| BackgroundRecent30years, with the application of hormone and immunodepressant, and increase of malignant tumor, a large area of burning, organ transplantation, hematonosis and HIV, which had been the major cause of these patient. Recent20years, except for Candida albicans, Aspergillus fumigatus and cryptococcus neoformans, another important pathogenic fungi——the infection rate of Trichosporon asahii (T.asahii) present upgrade tendency yearly. Trichosporon asahii is an opportunistic fungal pathogen that exists widely in the natural environment and colony in some organ of human. T.asahii generally enters the human body and carries diseases such as superficial skin infections, summer hypersensitivity pneumonia, and dissemination infections of the internal organs through built-in catheter, drainage, damaged burned skin, translocation of intestinal mucosa microbials, and respiratory tract inspiration. The patients of immunosuppressed, immunocompromised, or immunodeficient is the susceptible population of T.asahii infection, however, the reports about T.asahii disseminated infection has been more and more.It has shown by domestic and international research that T.asahii exhibits drug resistance to most antifungals, such as amphotericin, Flucytosine and caspofungin, and is sensitive to little antifungals, such as Pyrroles drug——Voriconazole, fluconazole and itraconazole. So its infection is difficult to treat and mortality rates is high. Current research data shows that the mortality rates of T.asahii invasive is still as high as80-90%. However, over the last ten years, the reports about T.asahii resistance to azole drugs are more and more. Susceptibility testing in vitro has indicated that some clinical isolates of T.asahii are not sensitve to Fluconazole. T.asahii resistance to azole make its treatment and prevention become more difficult. So, Azole resistance mechanisms of T.asahii become very important and very urgent. Currently azole resistance mechanisms of T.asahii are unclear, resistant strains is the premise and foundation to study the resistance mechanisms, resistant strains is acquired by clinical isolation and laboratory induced in vitro. It is difficult to acquire clinical isolates that clinical isolation is influenced by many factors, and it is more difficult to acquire the sensitive parents and resistant progeny of the same strain, which are influenced on the reliability of research. Acquired resistant strains by laboratory induced in vitro has the advantage of good handling, easy acquisition resistant progeny which has the same genotype as sensitive parents, this method has been extensively application in research of other fungus.In the past, the research of T.asahii focused to clinical isolates, no drug susceptibility research in vitro about environment isolates and clinical isolates, it is also no report about T.asahii resistant strain acquisition by antifungal agents induced in vitro. Firstly, this study determine the drug susceptibility of T.asahii isolated from different sources, screen out the sensitive clinical isolates and environment isolates, then induce the sensitive clinical isolates and environment isolates to acquire resistant progeny strains with Fluconazole by concentration cascade, lastly take on the recovery study of resistant progeny strains to observe their stability, lay on the foundation for studying the resistance mechanism of T.asahii.ObjectiveInvestigate the differences of drug sensitivity in vitro of T.asahii from different sources, screen out the most sensitive clinical isolates and environment isolates, induce the sensitive clinical isolates and environment isolates with Fluconazole, acquire the resistant progeny strains, proceed the recovery study of resistant progeny strains to observe their stability.Methods1. The12T.asahii strains preserved in our laboratory are as the study object, prepare the suspensions, smear on the PDA medium, paste the E-test Reagent strip containing different antifungal agents on the PDA medium, after48h, determine and record the MIC value of every strain, pick out the most sensitive strain from different sources.2. The two sensitive strains were respectively and serially subcultured in PDA medium containing growing concentrations of Fluconazole. The susceptibilities of parental strains and their resistant filial generations to fluconazole were measured respectively by E-test. Up to the MIC>256μg/ml;3. The resistant filial generations of MIC>256μg/ml were subcultured18days in drug-free medium, the stability of fluconazole resistance of each generation were measured in MIC, observe their resistant stability.Results1. All experiment T.asahii strains were innate resistance to Fucytosine and Anidulafungin, their MIC exceed32μg/ml; All T.asahii had instability sensitivity to amphotericin B, the MIC of most strains exceed32μg/ml, the MIC of CBS2479, BZP07005R, BZP09001are0.25μg/ml,1.00μg/ml and1.00μg/ml; Yet all T.asahii were sensitivity to Pyrroles, especially to Voriconazole, the MIC of most strains are less than0.016μg/ml, the highest MIC of BZP09001is not less than0.125μg/ml; Environmental isolates were more sensitive to Pyrroles than clinical isolates, the MIC of all environmental isolates are less than0.006μg/ml. CBS2479and CBS8904are the most sensitive strain in the different sources strains.2. Fluconazole-susceptible T.asahii CBS2479/CBS8904(MIC=0.25μg/ml,MIC=1.5μg/ml) were successfully induced by fluconazole to generate fluconazole-resistant T.asahii CBS2479R/CBS8904R(MIC>256μg/ml); The MIC value was rasied to170-1024times than that of before induction.3. The resistant filial generations—T.asahii CBS2479R was maintaining a fluconazole resistance when it was subcultured in drug-free medium for18days consecutively; by contrast, the MIC of CBS8904decreased gradually to64μg/ml at the time of18th day, which decrease to4times than that of before recovery.ConclusionsThe T.asahii from different sources had different drug sensitivity to different antifungal agents, and clinical isolates and environmental isolates have significant differences, environmental isolates have higher drug sensitivity; Fluconazole can induce fluconazle-resistant sub-generation T.asahii regardless of their origins. Different types of drug-resistance parental strains show distinct stabilities of fluconazle-resisinduce... |
PDF Full Text Request