Investigations On The Growth Of Candida Albicans-Candida Glabrata Mixed Biofilm And Associated Resistance Mechanism

Objective:Explore the effects of different conditions on the formation of single biofilm,mixed biofilm by Candida albicans and Candida glabrata,and the relationship between quantity and virulence.Methods:In this study,eight C.albicans isolates as well as two C.glabrata strains were used to investigate the effects of culture medium(RPMI-1640,YPD,YND),incubation time(24 h,48 h,72 h,96 h),initial inoculum(C.glabrata: C.albicans = 2:1,1:1,1:2),and medium state(static and dynamic states)on viable cell enumeration and relative abundance in both Candida SB and MB.Two strains of C.glabrata and eight strains of C.albicans were used to test the number of SB / MB-forming cells at 24 and 48 hours,and the sensitivity test of MB to caspofungin,secreted aspartic proteinase(SAP)and phospholipase(PL)levels,efflux pump function,and ?-glucan exposure.XTT analysis and spot assay to detect the resistance of Candida to the antibacterial drug caspofungin,crystal violet staining to observe the total biofilm,ELISA kit to detect the content of secreted aspartic protease and phospholipase.The fluorescent image was observed with an inverted fluorescence microscope IX71(Olympus,Tokyo,Japan)and the fluorescent intensity was measured with BD Accuri TM C6 flow cytometer(Shanghai,China)at 488 nm excitation wavelength and 525 nm emission wavelength.Efflux related gene expression was detected by q RT-PCR.Results:In most cases,C.glabrata and C.albicans SB and MB flourished in RPMI-1640 at 24 h under dynamic state compared with other conditions.Except YPD medium,there were high proportions of preponderance of C.albicans over C.glabrata in MB comparedwith SB.High initial inoculum promoted corresponding Candida number in both SB and MB and its abundance in MB.Through this study,the declines of individual cell counting were observed in the 24-and 48-h Candida mixed biofilms compared with their single counterparts.However,the antifungal resistance to caspofungin,the SAP and phospholipase levels,the rhodamine 6G efflflux and the efflflux-related gene expressions were increased signifificantly(*p < 0.05;**p < 0.01;***p < 0.001)accompanying with reduced?-glucan exposure in the mixed biofilms by comparison with the single counterparts.Conclusion:We found that in most cases,these conditions had varying impacts on the populations of C.albicans and C.glabrata in their SB and MB,and C.albicans was preponderant over C.glabrata in MB.We demonstrate that C.albicans and C.glabrata can grow together and form mixed biofilm with competition,the virulence of the mixed biofilms is not weakened compared with their single counterparts in terms of the dramatic shrinkage of each Candida species population(at least within 48 h of incubation).