Genotyping And Antifungal Susceptibility Research Of Sporothrix Schenckii Isolates From Different Regions Of China

Objective：The purpose of our study was genotyping and phylogenetic analysis of differentparts of our country clinical isolates of sporotrichosis；In this study we aimed to studyof antifungal susceptibility of thermally dimorphic fungus—Sporothrix globosa whichwere respectively in the hypha phase (30℃,35℃) and yeast (35℃) for8kinds ofantifungal drugs,then compared with the drug sensitivity of different parts ofSporothrix globosa.Methods：1. We took four methods, including Cetyltrimethyl Ammonium Bromide (CTAB)method, Guanidine thiocyanate boiling method, alkaline lysis method and DNAextraction kit, to extract DNA from the samples which were choosen5strains fromChangchun, Guangzhou, Chongqing, Chengdu, Beijing, Nanjing.Then by testing itsnucleic acid concentration,purity and comparing stripe brightness of the productswhich were through PCR amplification,we could choose the best method.2. We amplified the calmodulin-encoding (CAL) gene of43isolates,then thesequences were sent to Genbank database and detected the molecularidentification,subsequently with15isolates reported in the literature were used forneighbor-joining analysis performed using MEGA5.0software3. According to a modified CLSI mierodilution method M-38A and M27-A2which were slightly modified combing with the actual situation,we tested thesusceptibility of these isolates which respectively in the hypha phase (30℃,35℃)and yeast (35℃) to flueonamle, itraeonazle, terbinafine, ketoconazole, econazole, miconazole, voriconazole and amphotericin B. Based on these datas,we calculated theminimum inhibitory concentration (MICs)、MIC50、MIC90and GM，and comparedthe difference via the statistical software.Results：1. We compared the nucleic acid concentration and purity from four methods,including Cetyltrimethyl Ammonium Bromide (CTAB) method, Guanidinethiocyanate boiling method, alkaline lysis method and DNA extraction kit. Itconcluded that the best method was alkaline lysis method.2. A fragment of the CAL gene that used the primers CL1and CL2A to amplifywas approximately770base pairs (bp).The sequences of all isolates revealed a highlevel of sequence similarity with previously reported S. globosa strains. And by usingneighbor joining(NJ)method the phylogenetic tree of the CAL locus was set to showsix well-defined as previously described and all clinical isolates originated in China inthis study were clustered with group S. globosa.Besides the above, S. globosa couldbe further subdivided into two sub-clades.3. In Vitro Susceptibilities of Isolates of Sporothrix globosa(1) Regardless of the mycelial phase or yeast phase, TBF both showed the bestantifungal activity, MICs rang were0.03to0.25μg/ml, GM was0.04μg/ml, followedby MCZ, ECZ, KCZ, MICs respectively rangs were0.03-1.0μg/ml,0.03-1.0μg/ml,0.03-4.0μg/ml, while MICs could fluctuate with the different temperatures and phases.(2)SPSS statistical analysis software showed that the MICs of different regionswere no significant different (P>0.01). Compared with MICs of hypha phase (30℃,35℃) and35℃yeast phase’, they had significant differences(P<0.05), and the yeastphase showed higher MICs than hypha phase, and hypha phase30℃were higher MICsthan35℃. Conclusion：1. Alkaline lysis method is the best method that is easy to operate and time-savingto get high quality, concentration and purity of DNA of S. globosa.2. S.globosa is the dominating etiologic agent in different regions of China andmost of the clinical isolates come from China environment. There are no differencesamong the different regions.3. S. globosa strains of different parts for TBF are the most sensitive, followed byMCZ, ECZ, KCZ and ICZ, for FCZ, VCZ and AMB showing highMICs, regionaldifferences are not obvious. S. globosa is influenced by temperature and the phase offungi in vitro susceptibility test. Except for TBF, hypha phase MIC values aregenerally higher than yeast phase, MIC values in the hyphae30℃are highest,followed by35℃, yeast phase shows lowest.