Effects Of Microevolution On Virulence Factors And Drug Susceptibility Of Trichosporon Asahii

OBJECTIVEWe isolated two clinical strains of Trichosporon asahii(T.asahii)from the same patient successively(with an interval of 14 years),and two clinical strains made up an ideal model for the study of microevolution of Trichosporon asahii in the host.In this study,we aim to explore effects of microevolution on virulence factors and susceptibility of triazoles antifungal agents in Trichosporon asahii.METHODS1.The phenotypic characteristics of T.asahii orignal strain(TO)and microevolutionary strain(TEVO)were observed,including temperature tolerance,growth curve,general morphology,colony morphology,mycelium morphology,cell morphology and biofilm structure.2.The detection for extracellular enzymes and melanin:strains were cultured on the enzyme detection medium and melanin induction medium.3.Drug susceptibility testing and analysis of ERG11 gene expression:drug susceptibility testing was conducted in accordance with the M27-A3 protocol developed by the American society of clinical and laboratory standards(CLSI).The total RNA of two strains were extracted,and then the expression level of the ERG11 gene was detected by RT-qPCR method.The SPSS 21.0 software was used to analyze the experimental data.RESULTS1.The results of phenotypic characteristics of two strains were as follows.Both two strains could grow well under the room temperature and human physiological temperature.TO could grow under a wide range of temperature from 25? to 39?,however,TEVO could grow under a narrow range of temperature from 25? to 37?.TO showed a faster speed of growth and reached the plateau phase after 24 hours,TEVO's growth rate was slower,and it took 64 hours to reach the plateau phase.After 7 days of culture on PDA solid medium,the general morphology of both two strains were milky white with gyrus on the surface,but the number of surface gyrus of TEVO was less than that of TO,what's more,TO showed a powdery surface,while TEVO had a smooth surface.Cultured on solid PDA medium,colonies of TO became mature in 48 hours,while TEVO became mature after 72 hours.TO showed a radioactive villi on the colony's surface,while the surface of TEVO colony was smooth.The cell morphology of TO was dominated by mycelium cells with a small number of yeast cells.The cell morphology of TEVO was dominated by the yeast cells with a small number of mycelial cells.Cultured on the glass slide with PDA,TO s mycelia were longer with fewer branches,and they looked like wickers.However,TEVO s mycelia were thicker with many branches,and they looked like pine branches.TO formed a mature biofilm in 48 hours,while TEVO in 72 hours.TO's biofilm structure was mainly composed of long hyphaes with a small number of yeast cells,and the biofilm structure was looser.TEVO's biofilm structure was mainly composed of mycelia cells,pseudomycelia cells and yeast cells,a large number of lateral spores and free spores filled in the space between mycelia,and the biofilm structure was denser.2.The results of extracellular enzymes and melanin induction of two strains were as follows.An opaque halo was observed on day 1 and day 2 when TO and TEVO were cultured on bovine serum albumin medium.Pz values were 0.69(++++)and 0.90(+),respectively.After growing on tween agar medium for 3 days,an opaque halo appeared around the colonies of two strains,with Pz values of 0.80(++)and 0.82(++),respectively.An opaque halo appeared on the sheep blood medium on day 1 and day 2 respectively,and the Pz values were 0.63(++++)and 0.70(+++),respectively.TO was cultured on vitelline agar medium for 10 days,and an opaque halo was observed,with a Pz values of 0.79(+++),while no halo was observed around the colonies of TEVO after culture for 2 weeks.Both two strains were grown on DAase test agar medium for 2 weeks without an opaque halo.Cultured on the plate of melanin induction,TO3 colonies were milky white with brown edges,but the overall colonies of TEVO were brown.3.The results of drug susceptibility and expression level of ERG11 gene in two strains were as follows.TO showed a minimal inhibitory concentration(MIC)of 4ug/ml for fluconazole,0.5ug/ml for itraconazole and 0.125ug/ml for voriconazole,however,TEVO showed a MIC of 32ug/ml for fluconazole,4ug/ml for itraconazole and 0.5ug/ml for voriconazole.We regarded the TO strain as control group with a expression level of ERG11 gene of 1,and the expression level of ERG11 gene of TEVO was 3.20 times of that in TO.The SPSS21.0 software was used to analyze the result of the relative expression level of the ERG11 gene between the two strains,and then the result of t=34.135,p=0.000 demonstrated that the differences in the relative expression level of ERG11 gene between the two strains was statistically significant.CONCLUSION1.Under the influence of microevolution,the phenotypic characteristics of T.asahii changed a lot,temperature tolerance and growth rate decreased,the cell morphology of T.asahii changed from the dominantly mycelium cell to the dominantly yeast cell,the mycelia became shorter with more branchy than ever,the cell morphology of biofilm structure was more diversified and denser than before..2.Under the influence of microevolution,T.asahii remained the basic survival virulence factor of hemolysis activity,reduced the protease activity intensity,abandoned the capacity of producing phospholipase and improved the ability to produce melanin.3.Under the influence of microevolution,T.asahii can make adaptive changes in response to the internal environment of the host and the pressure of antifungal drugs The manifestation in phenotypic characteristics showed a increased resistance to triazoles,such as fluconazole and itraconazole.The manifestation in molecular mechanisms showed the amplication of the targets of triazoles to limit the efficacy of antifungal drugs,such as the upgradation of the expression of ERG 11 gene.The microevolution strategies in phenotypic characteristics and molecular were conducive to its adaption to host environment and coexistence with host.