Microsporum canis is considered one of the most common zoophilic dermatophyte species causing infections in animals and humans worldwide. However, molecular epidemiological studies on this dermatophyte are still rare. In this study, we aimed to analyse the population structure and relationships between M. canis strains (n = 66) collected in southern Italy and those isolated from symptomatic and asymptomatic animals (cats, dogs and rabbits) and humans. For subtyping purposes, using multilocus sequence typing (MLST) and multilocus microsatellite typing (MLMT), we first used a limited set of strains to screen for variability. No intraspecies variability was detected in six out of the eight reference genes tested and only the ITS and IGS regions showed two and three sequence genotypes, respectively, resulting in five MLST genotypes. All of eight genes were, however, useful for discrimination among M. canis, M. audouinii and M. ferrugineum. In total, eighteen microsatellite genotypes (A-R) were recognized using MLMT based on six loci, allowing a subdivision of strains into two clusters based on the Bayesian iterative algorithm. Six MLMT genotypes were from multiple host species, while 12 genotypes were found only in one host. There were no statistically significant differences between clusters in terms of host spectrum and the presence or absence of lesions. Our results confirmed that the MLST approach is not useful for detailed subtyping and examining the population structure of M. canis, while microsatellite analysis is a powerful tool for conducting surveillance studies and gaining insight into the epidemiology of infections due to this pathogen.

Department of Botany Faculty of Science Charles University 12801 Prague Czech Republic

Department of Veterinary Pathology and Microbiology University of Nigeria Nsukka 410001 Nigeria

Dipartimento di Medicina Veterinaria Università degli Studi Aldo Moro 70010 Bari Italy

Faculty of Veterinary Sciences Bu Ali Sina University Hamedan 6517658978 Iran

Laboratory of Fungal Genetics and Metabolism Institute of Microbiology of the Czech Academy of Sciences 14220 Prague Czech Republic

See more in PubMed

Uhrlaß S., Krüger C., Nenoff P. Microsporum canis: Current data on the prevalence of the zoophilic dermatophyte in central Germany. Hautarzt. 2015;66:855–862. doi: 10.1007/s00105-015-3697-7. PubMed DOI

Aneke C.I., Otranto D., Cafarchia C. Therapy and antifungal susceptibility profile of Microsporum canis. J. Fungi. 2018;4:107. doi: 10.3390/jof4030107. PubMed DOI PMC

Sharma R., De Hoog G.S., Presber W., Gräser Y. Virulent genotype of Microsporum canis is responsible for the majority of human infections. J. Med. Microbiol. 2007;56:1377–1385. doi: 10.1099/jmm.0.47136-0. PubMed DOI

Pasquetti M., Peano A., Soglia D., Min A.R.M., Pankewitz F., Ohst T., Gräser Y. Development and validation of a microsatellite marker-based method for tracing infections by Microsporum canis. J. Dermatol. Sci. 2013;70:123–129. doi: 10.1016/j.jdermsci.2013.01.003. PubMed DOI

Cafarchia C., Romito D., Capelli G., Guillot J., Otranto D. Isolation of Microsporum canis from the hair coat of pet dogs and cats belonging to owners diagnosed with M. canis tinea corporis. Vet. Dermatol. 2006;17:327–333. doi: 10.1111/j.1365-3164.2006.00533.x. PubMed DOI

Gräser Y., Frohlich J., Presber W., de Hoog G.S. Microsatellite markers reveal geographic population differentiation in Trichophyton rubrum. J. Med. Microbiol. 2007;56:1058–1065. doi: 10.1099/jmm.0.47138-0. PubMed DOI

Abdel-Rahman S.M. Strain differentiation of dermatophytes. Mycopathologia. 2008;166:319–333. doi: 10.1007/s11046-008-9108-1. PubMed DOI

Mochizuki T., Takeda K., Anzawa K. Molecular markers useful for intraspecies subtyping and strain differentiation of dermatophytes. Mycopathologia. 2017;182:57–65. doi: 10.1007/s11046-016-0041-4. PubMed DOI

Taylor J.W., Hann-Soden C., Branco S., Sylvain I., Ellison C.E. Clonal reproduction in fungi. Proc. Natl. Acad. Sci. USA. 2015;112:8901–8908. doi: 10.1073/pnas.1503159112. PubMed DOI PMC

Dobrowolska A., Debska J., Kozlowska M., Staczek P. Strains differentiation of Microsporum canis by RAPD analysis using (GACA)4 and (ACA)5 primers. Pol. J. Microbiol. 2011;60:145–148. doi: 10.33073/pjm-2011-020. PubMed DOI

Čmoková A., Rezaei-Matehkolaei A., Kuklová I., Kolařík M., Shamsizadeh F., Ansari S., Gharaghani M., Miňovská V., Najafzadeh M.J., Nouripour-Sisakht S. Discovery of new Trichophyton members, T. persicum and T. spiraliforme spp. nov., as a cause of highly inflammatory tinea cases in Iran and Czechia. Microbiol. Spectr. 2021;2:e0028421. doi: 10.1128/Spectrum.00284-21. PubMed DOI PMC

Čmoková A., Kolařík M., Dobiáš R., Hoyer L.L., Janouškovcová H., Kano R., Kuklová I., Lysková P., Machová L., Maier T., et al. Resolving the taxonomy of emerging zoonotic pathogens in the Trichophyton benhamiae complex. Fungal Divers. 2020;104:333–387. doi: 10.1007/s13225-020-00465-3. DOI

de Valk H.A., Meis J.F.G.M., Klaassen C.H.W. Microsatellite based typing of Aspergillus fumigatus: Strengths, pitfalls and solutions. J. Microbiol. Methods. 2007;69:268–272. doi: 10.1016/j.mimet.2007.01.009. PubMed DOI

de Groot T., Puts Y., Berrio I., Chowdhary A., Meis J.F. Development of Candida auris short tandem repeat typing and its application to a global collection of isolates. MBio. 2020;11:e02971-19. doi: 10.1128/mBio.02971-19. PubMed DOI PMC

Pritchard J.K., Stephens M., Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155:945–959. doi: 10.1093/genetics/155.2.945. PubMed DOI PMC

Rezaei-Matehkolaei A., Makimura K., de Hoog G.S., Shidfar M.R., Satoh K., Najafzadeh M.J., Mirhendi H. Multilocus differentiation of the related dermatophytes Microsporum canis, Microsporum ferrugineum and Microsporum audouinii. J. Med. Microbiol. 2012;61:57–63. doi: 10.1099/jmm.0.036541-0. PubMed DOI

Gräser Y., Scott J., Summerbell R. The new species concept in dermatophytes—a polyphasic approach. Mycopathologia. 2008;166:239–256. doi: 10.1007/s11046-008-9099-y. PubMed DOI

White T.C., Oliver B.G., Gräser Y., Henn M.R. Generating and testing molecular hypotheses in the dermatophytes. Eukaryot. Cell. 2008;7:1238–1245. doi: 10.1128/EC.00100-08. PubMed DOI PMC

da Costa F.V., Farias M.R., Bier D., de Andrade C.P., de Castro L.A., da Silva S.C., Ferreiro L. Genetic variability in Microsporum canis isolated from cats, dogs and humans in Brazil. Mycoses. 2013;56:582–588. doi: 10.1111/myc.12078. PubMed DOI

Watanabe J., Anzawa K., Mochizuki T. Molecular Epidemiology of Japanese isolates of Microsporum canis based on multilocus microsatellite typing fragment analysis. Jpn. J. Infect. Dis. 2017;70:544–548. doi: 10.7883/yoken.JJID.2016.424. PubMed DOI

Hubka V., Nováková A., Jurjević Ž., Sklenář F., Frisvad J.C., Houbraken J., Arendrup M.C., Jørgensen K.M., Siqueira J.P.Z., Gené J., et al. Polyphasic data support the splitting of Aspergillus candidus into two species; proposal of A. dobrogensis sp. nov. Int. J. Syst. Evol. Microbiol. 2018;68:995–1011. doi: 10.1099/ijsem.0.002583. PubMed DOI

Sklenář F., Jurjević Ž., Houbraken J., Kolařík M., Arendrup M.C., Jørgensen K.M., Siqueira J.P.Z., Gené J., Yaguchi T., Ezekiel C.N., et al. Re-examination of species limits in Aspergillus section Flavipedes using advanced species delimitation methods and proposal of four new species. Stud. Mycol. 2021;99:100120. doi: 10.1016/j.simyco.2021.100120. PubMed DOI PMC

Katoh K., Rozewicki J., Yamada K.D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2017;20:1160–1166. doi: 10.1093/bib/bbx108. PubMed DOI PMC

Martins W.S., Lucas D.C.S., de Souza Neves K.F., Bertioli D.J. WebSat-A web software for microsatellite marker development. Bioinformation. 2009;3:282–283. doi: 10.6026/97320630003282. PubMed DOI PMC

Schuelke M. An economic method for the fuorescent labeling of PCR fragments. Nat. Biotechnol. 2000;18:233. doi: 10.1038/72708. PubMed DOI

Alberto F. MsatAllele_1.0: An R package to visualize the binning of microsatellite alleles. J. Hered. 2009;100:394–397. doi: 10.1093/jhered/esn110. PubMed DOI

Huson D.H. SplitsTree: Analyzing and visualizing evolutionary data. Bioinformatics. 1998;14:68–73. doi: 10.1093/bioinformatics/14.1.68. PubMed DOI

Evanno G., Regnaut S., Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study. Mol. Ecol. 2005;14:2611–2620. doi: 10.1111/j.1365-294X.2005.02553.x. PubMed DOI

Ehrich D. AFLPdat: A collection of R functions for convenient handling of AFLP data. Mol. Ecol. Notes. 2006;6:603–604. doi: 10.1111/j.1471-8286.2006.01380.x. DOI

R Core Team . R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing; Vienna, Austria: 2016.

Excofer L., Smouse P.E., Quattro J.M. Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data. Genetics. 1992;131:479–491. doi: 10.1093/genetics/131.2.479. PubMed DOI PMC

Schneider S., Roessli D., Excofer L. ARLEQUIN: A Software for Population Genetics Data Analysis. Volume 2 University of Geneva; Geneva, Switzerland: 2000. Version 2.000.

Agapow P.M., Burt A. Indices of multilocus linkage disequilibrium. Mol. Ecol. Notes. 2001;1:101–102. doi: 10.1046/j.1471-8278.2000.00014.x. DOI

Burt A., Carter D.A., Koenig G.L., White T.J., Taylor J.W. Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis. Proc. Natl. Acad. Sci. USA. 1996;93:770–773. doi: 10.1073/pnas.93.2.770. PubMed DOI PMC

Nei M. Molecular Evolutionary Genetics. Columbia University Press; New York, NY, USA: 1987.

Kosman E. Nei’s gene diversity and the index of average differences are identical measures of diversity within populations. Plant Pathol. 2003;52:533–535. doi: 10.1046/j.1365-3059.2003.00923.x. DOI

Schonswetter P., Tribsch A. Vicariance and dispersal in the alpine perennial Bupleurum stellatum L. (Apiaceae) Taxon. 2005;54:725–732. doi: 10.2307/25065429. DOI

White T.J., Bruns T., Lee S., Taylor J.W., Innis M.A., Gelfand D.H., Sninsky J. PCR Protocols: A Guide to Methods and Applications. Academic Press Inc.; New York, NY, USA: 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics; pp. 315–322.

Gardes M., Bruns T.D. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol. Ecol. 1993;2:113–118. doi: 10.1111/j.1365-294X.1993.tb00005.x. PubMed DOI

Kumar M., Shukla P.K. Use of PCR targeting of internal transcribed spacer regions and single-stranded conformation polymorphism analysis of sequence variation in different regions of rRNA genes in fungi for rapid diagnosis of mycotic keratitis. J. Clin. Microbiol. 2005;43:662–668. doi: 10.1128/JCM.43.2.662-668.2005. PubMed DOI PMC

Glass N.L., Donaldson G.C. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 1995;61:1323–1330. doi: 10.1128/aem.61.4.1323-1330.1995. PubMed DOI PMC

Mirhendi H., Makimura K., de Hoog G.S. Translation elongation factor 1-α gene as a potential taxonomic and identification marker in dermatophytes. Med. Mycol. 2015;53:215–224. doi: 10.1093/mmy/myu088. PubMed DOI

Peterson S.W. Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. Mycologia. 2008;100:205–226. doi: 10.1080/15572536.2008.11832477. PubMed DOI

Carbone I., Kohn L.M. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia. 1999;91:553–556. doi: 10.2307/3761358. DOI

Kawasaki M., Anzawa K., Ushigami T., Kawanishi J., Mochizuki T. Multiple gene analyses are necessary to understand accurate phylogenetic relationships among Trichophyton species. Med. Mycol. J. 2011;52:245–254. doi: 10.3314/mmj.52.245. PubMed DOI

Schmitt I., Crespo A., Divakar P.K., Fankhauser J.D., Herman-Sackett E., Kalb K., Nelsen M.P., Nelson N.A., Rivas-Plata E., Shimp A.D., et al. New primers for promising single-copy genes in fungal phylogenetics and systematics. Persoonia. 2009;23:35–40. doi: 10.3767/003158509X470602. PubMed DOI PMC

Resolving Phylogenetic Relationships Within the Trichophyton mentagrophytes Complex: A RADseq Genomic Approach Challenges Status of 'Terbinafine-Resistant' Trichophyton indotineae as Distinct Species