Comparison Of Phenotypic And Phylogenic Characteristics Of Pathogenic Trichosporon And Interspecific Identifying Methods

For many years,Trichosporon is known as the pathogen of white piedra only. Recently,with the increasing number of immunosuppressive hosts,the proportion of invasive infection due to genus Trichosporon represented by T.asahii has increased obviously.Since early classification of this genus is not clear enough, and lack of biochemical data as well,it is difficult to identify accurately the strains to species level and to compare the pathogenicity and susceptibility to antifungal compounds interspecies.It is very important to have accurate interspcies identification,which can help us to understand the clinical features such as epidemiology,infectious location,clinical manifestation,and treatment strategies. In this research,Trichosporon inkin strain isolated from one case of vaginal colonization was identified,and further studies focusing on phenotype characteristics,phylogenetic evolution characteristics and means of interspecific identification of the human-pathogenic-related Trichosporon spp.were performed as well.Part 1.A case of vaginal colonization due to Trichosporon inkin was presented, meanwhile the clinical and experimental studies related to the strain were carried out.A 34-year-old female was presented with the increasing of vaginal discharge and local abnormal odor for two months.Gynecological examination revealed white homogeneous secretions attached to mucous membrane of the vagina. Nugent scores of vaginal discharge were 5 to 6 and stramineous,reductus,and yeast-like colonies were isolated twice.The strain was growing in 42℃.By the slide microculture of corn agar,appressorium on the top of hypha and typical sarcinae were observed.Yeast malt agar was the optimal growth medium of the strain.The urea enzyme test of the strain was positive,while the API 20C AUX biochemistry test and gene identification based on molecular sequencing were consistent with Trichosporon inkin.The isolate was sensitive to amphotericin B and azoles such as clotrimazole and fluconazole,while resistant to flucytosine and caspofungin.This is the first case of vaginal colonization by T.inkin in China. The accurate identification of the species relies on the aggregate analysis based on phenotype characters,physiological test,and molecular sequencing.Part 2.We studied the molecular characteristics and intraspecific genotyping of Trichosporon inkin strains isolated from different sources with internal transcribed spacer(ITS) and the intergenic spacer 1(IGS1) regions of rRNA gene sequencing,PCR-restriction fragment length polymorphisms(PCR-RFLPs),and random amplified polymorphic DNA(RAPD) of genomic DNA in this part.The sequences of rDNA-ITS/IGS1 regions were 100%identical in 3 strains,while restriction fragment patterns of rDNA-ITS/IGS1 showed high similarity intraspecies as well.The RAPD finger printing of the 3 isolates amplified by primer M13,OPA4 and OPA13 displayed intraspecific variability in some degree. Our researches reveal that the sequencings of ITS/IGS1 and PCR-RFLP of rRNA gene can be used to identify T.inkin,while RAPD of genomic DNA is more suitable for intraspecific genotyping.Part 3.14 strains of 8 species of Trichosporon were isolated from clinical specimens,which including T.asahii,T.asteroides,T.cutaneum,T.inkin,T. dermatis,T.domesticum,T.jirovecii,and T.debeurmannianum.To compare the differences of 8 species on biology,morphology,physiology and biochemistry,we used methods such as optimal growth medium test,single colony morphology by stereomicroscope,slide microculture of corn agar,temperature tolerance test, urea enzyme test,cycloheximide tolerance test,biochemical identification test, and antifungal susceptibility test in vitro.The results showed that yeast malt agar was the optimal growth medium for most species.There were no significant difference on general morphology of colonies among the eight species,but for single colony,each species had unique morphotype.Special structures such as appressorium,megaspores and sarcinae could be observed on some species. Hyphae configuration and budding model were helpful on the morphological identification.There were some differences on temperature and cycloheximide tolerant tests among the 8 species but were not enough to identify the all species. Accuracy of biochemical identification of T.asahfi and T.inkin by API 20C AUX reached 100%,but reliability were only 80%and 66.7%,respectively.Other species except T.asahii and T.inkin couldn't be identified accurately by biochemistry identification.All the strains were resistant to flucytosine and caspofungin,while most strains of T.asahii were resistant to amphotericin B, fluconazole and itraconazole.Most stains of Trichosporon spp.were susceptible to voriconazole except the 3 strains of T.dermatis.And 3 strains of T.dermatis were resistant to all azoles but suscepitible to amphotericin B.The evidences indicate that single colony morphology by stereomicroscope is effective to identify the 8 species,while accuracy of biochemical identification on genus Trichosporon is poor.Trichosporon spp.displayed different susceptibility to different antifungal compounds.Accuracy interspecific identification and antifungal susceptibility test in vitro are necessary and helpful to antifungal drug choice of invasive Trichosporon infection.Part 4.To study the molecular identification and phylogenetic evolution features of 8 species of Trichosporon by rDNA-ITS and IGS1 sequencings and phylogenetic tree construction,genotyping T.asahfi originated from China and comparing phylogenetic relations between the strains of T.asteroides and of T. japonicum.The results revealed that rDNA-ITS and IGS1 sequencings could identify 14 strains of 8 Trichosporon species,and phylogenetic tree based on rDNA-ITS could distinguish most human-pathogenic-related species of genus Trichosporon except T.asteroides and T.japonicum,while phylogenetic tree based on rDNA-IGS1 could differ all the pathogenic species.Most of T.asahii originated from China belonged to genotypeⅠandⅣ,while 2 strains were found to belong to the new genotypeⅦ.There are much confusion on the molecular identification of T.asteroides and T.japonicum,while the clinical strain of T. asteroides in our research may be a new species or variants between T. asteroides and T.japonicum.Part 5.In order to identify the different species by non-sequencing method,we constructed the multi-restriction fragment patterns of rDNA-ITS and rDNA-IGS1 regions of 8 species of Trichosporon,and then cluster analysis were held too.The results revealed that rDNA-ITS-multi-RFLP could identify the 8 species into 4 subgroups,while rDNA-IGSI-multi-RFLP could effectively identify all the 8 species,and even some genotypes of T.asahii which have relative long phylogenetic distance.The results indicate that rDNA-ITS/IGS1-multi-RFLP is an accurate,rapid,convenient and inexpensive method on interspecific identification of genus Trichosporon,which can be widely used in clinical routine laboratory.