Persoonia 48, 2022: 203 – 218 ISSN (Online) 1878-9080 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE https://doi.org/10.3767/persoonia.2022.48.06 Host-driven subspeciation in the hedgehog fungus, Trichophyton erinacei, an emerging cause of human dermatophytosis A. Čmoková1,2, M. Kolařík2, J. Guillot 3,4, V. Risco-Castillo 3,5, F.J. Cabañes6, P. Nenoff 7, S. Uhrlaß7, R. Dobiáš 8,9, N. Mallátová10, T. Yaguchi11, R. Kano12, I. Kuklová13, P. Lysková14, K. Mencl15, P. Hamal16, A. Peano17, V. Hubka1,2,11,* Key words Abstract   Trichophyton erinacei is a main cause of dermatophytosis in hedgehogs and is increasingly reported from human infections worldwide. This pathogen was originally described in the European hedgehog (Erinaceus epizootic fungal infections europaeus) but is also frequently found in the African four-toed hedgehog (Atelerix albiventris), a popular pet ani- microsatellite typing mal worldwide. Little is known about the taxonomy and population genetics of this pathogen despite its increasing multigene phylogeny importance in clinical practice. Notably, whether there are different populations or even cryptic species associated population genetics with different hosts or geographic regions is not known. To answer these questions, we collected 161 isolates, skin infections performed phylogenetic and population-genetic analyses, determined mating-type, and characterised morphology Trichophyton benhamiae complex and physiology. Multigene phylogeny and microsatellite analysis supported T. erinacei as a monophyletic species, zoophilic dermatophytes in contrast to highly incongruent single-gene phylogenies. Two main subpopulations, one specific mainly to Atelerix and second to Erinaceus hosts, were identified inside T. erinacei, and slight differences in the size of microconidia and antifungal susceptibilities were observed among them. Although the process of speciation into two lineages is ongoing in T. erinacei, there is still gene flow between these populations. Thus, we present T. erinacei as a single species, with notable intraspecies variability in genotype and phenotype. The data from wild hedgehogs indicated that sexual reproduction in T. erinacei and de novo infection of hedgehogs from soil are probably rare events and that clonal horizontal spread strongly dominates. The molecular typing approach used in this study represents a suitable tool for further epidemiological surveillance of this emerging pathogen in both animals and humans. The results of this study also highlighted the need to use a multigene phylogeny ideally in combination with other inde- pendent molecular markers to understand the species boundaries of dermatophytes. Citation: Čmoková A, Kolařík M, Guillot J, et al. 2022. Host-driven subspeciation in the hedgehog fungus, Trichophyton erinacei, an emerging cause of human dermatophytosis. Persoonia 48: 203 – 218. https://doi.org/10.3767/persoonia.2022.48.06. Effectively published online: 5 June 2022 [Received: 6 January 2022; Accepted: 20 April 2022]. INTRODUCTION In the last decade, pathogens from the T. benhamiae complex have attracted considerable attention mainly due to the epizootic / Trichophyton erinacei and other species from the Trichophyton epidemic spread of dermatophytosis caused by T. benhamiae benhamiae complex are mainly zoophilic fungal pathogens that var. luteum in guinea pigs and humans in Europe (Berlin et al. are frequently transmitted to humans. These zoonotic infections, 2020, Čmoková et al. 2020). Similarly, the hedgehog pathogen usually manifesting as highly inflammatory tinea corporis or T. erinacei is increasingly reported as a cause of dermatophy- tinea capitis, are common in pet owners and contribute signifi- tosis in hedgehog breeders or owners, but with a lower inci- cantly to numerous occupational infections in farmers, workers dence. Unlike T. benhamiae var. luteum, the growing incidence in livestock production, laboratory workers, pet shop workers, of T. erinacei is not due to the emergence of a new virulent and other professions that require contact with animals (Halsby pathogen but rather to changes in preferences breeders have et al. 2014, Hubka et al. 2018b). who increasingly buy hedgehogs as pets (Fig. 1). A worldwide 1 Department of Botany, Faculty of Science, Charles University, Prague, 9 Institute of Laboratory Medicine, Faculty of Medicine, University of Ostrava, Czech Republic; Ostrava, Czech Republic. corresponding author e-mails: vit.hubka@gmail.com, 10 Laboratory of Medical Parasitology and Mycology, Hospital České Budě­ hubka@biomed.cas.cz. jovice, České Budějovice, Czech Republic. 2 Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology, 11 Medical Mycology Research Center, Chiba University, Chiba, Japan. Czech Academy of Sciences, Prague, Czech Republic. 12 Teikyo University Institute of Medical Mycology (TIMM), Tokyo, Japan. 3 Dynamyc Research Group EA 7380, Ecole Nationale Vétérinaire d’Alfort, 13 Department of Dermatology and Venereology, First Faculty of Medicine, UPEC, USC ANSES, Maisons-Alfort, France. Charles University and General University Hospital in Prague, Prague, 4 Department of Dermatology, Parasitology, Mycology, Ecole Nationale Czech Republic. Vétérinaire, Agroalimentaire et de l’Alimentation, Oniris, Nantes, France. 14 Laboratory of Mycology, Department of Medical Microbiology Prague and 5 Ecole Nationale Vétérinaire d’Alfort, Biopole Alfort, Service de Parasitologie- Kladno, Prague, Czech Republic. Mycologie, Maisons-Alfort, France. 15 Pardubice Regional Hospital, Pardubice, Czech Republic. 6 Veterinary Mycology Group, Department of Animal Health and Anatomy, 16 Department of Microbiology, Palacký University and University hospital, Autonomous University of Barcelona, Barcelona, Spain. 7 Laboratory of Medical Microbiology, Mölbis, Germany. Olomouc, Czech Republic. 8 Public Health Institute in Ostrava, Ostrava, Czech Republic. 17 Department of Veterinary Sciences, University of Turin, Turin, Italy. © 2022 Naturalis Biodiversity Center & Westerdijk Fungal Biodiversity Institute You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at https://creativecommons.org/licenses/by-nc-nd/4.0/. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 204 Persoonia – Volume 48, 2022 distribution of T. erinacei is documented by numerous tinea (Smith & Marples 1964). The prevalence in A. albiventris ranged cases from Europe (English et al. 1962, Romano et al. 2001, between 22 – 38 % in wild animals in Africa (Gregory & English Schauder et al. 2007, Weishaupt et al. 2014, Perrier & Monod 1975, Gregory et al. 1978) and 39 – 50 % in pet hedgehogs in 2015, Abarca et al. 2017, Kargl et al. 2018, Lysková et al. 2018, Japan and Spain (Takahashi et al. 2003, Abarca et al. 2017). Rivaya et al. 2020), Asia (Mochizuki et al. 2005, Lim et al. 2009, Hedgehogs are also the source of other potentially zoonotic Rhee et al. 2009, Hsieh et al. 2010, Hui et al. 2017, Choi et al. bacterial, viral and fungal pathogens. Among these, Staphylo­ 2018, Kim et al. 2018, Al Masaoodi et al. 2020, Ogawa et al. coccus aureus, and especially methicillin-resistant S. aureus 2020, Watabe et al. 2021), America (Rosen 2000, Concha et al. (MRSA) strains, belong to the most feared pathogens which 2012, Eid et al. 2014, Alejandra et al. 2018, Frantz et al. 2020, have a high prevalence in hedgehogs (Bengtsson et al. 2017, Rodríguez-Silva et al. 2021, Walsh et al. 2021) and Africa (Drira Rasmussen et al. 2019). Interestingly, it was demonstrated that et al. 2015). Most cases described in literature were transmitted dermatophyte-infected hedgehogs provide a natural selective from pet African pygmy hedgehogs (syn. four-toed hedgehog; environment for MRSA because T. erinacei produces two anti­ Atelerix albiventris). However, T. erinacei is also reported in wild biotics from the penicillin class (Dube et al. 2021, Larsen et European hedgehogs, i.e., West European hedgehogs (Eri­ al. 2022). Specific lineages of MRSA derived from hedgehogs naceus europaeus) and Northern white-breasted hedgehogs have existed even before the era of antibiotics and their spread (Erinaceus roumanicus), which may represent risks for people, between livestock and humans has been confirmed (Larsen especially in wildlife rehabilitation centres or for dogs that may et al. 2022). occasionally encounter hedgehogs in places of their hibernation The geographic distribution of T. erinacei follows the distribution (Le Barzic et al. 2021). Infections due to T. erinacei most often of hedgehogs (Erinaceidae) in Afro-Eurasia, with the highest manifest as tinea manuum or tinea corporis localized on fingers, diversity in Asia, where four of five genera occur, while the hands, wrists, or forearms (Fig. 2). Tinea faciei, kerion type of fifth genus Atelerix is found exclusively in Africa. Only two tinea capitis, tinea barbae and onychomycosis have also been species of the genus Erinaceus are widespread in Europe, i.e., documented in the abovementioned case reports. E. europaeus occurring in Western Europe and E. roumanicus Similar to T. benhamiae var. luteum in guinea pigs (Berlin et al. in Eastern Europe (He et al. 2012), with the contact zone in 2020), available epidemiological surveys showed high infection Central Europe (Curto et al. 2019). Imperceptible morpholo­ rates of T. erinacei in hedgehogs. The prevalence in E. euro­ gical differences between these two species, especially in the paeus ranged between 20 – 30 % in wild hedgehogs in France, contact zone, make exact identification difficult for non-experts Poland and Great Britain (English & Morris 1969, Gnat et al. (Černá Bolfíková et al. 2020). In addition to natural habitats, 2021, Le Barzic et al. 2021) and up to 45 % in New Zealand hedgehogs have been introduced in many non-native areas. Fig. 1   Interest in hedgehogs as pet animals (solid line) compared to interest in hedgehogs in general (dotted line). Data were obtained from the Google Trends and show relative interest scaled on a range of 0 to 100 during 2005 – 2021 (measured as number of searches during half-year). Data are shown for four countries, and key words are listed in parentheses. A. Čmoková et al.: Population biology of Trichophyton erinacei 205 Fig. 2   Clinical manifestation of human (a – e) and animal (f– h) infections caused by Trichophyton erinacei. a. Tinea corporis on the left forearm (26-yr-old woman), erythaematous lesion covered with pustules, contact with a pet hedgehog (Atelerix albiventris) shown on the same picture, isolate IDE 740/19; b. tinea faciei (20-yr-old woman), erythaematous, scaly, sharply demarcated lesion on the left cheek, contact with a pet hedgehog (A. albiventris), isolate CCF 5775; c– d. erythaematous, scaly lesion on the little finger (20-yr-old man), contact with a pet hedgehog (A. albiventris), isolate SK 1826/19; e. tinea corporis (24-yr-old woman) on the outer side of the right thigh, lesion approximately 5 cm diam, pustules in the centre, contact with pet hedgehog (A. albiventris), iso- late CCF 5209; f. hedgehog (Erinaceus europaeus) with erythematous dermatitis and scaly lesions above the muzzle; g – h. alopecia, erythema and pustules affecting head and limbs of a 4-yr-old Bull terrier, isolate CBS 124411. The genus Erinaceus has been introduced to New Zealand, Erinaceus and Atelerix hedgehogs have been reported to be while A. albiventris and Hemiechinus auratus are considered carriers of T. erinacei isolates with different genotypes and invasive species in Spain. Worldwide, A. albiventris, A. algirus mating type gene idiomorphs, respectively (Takashio 1979, and their hybrids are kept as pets (Reeve 1994, He et al. 2012) Takahashi et al. 2003, Schauder et al. 2007, Heidemann et al. or as research animals in laboratories (Lawn et al. 1995). 2010), suggesting that partially isolated populations co-evolved 206 Persoonia – Volume 48, 2022 with different hosts. However, whether the level of differentiation and Cell Cultures (DSMZ), Leibniz Institute, Braunschweig, of these populations could meet the criteria for delimitation of Germany. separate species is not known. To answer this question, we col- lected T. erinacei isolates from different hedgehog genera Molecular studies and cases of human infections. The population structure and DNA was extracted from 7-d-old colonies using the Quick- characteristics (e.g., genotype diversity and random mating) DNATM Fungal/Bacterial Miniprep kit (Zymo Research, USA). were examined using multilocus microsatellite typing (MLMT) Amplification of the ITS rDNA region (ITS1-5.8S-ITS2 cluster) and multilocus sequence typing (MLST) approaches. The was performed using the primer set ITS1F and ITS4 (White association between population structure, host, geographic et al. 1990, Gardes & Bruns 1993), the partial gapdh gene origin, antifungal susceptibility, mating-type gene distribution encoding glyceraldehyde-3-phosphate dehydrogenase using and phenotype was investigated to reveal patterns suggestive primers GPDF and GPDR (Kawasaki et al. 2011), the partial of biological divergence or (sub)speciation. tubb gene encoding β-tubulin using primers Bt2a and Bt2b (Glass & Donald­son 1995), and the partial tef1-α gene encoding MATERIALS AND METHODS translation elongation factor 1-α using primers EF-DermF and EF-DermR (Mirhendi et al. 2015). Polymerase chain reaction Source of isolates (PCR) conditions were described by Sklenář et al. (2021). PCR products were purified using a Sap-Exo Kit (Jena Bio­ The isolates (n = 161) were collected from humans and hedge- science, Germany) and subsequent automated sequencing hogs and obtained in collaboration with clinical laboratories, was performed at BIOCEV (Vestec, Czech Republic) using hospitals, universities and culture collections (Table S1): both terminal primers. The DNA sequences obtained in this National Veterinary School of Alfort (France); Autonomous study (unique genotypes only) were deposited into the Gen- University of Barcelona (Spain), Laboratory for Medical Micro- Bank database (www.ncbi.nlm.nih.gov) under the accession biology (Germany); College of Veterinary Medicine, College numbers listed in Table 1. of Bioresource Sciences, Nihon University (Japan); University A fragment of the MAT1-1-1 gene encoding a protein with an of Turin (Italy); BCCM/IHEM Fungi Collection: Human and alpha-domain motif was amplified with the primer pairs MF3 Animal Health (Belgium); CBS culture collection housed at the and MF6 or MF3 and MF4, and a fragment of the MAT1-2-1 Westerdijk Institute (The Netherlands); and various institutions gene encoding the high mobility group (HMG) domain was in the Czech Republic, i.e., Institute of Public Health in Ostra- amplified with the primers TmHMG3S and TmHMG3R (Kano va and Ústí nad Labem, Hospital České Budějovice, General et al. 2012, Symoens et al. 2013). Several PCR products of University Hospital in Prague, University Hospital in Pilsen, and each MAT idiomorph were subjected to DNA sequencing for the University Hospital Olomouc. verification of specificity, and the remaining samples were visu- Selected isolates were deposited into the Culture Collection of alized on an electrophoretogram (1 % agarose gel with 0.5 μg/ Fungi (CCF), Department of Botany, Charles University, Prague, mL ethidium bromide) and identified according to the specific Czech Republic; and German Collection of Microorganisms lengths of PCR products. Table 1   Sequence accession numbers to unique multilocus genotypes of Trichophyton erinacei. MLST genotype Representative Accession numbers Corresponding strain ITS (genotype) tef1-α (genotype) gapdh (genotype) tubb (genotype) MLMT genotype(s) G1 (35 strains) CCF 5209 MZ314454 (a) MZ320337 (a) MZ320332 (a) MZ320327 (a) M1-M3 G2 (1 strain) CBS 124411 MZ314453 (b) MZ320336 (a) MZ320331 (a) MZ320326 (a) M4 G3 (4 strains) DSM 104923 MZ314456 (c) MZ320339 (a) MZ320334 (a) MZ320329 (a) M5-M6 G4 (8 strains) CBS 511.73T LR794136 (a) LR794267 (b) LR794242 (a) LR794292 (a) M7-M9 G5 (23 strains) CCF 6427 MZ314455 (a) MZ320338 (a) MZ320333 (b) MZ320328 (a) M10 MLST, multilocus sequence typing; MLMT, multilocus microsatellite typing Table 2   Characteristics of alignments, partition-merging results and best substitution model for each partition according to Bayesian information criterion. Alignment Length Variable Parsimony Phylogenetic Partitioning scheme (substitution model) (bp) position informative method sites ITS, gapdh, tubb & tef1-α 2278 243 154 Maximum likelihood Six partitions: ITS1 & ITS2 (TrN+G); 5.8S (K80); 1st codon positions (tree: Fig. 3) (ML) of gapdh & tef1-α (F81+I); 1st codon positions of tubb & 2nd codon positions of gapdh, tubb & tef1-α (JC); 3rd codon positions of gapdh, tubb & tef1-α (HKY+I); introns of gapdh, tubb & tef1-α (K80+I+G) ITS, gapdh, tubb & tef1-α 2278 243 154 Bayesian inference Five partitions: ITS1 & ITS2 & 3rd codon positions of gapdh, tubb & (tree: Fig. 3) tef1-α (HKY+G); 5.8S (K80); 1st codon positions of gapdh & tef1-α (F81+I); 1st codon positions of tubb & 2nd codon positions of gapdh, tubb & tef1-α (JC); introns of gapdh, tubb & tef1-α (K80+I+G) ITS (tree: Fig. 8) 592 88 53 ML Two partitions: ITS1 & ITS2 (TrN+G); 5.8S (K80) gapdh (tree: Fig. 8) 563 54 31 ML Three partitions: 1st codon positions (F81+I); 2nd codon positions (JC); 3rd codon positions & introns (HKY+G) tubb (tree: Fig. 8) 463 41 24 ML Three partitions: 1st & 2nd codon positions (JC); 3rd codon positions (HKY+I); introns (K80) tef1-α (tree: Fig. 8) 660 60 46 ML Three partitions: 1st & 2nd codon positions (JC); 3rd codon positions (HKY); introns (K80+G) A. Čmoková et al.: Population biology of Trichophyton erinacei 207 Subtyping of T. erinacei using microsatellite markers was per- association IA is estimated to be nearly zero or zero if no link- formed using the previously developed typing scheme for spe- age is noticed between the alleles of different loci (randomly cies from the T. benhamiae clade (Čmoková et al. 2020). The distributed alleles); in that case random mating (panmixis) is reaction volume of 5 μL for PCR contained 50 ng of DNA, 0.5 μL suggested. The values observed in the examined populations of the mixture of primers and 2.5 μL of Multiplex PCR Master were compared to those artificially generated by the 10 000 ran- Mix (Qiagen, Germany). The PCR conditions were chosen domisations. The null hypothesis of recombination and linkage according to the manufacturer’s recommendations. The PCR equilibrium is supported when observed and simulated values products (diluted in water 1 : 25) were mixed with 10 μL of are close (p > 0.05), while significant differences between val- deionized formamide and 0.2 μL of the GeneScan™ 600 LIZ ues support linkage disequilibrium and clonality. size standard, denatured for 5 min at 95 °C, and subsequently Genetic diversity within population(s) was calculated from the analysed using an ABI 3100 Avant Genetic Analyzer. frequencies of genetically distinct individuals using Nei’s geno- type diversity (Dg), and from the frequencies of alleles at indivi- Phylogenetic analysis dual loci using Nei’s gene diversity (D) (Nei 1987, Kosman 2003). Alignments of the ITS, gapdh, tubb and tef1-α regions were To obtain diversity values comparable between the clusters, performed using the FFT-NS-i option implemented in the the effective number of genotypes (Geff) (Parker Jr 1979) was MAFFT online service (Katoh et al. 2017). The alignments were calculated based on the number of equally abundant genotypes trimmed, concatenated and then analysed using maximum necessary to reflect the value of a diversity measure. All three likelihood (ML) and Bayesian inference (BI) methods. Suit- population indices (Dg, D, Geff) were calculated from the binary able partitioning schemes and substitution models (Bayesian data matrix using script AFLPdat (Ehrich 2006) in R v. 3.0.2. information criterion) for the analyses were selected using a greedy strategy implemented in PartitionFinder v. 2 (Lanfear et Phenotypic studies al. 2017) with settings allowing introns, exons, codon positions At least two randomly selected isolates from each MLST geno- and segments of the ITS region to be independent datasets. type (if available) were subjected to a more detailed analysis of The optimal partitioning schemes for each analysed dataset macromorphology on Sabouraud glucose agar (SGA, Oxoid, along with basic alignment characteristics are listed in Table 2. Basingstoke, UK), malt extract agar (MEA, Oxoid, Basingstoke, ML trees were constructed with IQ-TREE v. 1.4.4 (Nguyen UK) and potato dextrose agar (PDA, Himedia, Mumbai, India) et al. 2015) with nodal support determined by ultrafast boot- at 25 °C. In addition, the growth on SGA at 30 and 37 °C was strapping (BS) with 100 000 replicates. Trees were rooted with measured. The micromorphological features were measured Trichophyton rubrum. Bayesian posterior probabilities (PP) were in the same isolates and were recorded from cultures growing calculated for the concatenated dataset using MrBayes v. 3.2.6 on MEA after 14–21 d of cultivation. The dimensions of conidia (Ronquist et al. 2012). The analysis ran for 10 7 generations, were recorded at least 25 times for each isolate. two parallel runs with four chains each were used, every 1000th The macromorphology of the colonies was documented using tree was retained, and the first 25 % of trees were discarded an Olympus SZ61, and colony colours were determined using as burn-in. The convergence of the runs and effective sample the hexadecimal colour codes assigned according to the web- sizes were checked in Tracer v. 1.6 (http://tree.bio.ed.ac.uk/ site https://coolors.co. Micromorphology was documented using software/tracer). an Olympus BX-51 microscope. Statistical differences in the size of spores were tested with one-way analysis of variance Statistical analysis of microsatellite data (ANOVA) followed by Tukey’s honestly significant difference Allele and binary data matrices were created using GeneMarker (HSD) test in R v. 3.3.4 (R Core Team 2016). v. 1.51 (SoftGenetics, LLC, State College, PA, USA). The allele data matrix was reduced to ≤ 5 samples from each sampling site Statistical analysis of the antifungal susceptibility data to minimize the effect of uneven sampling and analysed using The raw data published by Shamsizadeh et al. (2021) were a Bayesian model-based clustering algorithm with a clustering used for statistical analysis. The isolates were divided into two number (K) ranging from 1 to 10 in the software STRUCTURE groups according to their assignment to the two clusters based (Pritchard et al. 2000). The admixture model and burn-in of on STRUCTURE analysis. Statistical differences in antifungal 100 000 iterations followed by 1 M additional Markov chain susceptibilities to eight antifungals (amorolfine, ciclopirox, efin- Monte Carlo iterations were used to calculate 10 simulation aconazole, itraconazole, luliconazole, griseofulvin, terbinafine runs. The optimal clustering number (K) was estimated by cal- and tavaborole) were tested with one-way analysis of variance culating ΔK (Evanno et al. 2005) using the script structure-sum (ANOVA) followed by Tukey’s honestly significant difference test (Ehrich 2006) in R v. 3.3.4 (R Core Team 2016). (Tukey’s HSD) in R v. 3.3.4 (R Core Team 2016). To estimate the similarities between individuals, Jaccard’s simi­ larity coefficient was calculated on the previously generated RESULTS binary matrix using the program FAMD (Schlueter & Harris 2006). A neighbour-joining tree based on Jaccard’s similarity Genotyping coefficient matrix was constructed and genetic distances were In total, 161 isolates of T. erinacei were subjected to multilocus calculated using the same software. Genetic distances were microsatellite typing (MLMT). Seven out of 10 markers, devel- used for the construction of the NeighborNet network in Splits­ oped by Čmoková et al. (2020), were successfully amplified Tree v. 4 (Huson & Bryant 2006). in T. erinacei (Table S1). A total of 71 samples were selected The degree of gene flow among clusters was estimated for for multilocus sequence typing (MLST) using four genes (ITS, seven loci on the clone-corrected dataset using a pairwise gapdh, tubb and tef1-α). The reduction in number was at the fixation index (FST) calculated in Arlequin (Schneider et al. expense of samples from France, where we observed a high 2000). The degree of clonality or recombination within popu- degree clonality among samples using MLMT. lations was estimated by calculating the index of association Using the MLST approach, we observed a single genotype in (IA) in MultiLocus v. 1.3 (Agapow & Burt 2001) using a binary tubb, two in gapdh and tef1-a, and three in ITS, which result- matrix (uncorrected and clone corrected dataset) and a matrix ed in five multilocus genotypes designated G1– G5 (Fig. 3). based on both microsatellite and sequence data. The index of Microsatellite markers showed higher levels of polymorphism 208 Persoonia – Volume 48, 2022 CBS 196.26T Host 100/1.00 IHEM 5470 T. concentricum IHEM 13435 Atelerix Human (infected from Atelerix ) 100/0.97 var. benhamiae IHEM 4710T & 16 others Erinaceus 100/1.00 var. benhamiae USA 3355 & 3360 T. benhamiae Human (infected from Erinaceus ) Human (not specified source) var. luteum IHEM 25068T & 235 others -/- dog 99/0.99 DMF 3061 88/0.95 DMF 1658 T. japonicum IHEM 17701T & 17 others Geography -/- Europe 95/1.00 IHEM 22725T & 38 others T. europaeum France 96/0.97 strain 237 Czechia strain T289 & 2 others Germany 99/1.00 CCF 6543T Spain strain SH1 & 20 others strain 72 T. persicum UK -/0.97 strain Ahv 376 Netherlands 92/0.99 strain KH-M4 & 8 others Italy CCF 6259T T. spiraliforme Non-Europeancountries strain 361 Japan strain 363 100/1.00 New Zealand CBS 220.25T T. eriotrephon Ivory Coast 70/- CCF 6545 Kenya IHEM 24340 CBS 365.53T MAT idiomorph 100/1.00 CCF 4613 CCF 4889 T. verrucosum CCF 4612 MAT1-1-1 MAT1-2-1 HOST GEO MAT MLMT MLST 74/0.97 CCF 5727 M1 GE 207355/17 M1 Microsatellite cluster 1 CCF 6358 M1 GE 215597/17 M1 CCF 6560 M2 IDE 740/19 M1 CCF 5775 M1 CCF 6504 M1 CCF 6564 M1 CCF 5929 M1 CCF 5863 M1 MLST genotype G1 CCF 5930 M1 GE 215596/17 M1 GE 201435/13 M1 CCF 5931 M1 CCF 5864 M1 CCF 6563 M2 DSM 103784 M1 PL 1388/19 M1 CCF 6347 M1 ME 1293/19 M1 T159SP M1 98/1.00 IHEM 25164 M3 T140SP M1 92/1.00 SK 1826/19 M1 T134SP M1 CCF 6332 M1 M. c. 1 T128SP M1 T127SP M1 CLIS 3198/20 M1 T138SP M1 T129SP M1 CCF 6399 M1 T135SP M1 0.005 CCF 5209 M1 G2 CBS 124411 M4 96/1.00 GE 200924/17 M5 T492 M6 G3 98/1.00 GE 205469/17 M5 DSM 104923 M5 CBS 474.76 M7 IHEM 19619 M7 92/1.00 IHEM 19621 M7 Microsatellite cluster 2 99/1.00 IHEM 25146 G4 M8 Trichophyton erinacei CBS 511.73T T1072 M7 M9 DSM 109031 M8 CBS 344.79 M7 CCF 6425 M10 T2003 M10 T1087 M10 T1326 M10 MLST genotype G5 T1426 M10 T1669 M10 T1863 M10 T2005 M10 T1265 M10 T258 M10 T1152 M10 T1340 M10 T950 M10 T1959 M10 T1269 M10 CCF 6427 M10 T2008 M10 T1337 M10 U52 M10 T1038 M10 T1458 M10 T2089 M10 CCF 6426 M10 99/1.00 CBS 363.35T 100/1.00 CBS 557.50 CCF 4831 T. bullosum 92/1.00 CCF 5730 99/1.00 IHEM 4032T IHEM 19628 T. africanum 100/1.00 IHEM 4033 Trichophyton rubrum CBS 202.88 Fig. 3   Multilocus phylogeny of Trichophyton benhamiae complex inferred with the maximum likelihood method based on the ITS rDNA, tef1-α, gapdh and tubb loci (alignment characteristics, partitioning scheme and substitution models are listed in Table 2). A total of 71 T. erinacei isolates were characterized, included in the phylogeny, and the following information was plotted on the tree and designated by different colours: host from which isolate was obtained (column HOST), geographic origin (GEO), mating type gene idiomorph (MAT), multilocus genotype based on seven microsatellite markers (MLMT; M1-M10) and assignment of isolates into cluster based on analysis in STRUCTURE software (cluster 1: red, cluster 2: yellow) and multilocus genotype based on 4 loci sequences (MLST; G1-G5). Maximum likelihood bootstrap values and Bayesian posterior probabilities are appended to the nodes; only support values higher than 70 % and 0.95, respectively, are shown. The ex-type strains are designated with a superscripted T and bold print. Trichophyton rubrum was used as the outgroup. A. Čmoková et al.: Population biology of Trichophyton erinacei 209 (10 genotypes, M1-M10) in comparison to MLST. The most 1 potent microsatellite locus was CT21b (5 genotypes), followed by TAG16 (4 genotypes), TCA16, TC17A, TC20 (3 genotypes 0.8 each) and CT21 (1 genotype, monomorphic). The plot in Fig. 4 mean genotypic diversity shows the dependence of genotype diversity as a function of an 0.6 increasing number of microsatellite markers. From the course of the curve, enough loci (n = 7) were used to uncover the genetic 0.4 diversity present in the examined set of T. erinacei strains. Bayesian approach-based analysis supported the distribution 0.2 of T. erinacei isolates (n = 161) into two clusters, designated here as cluster 1 and cluster 2, according to the ΔK peak 0 (Fig. 5). Cluster 1 consists of two MLMT genotypes (M1 and 1 2 3 4 5 6 7 M2) that correspond to MLST genotype G1 (except for IHEM number of microsatellite loci 25164, which belongs to cluster 2). This cluster contains isolates exclusively (n = 33) from African hedgehogs and T. benhamiae / T. japonicum / T. europaeum / T. erinacei their owners (Fig. 3). M1 genotype isolates exhibited only the MAT1-2-1 idiomorph and originated from Europe, while two M2 Fig. 4   Plot of mean genotypic diversity as a function of the number of micro- genotype isolates exhibited the MAT1-1-1 idiomorph and origi­ satellite loci. nated from Japan. The induction of a sexual state by crossing opposite mating-type strains of M1 and M2 genotypes was not successful on agar medium supplemented with Guizotia CCF 6347 GE 215596/17 CCF 5864 CCF 6332 T129SP CCF 5863 T127SP T128SP DSM 103784 GE 201435/13 GE 207355/17 CCF 6560 CCF 6563 CBS 124411 DSM 109031 IHEM 25146 T1072 CBS 511.73 IHEM 19621 IHEM 19619 CBS 474.76 CBS 344.79 IHEM 25164 GE 205469/17 DSM 104923 GE 200924/17 50 T492 U5 U86 CCF 6425 30 CCF 6426 T1063 T1070 Mean ΔK 10 T1007 T1038 0 2 4 6 8 10 cluster 1 K cluster 2 Fig. 5   Population structure plot for the distribution of Trichophyton erinacei isolates into two clusters (K = 2 according to the peak of ΔK; graph is shown in the lower left corner of the figure) based on seven microsatellite loci. The plot created with STRUCTURE software using Bayesian clustering is displayed vertically on the right side of the figure. Individual isolates (haplotypes) are represented by horizontal bars. The dataset was reduced to ≤ 5 samples from each locality to minimize the effect of uneven sampling. A neighbour-joining tree calculated from the multilocus microsatellite profiles using the Jaccard distance matrix measure in FAMD software is used for the comprehensive presentation of the results. 210 Persoonia – Volume 48, 2022 M4 (1) (1) (1) M8 (2) Italy (1) GE 218315/17 (2) Germany (1) CBS 124411 (1) (1) Kenya (1) M1 (32) (10) T1072 (1) Czechia (18) Germany (6) (21) Spain (8) CCF 5727 (32) (1) M9 (1) France (1) (1) CCF 6560 (2) (1) M7 (5) M2 (2) (1) CBS 474.76 (4) UK (3) Japan (2) (4) The Netherlands (1) (1) New Zealand (1) CCF 6425 (113) M10 (113) (113) IHEM 25164 (1) (1) M3 (1) France (113) Ivory Coast (1) M5 (3) (2) Germany (3) M6 (1) (1) (1) France (1) DSM 104923 (3) T492 (1) Isolation source Atelerix Erinaceus Dog Human (infected from Atelerix) Human (infected from Erinaceus) Human (not specified source) Fig. 6   Population structure of Trichophyton erinacei revealed by the analysis of seven microsatellite loci in 161 strains. The NeighborNet network was built with FAMD software and visualized in SplitsTree using the Jaccard index-based distance matrix. Geographic origin and host(s) are detailed separately for each multilocus genotype M1-M10. Subdivision of isolates into clusters based on STRUCTURE analysis (Fig. 5) is indicated by a colour gradient: cluster 1: red, cluster 2: yellow. The pie chart under the name of strain CBS 124411 indicates a genetic admixture between clusters. Detailed data associated with every isolate are listed in Table S1. abyssinica seeds at 20 °C (Symoens et al. 2013, Čmoková index-based distance matrix (Fig. 6). Genotypes M1 and M2 2015). Strain CBS 124411 showed a high level of admixture clustered together, and the next closest genotype was M4, a between clusters 1 and 2 (Fig. 5) but was assigned to cluster 2 transitional genotype between cluster 1 and 2. by STRUCTURE. This strain is the only representative MLMT More diversified cluster 2 consisted of eight MLMT genotypes genotype M4 (MLST genotype G2) and was isolated from a M3 – M10 corresponding to strains from the remaining MLST dog dermatophytosis (Fig. 2) in Italy, and transmission from genotypes G2– G5 (Fig. 3, 6). The cluster comprised mostly wild hedgehog of the genus Erinaceus is presumed. strains originating from hedgehogs of the genus Erinaceus in Similar grouping of microsatellite genotypes was observed Europe, except for two isolates from wild hedgehogs of the when the NeighborNet network was constructed with FAMD genus Atelerix in Kenya (IHEM 25146) and Ivory Coast (IHEM software and visualized in SplitsTree 4 using the Jaccard 25164). Three German and one French isolate belonged to the M5 and M6 genotypes, respectively, and MLST genotype G3, all isolated from Erinaceus hedgehogs or human infection 160 contracted from them. I A (observed) = 4.2 (p<0.001) The old strains isolated before 2000, including the ex-type strain 140 of T. erinacei CBS 511.73 and the ex-type strain of T. pro­liferans 120 CBS 474.76, mostly belonged to MLST genotype G4 and micro- 100 ­satellite genotypes M7– M9 (Fig. 3, 6). These strains were iso- lated from hedgehogs of the genera Erinaceus and Atelerix 80 (strain from Kenya, IHEM 25146) and included strains of both 60 mating types (mating experiments were not successful). The majority of strains from cluster 2 were segregated to the M10 40 genotype corresponding to MLST genotype G5. These strains 20 originated exclusively from Erinaceus hedgehogs from France, count 0 and all showed MAT1-1-1 gene idiomorph. 0.1 0.138 61, 161 84 184 5 IA 7 3 ,0 2 5 99 .115 0.115 30 38 46 92 92 23 69 76 2 00 9 3 6 0.1 0.11 0.2 0.25 .34 2 0.2 , 0.20 .09 0.09 .02 0.02 .06 .04 0.3 , 0.3 0.0 , 0.0 0.0 , 0.0 -0 , -0.1 0.2 , 0.2 0.2 0.2 0.2 , 0.2 0.0 , 0.0 . 0.0 , 0.0 Population structure 0.0 0.0 ,0 0. - -0 2, -0 0 , 38 15, ,- - 8, , - , 30 99 53 76 22 07 00 3, 69 46 23 1 6, 9, .16 .13 0.1 .04 Both clusters were genetically uniform as evidenced by low .06 0.1 -0 -0 -0 -0 -0 values of Nei’s gene diversity (D), especially in cluster 1 Fig. 7   Histogram of the simulated index of association (IA) for cluster 2 isolates calculated from 10 000 permutations of randomization tests under (D = 0.07) compared to cluster 2 (D = 0.22). The low Nei’s a null model of allelic recombination; the observed value of IA is 4.2, which genotype diversity index in cluster 1 (Dg = 0.01) and cluster 2 is out of the 95 % confidence interval of the simulated values. (Dg = 0.22) reflects that they consisted of several abundant A. Čmoková et al.: Population biology of Trichophyton erinacei 211 ITS tef1-α 97 99 T. concentricum T. concentricum 99 94 91 T. benhamiae T. benhamiae 0.01 88 0.01 81 T. japonicum 87 74 T. europaeum T. europaeum T. japonicum 85 96 T. spiraliforme T. persicum 43 98 92 T. spiraliforme T. persicum 75 100 T. bullosum 42 T. erinacei 99 94 T. africanum MLST genotypes G1, G4, G5 (66 strains) 67 T. erinacei CBS 124411 MLST genotype G2 97 MLST genotypes 53 82 100 G1-G3, G5 T. eriotrephon (63 strains) 49 DSM 104923 84 T492 GE 205469/17 MLST genotype G3 80 T. verrucosum GE 200924/17 86 99 T. eriotrephon T. verrucosum 63 CBS 344.79 IHEM 19621 100 T. bullosum 88 IHEM 19619 DSM 109031 91 T1072 MLST genotype G4 91 IHEM 25146 T. africanum CBS 511.73T CBS 474.76 T. rubrum CBS 202.88 T. rubrum CBS 202.88 gapdh tubb 92 T. persicum 90 T. concentricum T. benhamiae T. spiraliforme 81 0.01 0.01 98 T. spiraliforme 73 T. concentricum 92 T. japonicum T. benhamiae 90 T. japonicum T. europaeum T. europaeum CCF 6425 T1038 T950 38 T2089 T258 T1087 T1669 T1326 T1959 T2008 84 U52 MLST genotype G5 T1269 T1458 (23 strains) T. erinacei T1426 70 92 T1152 63 T2003 T1337 MLST genotypes G1-G5 76 T1863 (71 strains) T1340 T1265 T2005 CCF 6427 70 CCF 6426 62 97 T. eriotrephon T. verrucosum 93 75 T. persicum 67 T. verrucosum 98 T. africanum 98 99 T. bullosum T. eriotrephon 79 T. erinacei T. bullosum 96 83 MLST genotypes G1-G4 (48 strains) 78 T. africanum T. rubrum CBS 202.88 T. rubrum CBS 202.88 Fig. 8   Best scoring maximum likelihood tree based on single genes: ITS rDNA, tef1-α, gapdh and tubb (alignment characteristics, partitioning scheme and substitution models are listed in Table 2). Trichophyton erinacei strains are highlighted with violet colour; corresponding multilocus genotypes (MLST) from the multiple gene phylogeny (Fig. 3) are listed. Maximum likelihood bootstrap values are appended to the nodes; Trichophyton rubrum CBS 202.88 was used as the outgroup. 212 Persoonia – Volume 48, 2022 clones that were overrepresented due to uneven sampling. Single-gene phylogenies were highly incongruent, and genes Clonal reproduction prevails in both clusters, which is sup- with more genotypes did not resolve T. erinacei strains as a ported by a low effective number of genotypes (Geff = 1.1 and monophyletic group (Fig. 8). The isolates were divided into 1.3, respectively) in comparison with the observed number of three lineages in the ITS phylogeny. Four strains from Erina­ genotypes (G = 2 and 8, respectively). ceus hedgehogs, corresponding to MLST genotype G3, did not cluster with the majority of T. erinacei strains and were more The first cluster of T. erinacei consists of only two microsatellite closely related to T. verrucosum (Fig. 8). These strains had genotypes, which is not enough to determine linkage among three unique substitutions in the ITS region and another three markers and make conclusions about the presence of sexual substitutions that were only shared with strain CBS 124411 recombination in the dataset. The hypothesis about random (MLST genotype G2). The strain CBS 124411 was resolved on mating was rejected in cluster 2 by calculating the index of as- a long branch sister to the vast majority of T. erinacei strains sociation IA on clone uncorrected data (IA = 4.4, p < 0.01). Simi­ (n = 66), and its ITS sequence contained five unique substi- larly, sexual recombination was also rejected for the complete tutions that were not shared with any other T. erinacei isolate dataset of all T. erinacei samples based on microsatellite data (eight substitutions compared to the ex-type strain). (IA = 4.2, p < 0.01; Fig. 7) and for the dataset where MLMT data In the phylogeny based on the tef1-α gene, T. erinacei strains were combined with MLST data (data not shown). The hypoth- were divided into two groups. The majority of strains (n = 63) esis about random mating was not rejected on clone-corrected clustered with T. verrucosum and T. eriotrephon. Eight strains, data in either case (cluster 2 and the whole dataset) but not at corresponding to MLST genotype G4, also clustered with the significance level. We observed a relatively high proportion the species mentioned but were in a basal position to them. of fixed microsatellite alleles in both clusters, as evidenced by The tef1-α sequences of these strains associated with both the relatively high fixation index (FST = 0.85, p < 0.000001), Erinaceus and Atelerix hedgehogs contained 11 substitutions which means that there is only limited genetic information ex­ compared to the remaining T. erinacei strains. change between the clusters. The phylogeny based on the gapdh gene also divided isolates into two considerably remote groups. The French isolates from Phylogeny and position of T. erinacei in the T. benhamiae hedgehogs of the genus Erinaceus, corresponding to MLST complex genotype G5, clustered with T. verrucosum and T. eriotrephon, For the multigene phylogeny based on ITS, gapdh, tubb and while the remaining strains were resolved in the basal position to tef1-α loci, we used previously published alignments (Čmoková other T. benhamiae complex species. In total, these two groups et al. 2021) that were enriched by 71 T. erinacei isolates for differed by 11 substitutions in the gapdh gene. which we generated sequences in this study. The final alignment In contrast to single-gene phylogenies, the multigene phylogeny included 443 strains representing the entire species diversity of resolved T. erinacei as a monophyletic species (Fig. 3). Within the T. benhamiae complex. Detailed alignment characteristics the T. benhamiae complex, species were resolved into three for all phylogenies together with the partitioning schemes and major monophyletic clades, corresponding to T. benhamiae, substitution models are listed in Table 2. The isolation source T. erinacei and T. bullosum clades sensu Čmoková et al. (2020). and accession numbers for DNA sequences are available in Trichophyton erinacei formed an independent lineage within Table 1 and S1. The alignments were deposited in the Dryad the T. erinacei clade, which also encompassed T. verrucosum Digital Repository (https://doi.org/10.5061/dryad.pzgmsbcnv). and T. eriotrephon. T. concentricum T. persicum T. benhamiae T. spiraliforme var. luteum T. erinacei T. eriotrephon T. japonicum T. europaeum Fig. 9   Population structure of the Trichophyton benhamiae complex revealed by the analysis of seven microsatellite loci in 483 strains (Table S1). Tricho­ phyton verrucosum, T. bullosum and T. africanum were not included because the markers could not be amplified in them. The NeighborNet network was built with FAMD software and visualized in SplitsTree using the Jaccard index-based distance matrix. Trichophyton erinacei strains are indicated by violet colour. A. Čmoková et al.: Population biology of Trichophyton erinacei 213 SGA MEA PDA CCF 5727 MLST genotype G1 CCF 5930 CCF 6399 CCF 6563 CBS 124411 G2 DSM 104923 G3 CBS 344.79 G4 CBS 474.76 U1543 G5 CCF 6427 Fig. 10   Overview of the macromorphology of the Trichophyton erinacei isolates representing different multilocus genotypes. The isolates were cultivated on Sabouraud glucose agar (SGA), malt extract agar (MEA) and potato dextrose agar (PDA) for 14 d at 25 °C; obverse and reverse of colonies are shown. In addition to DNA sequence markers, we also constructed a strains (if available) of each MLST genotype were selected and phylogenetic network based on seven microsatellite markers cultivated on SGA, MEA and PDA media at 25 °C for 7 d with a across the members of T. benhamiae and T. erinacei clades subsequent measurement of micromorphological characteris- (except for T. verrucosum). In this network shown in Fig. 9, tics after 2 wk of cultivation. In total, 26 strains were examined. all T. erinacei strains clustered together in agreement with the There were no statistically significant differences in the growth results of the multigene phylogeny. The relationships between rates or visible differences in the macromorphology of colonies species resulting from this analysis were also similar to phylo­ (Fig. 10), which could be linked to MLST genotypes or micro­ geny (Fig. 3). Trichophyton eriotrephon was the most closely satellite clusters. Colonies of strains from cluster 1 reached related species to T. erinacei, followed by a group of species 22 – 26 mm diam (∅ = 24 mm) on MEA, 23–35 mm diam (∅ = from the T. benhamiae clade. 26 mm) on SGA and 17– 32 mm diam (∅ = 21 mm) on PDA in 7 d at 25 °C. Colonies of strains from cluster 2 reached 19 – Phenotypic analysis 45 mm diam (∅ = 28 mm) on MEA, 21– 43 mm diam (∅ = 29 All T. erinacei stains were initially examined on MEA at 25 °C. mm) on SGA, and 11– 35 mm diam (∅ = 24 mm on PDA in 7 d For more detailed morphological examination, at least two at 25 °C. In particular, the strains isolated before 2000 showed 214 Persoonia – Volume 48, 2022 3.5 width of microconidia (μm) 3 3.3 2.8 2.5 2.3 2 1.8 1.5 1.3 1 0.8 8.5 8 length of microconidia (μm) 7.5 7 6.5 6 5.5 5 4.5 4 3.5 2.5 3 1.5 2 GE 200924/17 GE 205469/17 cluster 1 cluster 2 GE 201435/13 GE 215597/17 DMS 103784 DMS 109031 IHEM 25164 CBS 474.76 CBS 124411 CBS 511.73 CCF 5929 CCF 5864 CCF 6426 CCF 6427 CCF 5930 CCF 6425 CCF 6399 CCF 6563 CCF 5775 CCF 5727 CCF 5931 T2008 U1427 U1545 U1543 T1893 MLST genotype G1 G2 G3 G4 MLST genotype G5 Microsatellite cluster 1 Microsatellite cluster 2 Fig. 11   Length and width of microconidia in Trichophyton erinacei isolates representing different microsatellite clusters/multilocus genotypes (MLST). The horizontal lines in boxplots indicate the mean value and interquartile range, whiskers span the 5 % and 95 % percentiles, and dots indicate extreme outliers. cottony colonies and poor sporulation and thus could not be produce larger quantities of spores, while conidiophores of properly examined. Typical strains were characterized by finely cluster 2 were less extensively branched with conidia mostly to coarsely granular or powdery colonies that were flat, centrally sessile or borne along the conidiogenous hyphae on short raised or umbonate. Colony colour varied in individuals from protrusions or short lateral branches. The size and shape of white (#F2F3F4) to yellowish white (#F0EAD6). The reverse microconidia also differed between clusters. Microconidia of of colonies was pale-yellow (#F3E5AB) or brilliant yellow cluster 1 strains were significantly wider and shorter (Fig. 11) (#FADA5E) on all examined media after 14 d of cultivation. than microconidia of cluster 2 (p < 0.0001) and were rather In some strains, the reverse colour in the centre of colonies pyriform, 2.2 –7.4 (3.9 ± 0.7) × 1.4 – 3.5 (2.3 ± 0.3) µm, while turned brown (Fig. 10). conidia of cluster 2 were predominantly clavate, 2.5–8.7 (4.2 ± The dimensions and shape of microconidia and degree of 0.8) × 1– 3.4 (2.1 ± 0.3) µm. Although sizes were significantly sporulation differed slightly between isolates from cluster 1 different, there was a large overlap in the dimensions, and and cluster 2. Cluster 1 isolates (n = 11) exhibited extensive significant interindividual differences between strains from the production of microconidia on all media compared to cluster 2 same cluster were detected. Therefore, the observed differen­ isolates (n = 15), which did not sporulate so intensively, possibly ces could not be used in the differentiation of clusters in practice. a consequence of the slightly different arrangement of conidio­ Macroconidia were absent in most T. erinacei isolates. When phores. Conidiophores of cluster 1 strains showed mostly a present, they were rather sparse to rare, cigar-shaped or pyramidal (grape-like) arrangement and therefore tended to clavate, with a tapering rounded apex and truncate base, and Table 3   Comparison of clinical and epidemiological data associated with strains representing different populations of Trichophyton erinacei (cluster 1 and cluster 2). Source Clinical data Population1 cluster 1 cluster 2 Human Number of samples 23 7 Sex women 86 % women 75 % Age range (median) 15 – 46 (26) 2 – 28 (23) Site of infection upper extremities (59 %), face (13 %), trunk (13 %) upper extremities (100 %) Contracted from: Erinaceus / Atelerix / unknown 0 / 22 / 1 2/0/5 Animal number of samples 11 120 Erinaceus / Atelerix / dog 0 / 11 / 0 117 / 2 / 1 Total number of samples 34 127 1 Delimited by STRUCTURE software. A. Čmoková et al.: Population biology of Trichophyton erinacei 215 ciclopirox terbinafin tavabarole 0.0625 8 0.5 0.0312 0.25 4 MIC (μg/ml) 0.125 0.0156 0.0625 0.00781 2 1 cluster 1 cluster 2 cluster 1 cluster 2 cluster 1 cluster 2 Fig. 12   Differences in antifungal susceptibilities in Trichophyton erinacei isolates from cluster 1 and cluster 2. Significant differences in susceptibility to ci- clopirox, terbinafine and tavaborole were found (ANOVA, p < 0.05) while no statistically significant differences were found in susceptibility to the remaining five antifungals (see Results). The dots in the graph represent susceptibilities of individual isolates and geometric mean values are indicated by horizontal lines. usually consisted of 2 – 8 cells (median 4), 25.5 – 33.5 (26.8 ± 0.00781–0.0625 μg/mL; median 0.0156) than cluster 2 strains 3.5) × 4.3–6 (5 ± 0.6) μm. Chlamydospores were rarely present. (0.0156 – 0.0625 μg/mL; median 0.0625) (ANOVA, p < 0.05). No spiral hyphae were found even after 21 d of incubation. Strain CBS 474.46, ex-type of T. proliferans, was characteristic DISCUSSION by more intensive production of macroconidia and poor produc- tion of microconidia. The shape of its macroconidia was also Hedgehogs have become fashionable pets in many countries different from the shape of macroconidia of the other examined worldwide. These animals are a potential source of many patho- strains, which were thin and long or flask-shaped, with pointed gens with zoonotic potential, including bacteria, viruses and ends, and consisted of 2 – 6 cells (median 4), 10 –72.5 (39 ± fungi (Riley & Chomel 2005, Ruszkowski et al. 2021). Despite 19) × 2.5 – 5.5 (3.5 ± 1) μm. These macroconidia are often these facts, hedgehog-borne infections are generally of low produced intercalarily in vegetative hyphae. public awareness. Together with the high prevalence of T. eri­ nacei in hedgehogs, this low public awareness contributes to a Clinical picture and epidemiology significant increase in reported cases in recent years. Despite Clinical and epidemiological data associated with strains exami- the growing importance of T. erinacei as a cause of zoonotic ned in this study are summarised in Table 3. The spectrum of dermatophytoses, little is yet known about its taxonomy and di- patients and clinical manifestations of human and animal infec- versification of populations depending on the different hedgehog tions due to T. erinacei did not differ between the two clusters. hosts and the geographical origin of isolates. In this study, we The human patients were within the range of 2 – 46 yr (median collected the largest set of isolates from different hosts to date 23 and 26 yr, respectively) and were predominantly women and examined population genetics using 11 genetic markers (75 and 86 %, respectively). Infections usually manifested to address the questions mentioned. as tinea corporis (Fig. 2) localised on the upper extremities Based on microsatellite data, the isolates of T. erinacei are (predominantly hands), face and trunk. Infections in animals distributed into two main subpopulations (clusters 1 and 2) with (mostly young individuals) were frequently asymptomatic or limited gene flow between them. The random mating hypothesis manifested as scaly skin or spine loss (Table S1). was rejected (IA) for the whole species T. erinacei as well as Most human patients infected with isolates from cluster 1 for cluster 2 which is more diversified than cluster 1. However, (~ 96 %, 22 out of 23) reported contact with pet hedgehogs this does not mean that there are no occasional recombination of the genus Atelerix. The animal strains from cluster 1 were events between strains within and between subpopulations, all isolated from Atelerix hedgehogs (n = 11). Most isolates rather reflecting that isolates examined in the present study (~ 80 %, n = 101 of 127) belonging to cluster 2 were obtained spread clonally between hosts from which they were isolated. from European hedgehogs (Erinaceus spp.) or were isolated Most notably, isolates belonging to cluster 1 (MLMT genotypes directly from Erinaceus hedgehogs (n = 2). Cluster 2, however, 1 and 2 / MLST genotype G1) spread clonally between pet Afri­ also contained two samples from wild African hedgehogs of the can hedgehogs, while cluster 2 strains spread predominantly genus Atelerix from Africa. between Erinaceus wild hedgehogs. The isolates of cluster 2 were more diverse in terms of genetic variability and ecology. Antifungal susceptibility Most of the isolates were obtained from European hedgehogs The antifungal susceptibility to tavaborole, ciclopirox and ter­ (Erinaceus) admitted to wildlife rehabilitation centres in France binafine differed significantly (Fig. 12) between isolates from (Le Barzic et al. 2021), and except for two strains (T1072 cluster 1 (n = 10) and cluster 2 (n = 14), while the results from and T492), all belonged to a single clone (MLMT genotype the remaining antifungal drugs (amorolfin, griseofulvin, luli- 10 / MLST genotype G5). The remaining minority of strains conazole, itraconazole and efinaconazole) showed no signifi- of cluster 2 were obtained from both Erinaceus and Aleterix cant differences. Cluster 1 strains were significantly less sus- hedgehogs from different countries and belonged to diverse ceptible to tavaborole, with MICs ranging between 1– 8 μg/mL MLST and MLMT genotypes (Fig. 3, 6). (median 4) than cluster 2 strains (2 – 4 μg/mL; median 2) From a genetic point of view, we can observe several signs (ANOVA, p < 0.001). In contrast, cluster 1 strains were more of sexual reproduction/recombination in the population of susceptible to ciclopirox with lower MICs (0.0625 – 0.5 μg/ T. erinacei. First, both mating type genes were present among mL; median 0.1875) than the MICs from cluster 2 strains isolates representing both clusters. Second, we observed incon- (0.125–0.5 μg/mL; median 0.5) (ANOVA, p < 0.005). Similarly, gruences between DNA sequence data and microsatellite data, cluster 1 strains were more susceptible to terbinafine (MICs and some isolates had significant admixture levels between 216 Persoonia – Volume 48, 2022 microsatellite clusters (CBS 124411; Fig. 5). The intraspecific dataset such as isolates from wild African hedgehogs of the genetic variability in T. erinacei is higher than the intraspecific genus Atelerix, as they seem to represent a connecting link genetic variability in other species of the T. benhamiae complex between isolates of clusters 1 and 2. Two African strains were in- (Čmoková et al. 2020, 2021). Although the number of MLMT cluded in this study, IHEM 25164 from the Ivory Coast (Gregory genotypes was comparable to the number of MLMT genotypes et al. 1978) and IHEM 25146 from Kenya (Gregory & English of T. europaeum, this species showed no variability in the DNA 1975). Strain IHEM 25164 showed a conflicting classification sequence data. A relatively high number of MLST genotypes in terms of various molecular markers – MLST genotype G1 detected among T. erinacei strains have also been observed typical for cluster 1 but the strain was placed in cluster 2. in another sexual dermatophyte species, T. mentagrophytes, Strain IHEM 25146 was the only verified isolate from the ge- which also includes isolates of both mating types (Suh et al. nus Atelerix among strains of cluster 2. Encompassing more 2018, Tang et al. 2021). Specific MLST genotypes in T. erinacei samples from wild African hedgehogs would probably result in are substantiated by a relatively large number of substitutions a better understanding of the population structure and gene that are present in certain strains, contributing to the polyphy- flow between subpopulations of T. erinacei. Additionally, future letic nature of T. erinacei in the single-gene phylogenies (Fig. 8) collections of strains should be performed in collaboration with and highlighted the pitfalls of constructing phylogeny in species zoologists to clearly identify the host at the species level. For groups where both sexual (T. erinacei) and clonal (e.g., T. ver­ instance, European and African hedgehogs sampled in hybrid rucosum and T. eriotrephon) species are present. In general, zones may be either E. europaeus or E. roumanicus and A. albi­ the observed incongruence between single gene genealogies ventris or A. algirus, or respective hybrids between mentioned could be explained by past recombination/hybridization events pairs of species. Clear identification of hosts would allow us to or sharing of an ancestral polymorphism caused by incomplete study the extent to which the pathogen is transmitted between lineage sorting, typical for recently diverged species (Hubka et hedgehog species and how the ecological barrier of the host al. 2018a, Steenkamp et al. 2018, Matute & Sepúlveda 2019, species may influence the population genetics of the pathogen. Kandemir et al. 2020). However, both the multigene phylogeny The molecular markers used in this study represent powerful and network constructed from multilocus microsatellite data tools for surveillance of infections due to T. erinacei and pro- resolved T. erinacei as a monophyletic species (Fig. 3, 9). vide for the first-time insight into the population structure and In summary, we detected some level of substructuring into two spreading strategy of this emerging pathogen. As the popularity subpopulations in T. erinacei that are associated with different of hedgehogs as pet animals increases, an increasing number hedgehog genera. These populations are not completely sepa- of human infections can be expected in the future. The spread rated and there is a certain gene flow between them, probably of these infections underscores the need for closer collabora- due to the occasional rare transmission of pathogen between tion between veterinarians, dermatologists, epidemiologists, different hedgehog hosts. We also found minor differences in and public health personnel to set up appropriate preventive the micromorphology and antifungal susceptibilities between measures. these two subpopulations, which are not large enough to recog­ nise them as separate taxonomic entities, also supported by a Acknowledgements   This research was supported by the Czech Ministry coherent ecology strongly linked to hedgehogs and by similar of Health (grant NU21-05-00681), Charles University Research Centre growth parameters of strains belonging to different populations. program no. 204069 and Czech Academy of Sciences Long-term Research The soil environment enriched by keratin sources is necessary Development Project (RVO: 61388971). We are grateful to Radek Zmítko for the sexual reproduction of dermatophyte species (Weitzman for the help with graphical adjustments of analysis outputs. We thank Milada & Summerbell 1995, Tang et al. 2021). According to this theory, Chudíčkova, Soňa Kajzrová, Lenka Zídková and Adéla Kovaříčková for their anthropophilic and some zoophilic dermatophytes lost their invaluable assistance in the laboratory. Vit Hubka is grateful for the support from the Japan Society for the Promotion of Science – grant-in-aid for JSPS sexuality due to lack of contact with soil habitat. Only some zoo- research fellow (grant no. 20F20772). The research reported in this publica- philic Trichophyton species are believed to retain their ability to tion was part of the long-term goals of the ISHAM working group Onygenales. reproduce sexually, namely, T. africanum, T. benhamiae, T. eri­ nacei and T. mentagrophytes (Hubka et al. 2018b, Čmoková Declaration on conflict of interest   The authors declare that there is no et al. 2020, Metin & Heitman 2020, Tang et al. 2021). As far as conflict of interest. we know, T. erinacei has not been cultivated directly from soil. 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