| Background and ObjectiveTrichosporon spp. exsit widely in nature, comprising 50 species, of which 16 species can cause human infection.The number of infections caused by Trichosporon spp. account for 10% of the disseminated fungal infections.Trichosporon asahii is the common causative agent of Trichosporon spp. infections,and can lead to systemic and fatal trichosporonosis,especially in immunocompromised and immunodeficient hosts,such as people suffering from neutropenia,organ transplantation,hematologic malignancies, extensive burns or AIDS.With the widespread use of antifungal drugs, especially of azoles, the antifungal resistance of T. asahii has been increasingly reported.This has made clinical doctors difficult to deal with trichosporonosis,and the development of new antifungal agents often require long period.Therefore,it is significant to explore the fluconazole resistance mechanisms and find out new targets of antifungal agents.These need to establish a stable azole-resistant model of T. asahii.Resistant models acquired by induction in vitro often present low success rate and instable resistance,so it is imperative to establish a method which can acquire stable resistant strains successfully.In this study, we obtained fluconazole-resistant model of T. asahii which were cultured in mediums containing methylprednisolone and fluconazole,and evaluated the success rate and stability.Methods1.16 clinical and environmental strains of T. asahii were tested the susceptibility against fluconazole in vitro by CLSI M27-A3;2.Based on the susceptibility results,the fluconazole sensitive strains were induced resistance to fluconazole by individual fluconazole, individual methylprednisolone, and fluconazole combinated with methylprednisolone respectively,comparing with the control group;3.In order to observe the stability of azole-resistant strains, isolates found to exhibit FLC resistance were serially cultured 30 times in drug-free medium. After each subculture, antifungal susceptibility testing was performed;4.In order to measure the development of cross-resistance to other antifungal agents,the stable fluconazole-resistant strains were performed MICs assays with four antifungal agents,such as voriconazole,itraconazole, amphotericin B and caspofungin.Results1.16 strains of T. asahii were susceptible to FLC.The MICs range of these strains were from 0.125μg/mL to 4μg/mL;2.12 FLC resistant strains(75%) were obtained by single fluconazole induction, the MICs range were from 16μg/mL to 64μg/mL,both MIC50 and MIC90 were 64μg/mL.16 FLC strains(100%) were obtained by fluconazole induction combined methylprednisolone,the MICs were above 64μg/mL.The MICs range of strains in methylprednisolone group were from 0.25μg/mL to 4μg/mL,MIC50 was 0.5μg/mL,and MIC90 was 4μg/mL.The MICs range of strains in control group were from 0.5μg/mL to 4μg/m L,MIC50 was 1μg/mL,and MIC90 was 4μg/mL;3.After 30 passages of growth in drug-free medium, 6 strains(37.5%) presented fluconazole resistance in fluconazole group, and 9 strains(56.25%) presented fluconazole resistance in methylprednisolone combined fluconazole group;4.The MICs of AMB for 6 FLC resistant strains and their previous generations did not vary significantly and were at non-resistant level.The MICs of CAS were at non-susceptible level.The MICs of ITC and VRC for the strains paralleled the increases of FLC MICs.ConclusionsFluconazole induction combined methylprednisolone overmatch single fluconazole inductionin construction of in vitro fluconazole resistance model of T. asahii. Methylprednisolone can contribute to improve the successrate and stability of FLC resistant strains of T. asahii,... |
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