The Taxonomy And Terbinafine Resistance Of The Trichophyton Mentagrophytes Species Complex

First chapter:Taxonomy of the Trichophyton mentagrophytes/T.interdigitale species complexObjective:In order to define species limits,a taxonomic study was undertaken combining molecular,morphological,and physiological characteristics as evidence of classification.Methods:The study included 182 strains in total,of which 162 strains originated from patients,17 from animals and 3 from soil.Geographically,strains were obtained from Europe(n=63),India(n=46),China(n=49),Japan(n=2),Australia(n=21)and U.S.A.(n=1).Multilocus data comparison using tanglegrams,combining ITS,Tef1-?,and-HMG loci,was performed with random selection of 113 strains representing all genotypes.FastANI was used to compute pairwise Average Nucleotide Identity(ANI)values among all genomes of three genotypes.Differences between groups in anonymous markers need to be supported by phenotypic characteristics of morphology and physiology including urea hydrolysis,lipolytic activity,hair perforation,keratin degradation were performed.Results:Bootstrap support of the branches remained low due to the small number of mutations basing on ITS phylogenetic tree,however,bootstrap support of the branches high in HMG gene.Multilocus data comparison using tanglegrams,combining ITS,Tef1-a and HMG loci,it is suggested that phylogenetic trees own a good consistency.The FastANI values of all pairs exceeded 95%,which is generally is taken to indicate conspecificity.Physiologically,T.interdigitale and T.mentagrophytes are similar,and different from T.indotineae.Urea hydrolysis was found mostly positive in T.interdigitale and T.mentagrophytes,while most strains of T.indotineae strains were weakly positive or negative.The lipolytic abilities and hair perforation of T.mentagrophytes and T.interdigitale were very similar,and were higher than those of T.indotineae.Keratin degradation was significantly larger inT.interdigitale than in T.mentagrophytes and T.indotineae(p<0.01).Conclusion:Molecular characteristics show that T.mentagrophytes s.str.and T.interdigitale s.str.can be distinguished with difficulty from each other,but are unambiguously different from the Indian genotype,T.indotineae by sequences of the HMG gene.The entities were confirmed by multilocus analysis using tanglegrams.Phenotypic characters of morphology and physiology are not diagnostic,but statistically significant differences are observed between the molecular siblings.Trichophyton mentagrophytes represents the ancestral,homothallic cloud of genotypes with a probable geophilic lifestyle,while T.indotineae and T.interdigitale behave as anthropophilic,clonal offshoots.Second Chapter:Antifungal susceptibility and mutations in the squalene epoxidase gene in dermatophytes of the Trichophyton mentagrophytes species complexObjective:To investigate the molecular background of the squalene epoxidase(SQLE)gene in order to understand the risk of emergence and spread of multi-resistance in dermatophytes.Methods:Antifungal susceptibility for fluconazole,griseofulvin,itraconazole,ketoconazole,miconazole,naftifine,sertaconazole,and terbinafine was tested in 135 isolates from India,China,Australia,Germany and The Netherlands.Based on the latest taxonomic insights,strains were identified as three species:T.mentagrophytes s.str.(n=35),T.indotineae(n=64 representing the Indian clone)and T.intrdigitale s.str.(n=36).The terbinafine resistance strains were screened and detected the mutation of the squalene epoxidase(SQLE).13 strains of terbinafine resistance were choosed for evaluate the stability by serial transfer on drug-free medium.Results:High minimum inhibitory concentrations(MICs)of terbinafine(>16 mg/L)were found in 34(53%)T.indotineae isolates.These isolates showed an amino acid substitution in the 397th position of the SQLE gene.Elevated MICs of terbinafine(0.5 mg/L)were noted in 2(3%)T.indotineae isolates;these isolates lead to Phe415Val and Leu393Ser of the SQLE gene.Stability of the effect of the mutations was proven by serial transfer on drug-free medium.Substitutions of Lys276Asn and Leu419Phe were found in susceptible T.mentagrophytes strains.The double mutant Phe377Leu/Ala448Thr showed higher MIC values for triazoles.Conclusions:High MICs of terbinafine are as yet limited to T.indotinae,and are unlikely to be distributed through the T.mentagrophytes species complex by genetic exchange.