A Study On Phenotyping And Ribosome Genotyping Of Trichophyton Mentagrophytes

Trichophyton mengagrophytes is one of the cosmopolitan dermatophyte species most easily isolated from humans and animals. The clinical manifestations of tinea capitis, tinea unguium, and tinea corporis et al. caused by Trichophyton mengagrophytes are many and varied, often with obvious inflammation. Traditional fungal culture is still the main method for etiology typing of Trichophyton Mentagrophytes. However, phenotype is easily influenced by subjective and objective factors, and could not meet the need of scientific researches. In recent years, the genotyping at molecular biology level has been greatly developed, such as random primer PCR (AP-PCR), random amplified polymorphic DNA analysis (RAPD) and restriction analysis of mtDNA (RFLP). In the study, we tried to subtype Trichophyton Mentagrophytes within species by conventional method combined with molecular biological method of southern blotting, and explore the relationship between phenotype and genotype of Trichophyton Mentagrophytes. Objective: to subtype Trichophyton Mentagrophytes within species by conventional method combined with molecular biological method of southern blotting, and explore the relationship between phenotype and genotype of Trichophyton Mentagrophytes. Materials: One standard isolate was obtained from American Types Culture collection (ATCC0236). Thirty-five clinical isolates of Trichophyton mentagrophytes were adopted,14 of which were from DaLian, 5 from BeiJing, 5 from ShangHai, 3 from NanNing, 3 from ShenYang , 2 from HeBei, 2 from KunMing and 1 from Hang Zhou. Method:1.Phenotyping of Trichophyton Mentagrophytes is based on colonial morphology and structure under microscope, and identification by urease test, glucose rice flour agarose test and in vitro hair perforation test. 2. DNA was extracted using cetyltrimethyl ammonium Bromide (CTAB) method. The polymorphisms were detected by hybridization of EcoR1-digested Trichophyton Mentagrophytes genomic DNA with a probe amplified from the small-subunit rDNA and adjacent inernal transcribed spacer (ITS) regions. The probe was made using the standard isolate of Trichophyton Mentagrophytes as template, and a pair of dermatophyte-specific primers NS5: [5'-AACTTAAAGGAATTGACGG AAG-3'] and ITS4: [5'-TCCTCCGCTTATTGATATGC-3']. Then the probe was labeled with ECL by the random primer method. The band patterns obtained from results of Southern blotting were used as the basis for genotyping. Results: 1. The 35 clinical isolates were classified into 5 phenotypes according to the colonial morphology and structure under microscope: cottony, powdery, persicolor, granular and ulotrichy type. Among which, the ulotrichy, cottony and persicolor prevailed, and accounted for 71.43%; the granular was rare. Powdery type was seen exclusively in northern isolates, while the other four types distributed diversely, without obvious geographical differences. 2. Fourteen individual patterns (DNA type A to N) were recognized among 35 strains of T. mentagrophytes. Type A to D accounted for 62.86% of all strains. Southern strains were represented by Type A and C, no Type B or D was found; while northern strains were represented by Type B and D. Results suggested that two universal bands of 1.2kb and 0.8kb were seen in all the strains investigated; while the northern strains still had another 2.5kb universal band.Conclution: 1. The study for the first time typed T. mentagrophytes into five subtypes according to phenotype based on the colonial morphology and structure under microscope. 2. The study is the first intraspecies genotyping of T. mentagrophytes by hybridization of ribosome DNA with a probe and Southern blotting. 3. Fourteen individual patterns (DNA Type A to N) were recognized among 35 strains of T. mentagrophytes. Type A to D accounted for 62.86% of all the strains. 4. The DNA patterns of southern strains are obviously different from those of northern strains, exhibiting geographical differences.