Reseach On The Morphology, Mechanisms Of Antifungal Drug Resistance And Influencing Factors Of Trichosporon Asahii Biofilm

BackgroundTrichosporon is a genus of adelomycete,a class of blastomyces,Cryptococcales. Saccharomycetaceae that is common in nature and a member of the normal flora of the mouth, skin, and nails. It causes superficial and deep infections (systematic or disseminated trichosporonosis), It can found mainly in immunocompromised patients or patients with cancer, underlying hematologicmalignancies,respiratory insufficiency, chronic renal insufficiency, diabetes, cirrhosis and AIDS.The main predisposing conditions were antibiotic therapy, corticoids,immunosuppressive therapy, chemotherapy,mechanical ventilation, urethral catheterization, catheter,,transplant, granulocytopenia, surgical procedures and continuous ambulatory peritoneal dialysis. There is also report that disseminated trichosporonosis developed in a healthy woman at the site of an open lumbar discectomy. Trichosporon asahii is the most common cause of fatal disseminated trichosporonosis.It was reported that despite the use of antifungal drugs to treat trichosporonosis, infection is often persistent and is associated with high mortality. Antifungal susceptibility testing demonstrated a remarkable rise in the MICs of sessile T. asahii cells against clinically used amphotericin B, caspofungin, voriconazole, and fluconazole compared to their planktonic counterparts. There is also report in China that a remarkable rise in the MICs of sessile T. asahii cells compared to their planktonic counterparts.What causes the drug resistance of T. asahii?. How to prevent medical devices—associated infection by T. asahii?Objective1 To construct the asahii trichosporon(T. asahii) biofilm(BF)in vitro, the morphology characteristics were observed to have a better knowledge of the mechanisms of antifungal drug resistance.2 To examine the spatial structure of asahii trichosporon biofilms,a vital fluorescence technique was combined with optical analysis of sections in a confocal scanning microscope (CLSM) to have a better knowledge of the mechanisms of antifungal drug resistance.3 To study the ability of the formation of T. asahii biofilm(T.asahii BF) in vitro on different materials. Methods1 The biofilm of T. asahii was formed in vitro,its morphology characteristics were observed under inverted microscope and scanning electron microscope. The activity of biofilm was measured and quantitatively analyzed by XTT and viable-count.2 The distribution of Dead/Vital fungals at different time,spatial distribution of Dead /Vital fungals, activity and thickness of stained biofilm at different layers were observed under confocal laser scanning microscope (CLSM).3 The biofilm of T. asahii was formed in 24-well plate by PAT,polystyrene,PVC.The activity of biofolm was measured and quantitative analyzed by XTT. The characteristics of the morphology and development of T. asahii were observed respectively under inverted microscope and scanning electron microscope.The biofilm of T. asahii cultivated for 72h formed on PAT and polystyrene were observed under scanning electron microscope.Results1 The biofilm of T. asahii in vitro was formed on the surface of polystyrene materials. With time prolonged, the structure was changed from microcolonies to complex multi-layer structure, and was enveloped by the extracellular matrix.Meanwhile, the activity and quantity of the biofilm increased by XTT,which had good correlation with viable-count.2 The biofilm of T asahii in vitro was formed on the surface of PAT materials. There has no obvious difference between diferrent layers of the vitality of BF. Depth of the BF was 14.3μm～31μm.3 Biofilm was formed on the 3 support materials.And it was obvious that extensive biofilm was formed on PAT, polystyrene and PVC.There existed differences in the vitality of biofilm(F=14.743, P<0.01).After 48 h, T. asahii formed biofilms on support mrterials in the following order of strength:PAT=PVC>PS.There existed yeast cells, pseudohyphas and hyphae on PAT and PVC,but existed yeast cells only on polystyreneConclusions1 A biofilm can form in vitro,The structure of T. asahii BF is more complex than the planktonic ones.2 The low growth rate is not one of the mechanism of antifungal drug resistance of T. asahii biofilm.3 T.asahii can form biofilms on different support materials,but the ability to form biofilm is different.It's more easier for adhension on PAT and PVC than polystyrene,and the viability of pseudohyphas and hyphae is stronger than yeast cells.