabolic activity of the fungus inside biofilm.Confocal scanning laser microscopy(CSLM)was used to image and evaluate the effects of the drugs on biofilm-growing hyphal cells,and scanning electron microscopy(SEM)was used to examine the biofilm morphology.Results:A.fumigatus BF models were established successfully in vitro after being cultured for 24h and 48h.After affecting 48h,biofilm quantification by CV assay showed that:either in early or mature biofilm,there was no significant discrepancy between the untreated controls and the AMB groups(P>0.05);CRA alone were significantly less than the untreated controls and AMB groups(P<0.05),simultaneously,quantification of biofilms after exposure to CRA-AMB combinations were observed decrease further compared to the findings for CRA alone(P<0.05).XTT assay showed that there was no significant difference among untreated controls,the AMB groups and the CRA groups(P>0.05),when A.fumigatus biofilms were treated with CRA-AMB combinations,a reduction of the metabolic activity of hyphal cells were observed compared to the results for untreated controls and the AMB groups(P<0.05).Moreover,compared with the results obtained with the untreated controls and the AMB alone,the extracellular matrix(ECM)between the hyphae in the early and mature biofilms appeared to be obviously reduced after exposure to CRA by SEM observation,besides,ECM as well as hyphae within biofilms were reduced by CRA-AMB combinations treating,and some residual hyphae had been wizened.CSLM showed live/dead hyphae and conidia for A.fumigatus biofilms,the results showed that hyphae with treated with CRA or AMB alone were formed mainly green fluorescence inside metabolically active hyphae,after treating with CRA-AMB combinations,the hyphae were mostly dead with a red green fluorescence,furthermore,the hyphae of biofilm were decreased and thus the reticular structures of biofilms became sparse.Conclusions:Aspergillus fumigatus can form early and mature biofilms stably after 24h and 48h;the main active component of Honeysuckle(chlorogenic acid,CRA)would not achieve levels that would be effective against A.fumigatus biofilms,but chlorogenic acid can disrupt the early and mature A.fumigatus biofilms,when combined with AMB,it can significantly enhance the fungicidal activities of AMB to A.fumigatus biofilms in vitro.PART II THE INTERFERENCE MECHANISM OF CHLOROGENIC ACID ON ASPERGILLUS FUMIGATUS BIOFILM IN VITROObjective:To investigated the influences of chlorogenic acid on hydrophobin genes,drug efflux pump genes and azole target enzyme genes in A.fumigatus biofilm.Methods:A.f004 was applied as test stain as before.The experiments were divided into untreated controls and serially increasing concentrations of CRA(128?g/ml,256?g/ml,512?g/ml,1024?g/ml).Quantitative real-time PCR(qRT-PCR)experiment was used to measure the mRNA relative expressions of hydrophobin genes(RODA,RODB,RODC,RODD,RODE,RODF),drug efflux pump genes(AfuMDRl,AfuMDR2,AfuMDR3,AfuMDR4,atrF)and azole target enzyme genes(CYP51A,CYP51B)in A.fumigatus biofilm treated with CRA.Results:Hydrophobin genes expression analysis of A.fumigatus biofilm formation at different CRA concentrations.A significant effect on RODA gene expression was observed for A.fumigatus biofilm formation,with all CRA concentrations(128?1024?g/ml)tested.Similarly,RODB,RODC,RODD,RODE and RODF genes expressions showed a decreased at series increasing concentrations(256?1024?g/ml)of CRA with significant differences with respect to control treatments(P<0.05);a slight inhibitions of RODB,RODD and RODE was observed at 128?g/mL CRA compared with untreated controls(P<0.05).Dose-dependent suppression of gene expression was observed for all hydrophobin genes,with the lowest RODA expression at the highest concentration of CRA.Relative quantification of various pumps and resistance genes(AfuMDRl,AfuMDR2,AfuMDR3,AfuMDR4 and atrF)in A.fumigatus biofilm treated with CRA demonstrated a down-regulated expression to varying degrees in two concentrations(512?g/ml and 1024?g/ml)of CRA(P<0.05).As the results,gene expression was found to decreased with increase in concentration of CRA treatments.Following exposure to CRA at concentration of 1024?g/ml suppressed the expression level of AfuMDR3 most obviously.Compared to the untreated controls,AfuMDR2 and AfuMDR3 genes expressions with CRA at a concentration of 256?g/ml had a significance decrease(P<0.05).Pretreatment with CRA(concentrations of 512?g/ml and 1024?g/ml)resulted in consistently decreased in CYP51A and CYP51B genes expressions(P<0.05),the levels of CYP51A transcripts decreased obviously(P<0.01).Differences in the expression levels of CYP51A and CYP51B after exposure to CRA at a concentration of 256?g/ml are shown,CRA of 256?g/ml was observed slight suppressed the expression of CYP51A alone(P<0.05),while had no effect on CYP51B.Conclusions:Sub-minimum inhibitory concentration(sub-MIC)levels of CRA can induce suppression expressions of hydrophobin genes,drug efflux pump genes and azole target enzyme genes in Aspergillus fumigates biofilm,which performed a concentration-dependent decrease.