Treatment Of Candida Infection By Screening Small Molecule Drug Libraries And Combination Therapy

Objective:Candidiasis is a common fungal infection in hospitals,characterised by high morbidity and mortality.Candida albicans and Candida auris are the important pathogens of candidiasis.The limited number of available antifungal drugs（azoles,polyenes,echinocandins and pyrimidines）,and classic antifungal drugs have many drawbacks,including high resistance to clinically isolated Candida,narrow antifungal spectrum,adverse effects and poor therapeutic results.To find a safe and effective antifungal therapy has become an urgent problem.The common approaches to the problem of drug resistance are either the search for new antifungal drugs or the use of existing drug combinations.This project aims to provide a new theoretical basis for clinical Candida drug resistance from each of these approaches.Methods:1.Drug libraries screening to search for new antifungal agents:Preliminary screening of drug libraries:In this study,the primary screening of Candida albicans SC5314 strain was conducted from the small molecule drug libraries.Compounds that inhibited the growth of planktonic by at least 80%were identified as“hits”.Then,the antifungal activities of compound s identified as“hits”in the primary screening was confirmed by dose-response assays,XTT test,scanning electron microscopy and confocal laser scanning microscopy.2.Study on the activity of chlorhexidine acetate combined with fluconazole against Candida auris:Antiplanktonic activity was studied using the EUCAST methodology and time–kill test.Antibiofilm effectiveness was determined by the crystal violet method,checkerboard microdilution assay,scanning electron microscopy and confocal scanning laser mi croscopy.Results:1.584 small molecule compounds were screened against Candida albicans SC5314.We identified 42 candidates that showed greater than 80%fungal-growth inhibition at a concentration of 133μM.5 compounds（U73122,Disulfiram,BSK805,BIX01294,GSKJ4）were found effective against Candida albicans SC5314,Candida auris CBS10913 and fluconazole-resistant Candida auris CBS12373.Disulfiram showed the strongest antifungal activity against both biofilm and planktonic forms of tested strains.The BMIC₅₀ and BMIC₈₀ results（mg/L）for disulfiram,respectively,were as follows by species:Candida auris CBS10913（32,128）,Candida auris CBS12373（32,64）,and Candida albicans SC5314（128,>256）.Biofilm formation was further monitored by SEM.The control group of Candida albicans SC5314 showed a mature biofilm structure composed of yeast,pseudohyphae,and hyphae.Candida auris CBS12373 and Candida auris CBS10913 did not show multiple morphologies,growing only in elliptical,smooth,and regular yeast forms.After treatment with disulfiram,wrinkled and aggregated yeasts were seen in Candida albicans SC5314.Similar morphological changes in Candida auris CBS12373 and Candida auris CBS10913 were also observed with a poor biofilm structure.Confocal microscopy showed that the biofilm biomass was significantly reduced by disulfuram stimulation.2.The results indicated that the 80%minimal inhibitory concentrations for fluconazole alone against Candida auris was 2–32 mg/L and for chlorhexidine acetate was 2–8 mg/L.The combination of fluconazole with chlorhexidine acetate exhibited synergism with time kill test.In addition,the checkerboard microdilution assay presented that fluconazole was strongly synergistic with chlorhexidine acetate（FICI<0.1875）in inhibiting the growth of Candida auris biofilms.After treatment with FLC/CHX combination,the cells were destroyed and merged,showing aggregated and flattened cells.At the same time,the biofilm morphology was completely destroyed.CLSM images revealed that the number of livin g cells in FLC/CHX group was evidently inhibited,and the biofilm thickness was reduced from 40μm to 25μm.Conclusion:In summary,we identified five compounds that showed antifungal activity by screening small molecular libraries.Disulfiram inhibited the growth of both planktonic and biofilm formation in the tested strains of Candida.In addition,this study also confirmed the strong synergistic effect of chlorhexidine acetate and fluconazole in the treatment of Candida auris infection.These results have important significance for the treatment of drug resistance of Candida strains in clinic.However,this study has many limitations,because only a small number of Candida strains were tested,and most of the studies were limited to in vitro experiments,without fully considering the immune system,drug metabolism and other factors in vivo,so the potential mechanism of specific application in vivo still needs further research and analysis.