Establishment Of A In Vitro Flow Model Of Aspergillus Fumigatus Biofilm And The Effect Of Chlorogenic Acid On It

Objective:To screen out the best condition to establish a stable flow chamber model of aspergillus fumigatus biofilm in vitro.Make a comparison of biofilm formation ability between static and flow models of aspergillus fumigatus biofilm.Explore the effect of chlorogenic acid and its combination with amphotericin B on aspergillus biofilms in flow chamber system.Methods:Three of aspergillus strains(A.f.4,A.f.7,A.f.8),which were found to have the best biofilm formation ability was cultivated in the static system,under temperature of 25℃,30℃,37℃,with different medium,respectively,RPMI-1640,RPMI-1640+10%fetal bovine serum(FBS),DMEM,DMEM+10%FBS.Chosed the best conditions above and cultivated aspergillus fumigatus conidia with concentration of 1×10~5/ml,1×10~6/ml,1×10~7/ml in the flow chamber apparatus under these conditions.The growth of aspergillus fumigatus was observed under an optical microscope and fluorescence microscope,including spores adhesion,germination,and mycelial growth.Found out the optimal growth conditions for aspergillus fumigates formation in flow chamber system.A flow and static aspergillus biofilms model was established in vitro under the optional condition respectively.Biofilm extracellular matrix(ECM)was stained the with FITC-Canavalia ensiformis(FITC-Con A)dye at the defined time of 8h,16h,24h,48h,72h.Three-dimensional(3D)image of aspergillus fumigatus biofilm was observed and captured the under laser scanning confocal microscope(CLSM).Index of biofilm,including the substratum coverage,biomass,average thickness,roughness coefficient,average diffusion distance and surface area to biomass of different time points of two groups were calculated by COMSTAT software and compared with repeated measures analysis of variance.A flow system model of aspergillus fumigatus biofilm was established in vitro,dividing into four groups,control group(with fungi,but no dosing),chlorogenic acid group,AMB group,and a group combined chlorogenic acid with AMB.When cultivated for 48hours,drugs were added to different groups,while control group without drug.After 24 hours’interaction,biofilm morphology was observed under CLSM and3D pictures were taken and analyzed by COMSTAT software.ANOVA analysis was used to compare the substratum coverage,biomass,average thickness,roughness coefficient,average diffusion distance and surface area to biomass between each group.Results(1)A.f 4 strain grew well in the RPMI-1640 medium with the inoculation concentration of 1×10~6 conidia/ml in the flow chamber apparatus under 37℃.Under the light microscope and fluorescence microscope,we could see conidia adhere to the vector and germinate at 6-8h and branch at a 45° angle,hyphae extend normally,growing into a grid-like structure at 16h,and become a relatively dense three-dimensional structure with abundant extracellular matrix at 24h and it increased as time follow.Either in the static system or in flow system,we could see the aspergillus fumigatus biofilm develop with extracellular matrix increased under CLSM with ECM staining.COMSTAT quantitatively analysis showed that,in the static system,biomass of biofilm grew from(0.210±0.090)μm~3/μm~2 at 8h up to(18.602±1.123)μm~3/μm~2 at 72h;average thickness grew from(0.153±0.065)μm at 8h up to(26.342±1.064)μm at 72h;roughness coefficient from(1.973±0.016)at 8h decreased to(0.397±0.085)at 72h.In the flow system,biomass grew from(0.173±0.043)μm~3/μm~2 at 8h up to(7.634±1.721)μm~3/μm~2 at 72h;average thickness from(0.128±0.056)μm at 8h increased to(11.589±2.093)μm at 72h;roughness coefficient from(1.972±0.016)at 8h decreased to(0.656±0.158)at72h.Biomass,average thickness,substratum coverage and average diffusion distance increased as time prolonged.Otherwise,roughness coefficient and surface to biovolume ratio decreased with time.When compared each index between two models at a certain time piont,biomass,average thickness,average diffusion distance substratum coverage of biofilm was significantly lower in the flow system than that in the flow system,P<0.05;while roughness coefficient and surface to biovolume ratio of bioflm was lower in static system than that in flow system,the former with significantly difference,P<0.05.,but the latter had no significant difference,P>0.05.(2)When interaction with 48 hours’aspergillus biofilms biofilm for 24 hours’intervention.After ECM staining,we could see that,compared with the control group,AMB alone had no significant changes in ECM or mycelial density;in the chlorogenic acid group,mycelium was smooth with reduced ECM and space between hyphea became larger;in combination with chlorogenic acid and AMB,mycelium was significantly reduced with hardly ECM.COMSTAT software analysis showed that,biomass,average thickness and average diffusion distance between AMB group and the control group,has not statistically difference,P>0.05;while it was lower in CRA group with significant difference,P<0.05;and further reduced in combination with chlorogenic acid and AMB,P<0.05.And the roughness coefficient was versa.Conclusions:Our experiment has successfully established an in vitro biofilm flow model of aspergillus fumigatus with the flow chamber system.A.f4,1×10~6/ml conidia,37℃,RPMI-1640 medium is the best choice for this experiment modeling.Compared with the static model,Aspergillus fumigatus in flow model secreted less extracellular matrix.But the flow system model is much similar to the catheter-related biofilm infection in human body.In the flow system model,chlorogenic acid can destroy the extracellular matrix of the Aspergillus fumigatus biofilm,which can increase the penetration of AMB and enhance the fungicidal effect on Aspergillus fumigatus biofilm.