| Background:Sporotrichosis is a commonly chronic deep mycosis caused by the dimorphic fungus Sporothrix schenckii complex with world-wide distribution.It can be involved in skin and even systemic infection.Recent molecular phylogenetic studies suggested that S.schenckii represented a fungal complex comprising at least six closely related species,including S.schenckii sensu stricto,S.brasiliensis,S.globosa,S.luriei,S.mexicana and S.Pallida.Sporothrix globosa is the dominant species in China.However,there are differences in the phenotypic characteristics of isolates from China with diverse geographic distribution.And studies have shown a certain degree of genetic variability of S.globosa in China.So it is necessary to analyze the differences in genotyping,phenotypic characteristics and antifungal sensitivity.Objective:Phylogenetic and phenotypic analysis of clinical isolates of sporotrichosis in different areas of China were studied in this work.The purpose of our study was to assess the genetic diversity of S.globosa by amplified fragment length polymorphism(AFLP)and to discuss the correlation of AFLP profiles with clinical forms,geographic origins,in vitro drug susceptibility and growth rate of the S.globosa isolates in China.Methods:1.A total of 225 clinical isolates were included in this study,collecting between 2009 and 2013 from patients with sporotrichosis originating from eight provinces or municipalities in China.Calmodulin gene(CAL)of 225 clinical isolates were amplified and sequenced,then all the sequences were sent to Genbank database.Then all sequences were used for neighbor-joining analysis performed by MEGA 6.0 software.2.The reaction system of AFLP were optimizated by restriction enzyme Eco R I and Saq A I.64 pairs of primers were screened for selective amplification,and the primers with good DNA polymorphism were selected.All the 225 isolates were analyzed using the AFLP methods after the optimization.The raw data was analyzed using the NTSYS-pc version 2.10 software.3.All 225 isolates were inoculated on potato agar medium(PDA)at 30?,35?,37? for 21 days,then the colony diameters were measured at 7 days,14 days and 21 days respectively.Carbon assimilation experiments were tested for all clinical isolates.4.In vitro susceptibility tests were carried out for 8 drugs(fluconazole,itraconazole,voriconazole,terbinafine,amphotericin B,posaconazole,caspofungin and albaconazole)against mycelial phase of 43 S.globosa isolates with different AFLP genotypes,according to the M38-A2 program proposed by the Clinical and Laboratory Standards Institute(CLSI).These 43 isolates included group?(n=10)?group ?(n=10),group ?(n=2),group ?(n=9),group ?(n=4),group ?(n=2),group ?(n=3)and group ?(n=3),respectively.Results:1.After optimization the AFLP procedure was described below.Briefly,1000 ng of genomic DNA was digested with restriction enzymes Fast Digest Eco R?(1?l)and Fast Digest Saq A?(1?l)at 37? for 5 min,and then 8?l of enzyme solution was ligated to their respective adapters(1?l)with T4 DNA Ligase(2.5U)at 25? for 1h.Pre-amplification was performed in a total volume of 20?l,including 5?l of 1:20 diluted ligation products,2 m M Mg2+,0.2 m M of each d NTPs,1U Taq DNA polymerase and 1?l of 10 ?M each primers.Selective amplification was carried out in a 20?l reaction consisting of 5?l of 1:20 diluted pre-amplification products,1 m M Mg2+,0.l5 m M of each d NTP,1.5 U Taq DNA polymerase and 0.6?l of 10 ?M each primers.Moreover,selective amplification primers can use E4(GACTGCGTACCAATTCACT)and M1(GATGAGTCCTGAGTAACAA).2.225 isolates were identified as S.globosa by CAL gene,and the phylogenetic tree showed that S.globosa could be divided into three subgroups(named globosa ?,globosa ?,globosa III).A total of eight main distinct clustering groups(named from I to VIII)were identified by AFLP.Groups I(23%),II(41%)and IV(22%)were the most prevalent groups.Isolates from Changchun were separated into group II(35/60)and IV(25/60).The Siping isolates were found mainly in group II(23/31).Baicheng isolates(12/13)and most of the isolates from the Jilin city(18/20)were clustered into group I.Group III(n=2)and group VIII(n=3)included the Songyuan isolates alone.A significant association was found between the AFLP profiles and geographic origins,while no correlation with the clinical forms of sporotrichosis was found.3.The colony diameters varied from 16 to 42 mm at 30?,and 3 to 15 mm at 35? at the end point of cultivation for 21 days at PDA.When the culture temperature increased to 37?,most isolates(218/225)showed very limited growth,with the colony size varying from 1.5 to 5.5 mm in diameter,while only few isolates(7/225)could not growth at all.All isolates were able to assimilate glucose and sucrose but unable to assimilate raffinose.Against the mycelial phase of S.globosa isolates,terbinafine showed the best in vitro antifungal activity with MICs of 0.03~8?g/ml and a geometric mean(GM)of 0.05?g/ml,followed by posaconazole,albaconazole,caspofungin with MICs of 0.5~>16?g/ml,4~16?g/ml,0.25~>16?g/ml and geometric mean(GM)of 2.99?g/ml,8.26?g/ml,9.55?g/ml,respectively.Itraconazole,voriconazole,amphotericin B and fluconazole showed high MICs of 1~>16?g/ml,8~>16?g/ml,>16?g/ml,>64?g/ml.Statistical analysis showed that there were no significant differences in the MIC value of S.globosa isolates of different clinical forms,genotypes and geographical sources for every antifungal drugs Conclusion:1.Sporothrix globosa is the predominant pathogenic species in China.2.S.globosa could be divided into three subgroups by CAL gene.Globosa?is the most common subgroup in China,and globosa ? is found for the first time of S.globosa.3.Sporothrix globosa can be divided into eight main distinct clustering groups by AFLP using Eco R?-ACT and Saq A?-CAA primers.However,there was no correlation between the AFLP genotyping and the clinical forms of sporotrichosis.4.There was a significant difference in the distribution of the AFLP genotypes between northern and southern China,and the genotyping of Jilin Province isolates has a certain correlation with the distribution of cities in the province.5.Most S.blobosa showed limited growth in 37? with only a few isolates can not grow at all.All isolates were able to assimilate glucose and sucrose but unable to assimilate raffinose.Among the 8 antifungal drugs,TRB showed the best in vitro antifungal activity,followed by posaconazole,albaconazole,caspofungin.Itraconazole,voriconazole,amphotericin B and fluconazole showed high MICs.There were no significant differences in antifungal susceptibility of S.globosa isolates of different clinical forms,genotypes and geographical sources. |
PDF Full Text Request