Comprehensive Taxonomical Analysis of Trichophyton mentagrophytes/interdigitale Complex of Human and Animal Origin from India

Taxonomic delineation of etiologic agents responsible for recalcitrant dermatophytosis causing an epidemic in India is still debated. The organism responsible for this epidemic is designated as T. indotineae, a clonal offshoot of T. mentagrophytes. To evaluate the real identity of the agent causing this epidemic, we performed a multigene sequence analysis of Trichophyton species isolated from human and animal origin. We included Trichophyton species isolated from 213 human and six animal hosts. Internal transcribed spacer (ITS) (n = 219), translational elongation factors (TEF 1-α) (n = 40), ß-tubulin (BT) (n = 40), large ribosomal subunit (LSU) (n = 34), calmodulin (CAL) (n = 29), high mobility group (HMG) transcription factor gene (n = 17) and α-box gene (n = 17) were sequenced. Our sequences were compared with Trichophyton mentagrophytes species complex sequences in the NCBI database. Except for one isolate (ITS genotype III) from animal origin, all the tested genes grouped our isolates and belonged to the “Indian ITS genotype”, currently labeled as T. indotineae. ITS and TEF 1-α were more congruent compared to other genes. In this study, for the first time, we isolated the T mentagrophytes ITS Type VIII from animal origin, suggesting the role of zoonotic transmission in the ongoing epidemic. Isolation of T. mentagrophytes type III only from animal indicates its niche among animals. Outdated/inaccurate naming for these dermatophytes in the public database has created confusion in using appropriate species designation.


Introduction
Dermatophytosis due to Trichophyton species has reached an epidemic stage in the Indian subcontinent [1]. Probable reason for such a massive epidemic in India remains enigmatic. Taxonomic delineation of etiologic agents may provide leading evidence in understanding this ongoing epidemic. Labeling the etiological agent as T. mentagrophytes or T. interdigitale, or T. indotineae is still debated.
Two studies from India in 2018 attributed T. interdigitale (identified based on the classification of Trichophyton species described by de Hoog et al.) as the predominant species causing dermatophytosis [2][3][4][5]. In both studies, identity was confirmed as T. interdigitale (an anthropophilic species) based on the internal transcribed spacer (ITS) region of rDNA [4,5]. Nenoff et al. analyzed the ITS sequences of T. interdigitale and iterated that these isolates were T. mentagrophytes ITS genotype VIII (Indian genotype) and are zoophilic [6]. Chowdhary et al. indicated that rearing a pet in India is a rare phenomenon, and the zoophilic spread is questionable. Further, based on the concatenated ITS and β-tubulin sequences, they emphasized that the etiology was anthropophilic fungus [7]. Later, Nenoff et al. hypothesized that either zoophilic or geophilic Trichophyton species underwent "anthropization", developing high virulence under a suitable skin milieu. Based on in silico analysis, Pchelin et al. indicated that the Indian isolate included in their study was sister species to T. mentagrophytes and T. interdigitale [8]. Similarly, based on whole genome sequencing of the epidemic isolates, Singh et al. concluded that it was difficult to differentiate this epidemic strain from T. mentagrophytes/T. interdigitale complex [9]. Recently, Kano et al. isolated dermatophytes from patients of Indian origin in Japan and renamed them T. indotineae [10]. These isolates corresponded to Indian genotype VIII, exhibited high terbinafine minimum inhibitory concentration (MIC) and possessed unique clinical and mycological features [10]. Later, Tang et al. concluded that Indian genotype VIII differs from T. mentagrophytes sensu stricto and T. interdigitale sensu stricto [11]. They also highlighted that as the phenotypic and physiologic characteristics do not significantly vary from the name change has practical rather scientific justification [11].
The accurate identification of the Indian epidemic strain causing dermatophytosis is still uncertain. Hence, in the present study, we performed a multigene sequence of Trichophyton species isolated from human and animal origin and compared it with the published database to highlight the discrepancies in the labeling of the T. mentagrophytes/T. interdigitale complex in the GenBank database.

Fungal Strain and Growth Condition
A total of 213 Trichophyton mentagrophytes/T. interdigitale complex species isolated from dermatophytosis cases at our tertiary care referral hospital in India were used in this study. Six T. mentagrophytes/T. interdigitale complex species of animal origin (canine) included in the study were isolated at Indian Veterinary Research Institute (IVRI), Izatnagar, India. The isolates were revived on Sabouraud Dextrose Agar (SDA) (HiMedia, Mumbai, India) containing chloramphenicol (0.05 mg/L) and cycloheximide (0.5 mg/L) and incubated at 28 • C.

Phylogenetic Analysis
All the sequences of ITS, TEF 1-α, BT, LSU, CAL, HMG and α-box used previously in the literature from 2017 for identifying Trichophyton mentagrophytes/T. interdigitale complex was retrieved from the NCBI database [3,[6][7][8][11][12][13][14]. T. benhamiae (CBS 623.66) was used as the outgroup. In the present study, for barcoding, the ITS region, whole ITS1, 5.8 regions and ITS2 of KT354634 GenBank accession having 593 base pairs was used [12,15]. Retrieved sequences from the NCBI database and our sequences were aligned using multiple sequence alignment modes in ClustalX2 software. We exported the aligned sequences to Molecular Evolutionary Genetics Analysis software version X (MEGA X) [16]. The neighbor-joining tree was constructed with 1000 bootstrapping replicates using the Kimura 2 parameter model. Modification of the phylogenetic tree graphical representations was done by the Fig  Tree (version 1.4.4) and Dendroscope 3 software (version 3.7.5) [17,18].

Highlighting the Discrepancies in the Database
From 2017 to date, the sequences of the isolates used to delineate the taxonomy of T. mentagrophytes/T. interdigitale complex (n = 457) was retrieved to verify the identity submitted in the GenBank database. The retrieved sequences of ITS gene (n = 374), TEF1-α (n = 184), BT (n = 52), LSU (n = 50), CAL (n = 69) and HMG transcription factor (n = 102) were validated as per the phylogenetic analysis of ITS gene in the present study.

Results
Phylogenetic analysis of the ITS gene revealed that, except for one isolate from animal origin, all other isolates [human (n = 213) and animal origin (n = 5)] belonged to T. mentagrophytes ITS genotype VIII. The remaining isolate from animal origin belonged to ITS genotype III. The phylogenetic tree constructed based on the Neighbor-Joining method using Kimura 2-parameter model had a total of 15 clusters for T. mentagrophytes with previously defined genotypes III, III*, IV, V, VII, VIII, IX, XI, XII, XIII, XV-XVI, XVIII, XXII and an undefined genotype (KMU 5471, AB617775). Whereas T. interdigitale consisted of a cluster previously defined as Type I, II and II* and T. interdigitale genotype X (Table 1) (Supplementary Figure S1). Like the ITS gene, the TEF 1-α gene also clustered all our isolates [human (n = 34) and animal (n = 5) origin] together along with the T. mentagrophytes ITS genotype VIII isolates. In contrast, the remaining isolate from animal origin (PGI-IVRI B24-A) clustered with T. mentagrophytes ITS genotype III, III*, V and T. interdigitale. Phylogenetic analysis based on TEF 1-α divided isolates into six clusters (cluster 1: T. mentagrophytes ITS genotype VIII; cluster 2: T. mentagrophytes ITS genotype III, III*, V and T. interdigitale; cluster 3: T. mentagrophytes ITS genotype IV; cluster 4: T. mentagrophytes ITS genotype IV; cluster 5: T. mentagrophytes ITS genotype III, VIII and T. interdigitale; cluster 6: T. mentagrophytes genotype III*, VII and IX) (Supplementary Figure S2).
The phylogenetic tree based on the BT gene (n = 39) also clustered all our isolates and isolates belonging to T. mentagrophytes ITS genotype IV, VIII and T. interdigitale. The remaining isolate from animal origin (PGI-IVRI B24-A) formed a separate cluster and did not merge with other genotypes. Based on the BT gene, all the analyzed isolates were grouped into four clusters (cluster 1: T. mentagrophytes type IV, VIII and T. interdigitale; cluster 2: PGI-IVRI B24-A; cluster 3: T. mentagrophytes type III, III*, V, VII; and cluster 4: T. mentagrophytes type III*, IV and T. interdigitale (Supplementary Figure S3).
A comprehensive analysis of the labeling of submitted sequences in the GenBank database revealed discrepancies. The accurate identification of the isolates was considered based on our phylogenetic analysis (ITS gene). Based on the analysis, a large number of 4 ITS sequences deposited in the GenBank databases were mislabeled, i.e., T. mentagrophytes was named as T. interdigitale or vice versa (Supplementary Table S2).

Discussion
The identity of the pathogen responsible for the dermatophytosis epidemic in India is under scrutiny. The ongoing outbreak of superficial dermatophytosis is mainly due to Trichophyton spp. The Trichophyton mentagrophytes/T. interdigitale complex is the principal etiological agent responsible, and resistance in these isolates commonly occurs and leads to treatment failure. Thus finding the etiological agent may provide better insight into therapeutic management [9]. The whole-genome sequence of Indian Trichophyton spp. (D15P135) formed a conspecific clade distant from T. mentagrophytes and T. interdigitale [8]. Based on the ITS phylogenetic tree, Nenoff et al. designated the Indian isolates as ITS genotype VIII [6]. Several other studies have also reported that T. mentagrophytes ITS type VIII is most common in India [9,13,14,20] and second most common in Iran [14]. Apart from India and Iran, other countries (i.e., Japan, Germany, Oman and France) also reported T. mentagrophytes ITS genotype VIII [10,11,19].
Further, based on whole-genome analysis, Singh et al. showed that all the clinical isolates of India belonged to T. mentagrophytes type VIII and had only 42 SNPs between any two isolates. They could not conclusively differentiate T. mentagrophytes from T. interdigitale [9]. Recently, Kano et al. described two isolates of Indian origin resistant to terbinafine as a new species, T. indotineae, based on the ITS gene sequence. However, later Tang et al. performed multigene phylogeny (ITS, TEF 1-α and HMG gene) of many isolates (n = 182) and verified that it was indeed a new species. In the present study, we found that all the clinical (human) isolates formed a cluster with T. mentagrophytes ITS genotype VIII (D15P135; accession no KY761968).
Nenoff et al., in 2019, introduced a total of nine genotypes, including T. interdigitale (genotype I and II) and T. mentagrophytes (genotype III to IX) based on ITS sequences [19] and indicated that Indian origin isolates were T. mentagrophytes ITS genotype VIII. Further, Taghipour et al. analyzed their sequences including the sequences of IX genotypes (described by Nenoff et al.) and described more genotypes (genotype X to XXIV) [14]. Recently, Nenoff et al. also introduced one more genotype (genotype XXV) [13] [3]. However, Type isolates defined as T. mentagrophytes CBS 304.38 T (MF898372) by de Hoog et al. [3] and JF731044 (T. mentagrophytes ITS genotype VIII) by Nenoff et al. [6] were also clustered with our isolates [3]. Therefore, the BT sequences appear to have less discriminatory power than ITS sequences, leading to Chowdhary et al. concluding all the isolates as T. interdigitale. [7] LSU gene could not even differentiate the type strain of T. mentagrophytes KT155300 (CBS 646.73 T ) from T. interdigitale (type and neotype strains), thereby questioning their validity in identifying these species correctly. Like Tang et al. and Nenoff et al., ITS and TEF 1alpha were more congruent, with more diversity in the ITS gene [11,13].
In the present study, we found many previously designated isolates with one genotype belonging to other genotype clusters (Supplementary Table S2). For example, the sequence of isolates described as genotype VI (DP15P161, D15P156) clustered with genotype IV isolates in our study [12,13]. Similarly, the new genotype introduced by Taghipour et al., i.e., genotype XVII (RezRaf-277, MK312990), clustered with genotype III* [12]. The other genotypes by Taghipour Table S2) [12]. The new genotype XXV introduced by Nenoff et al.
(218292/17, MN886815; 201341/18, MN886816) clustered with genotype III* [13]. Further, as per Nenoff et al., T. interdigitale II* and X isolates form a separate group from genotypes I and II. Still, in the present study, these isolates belong to a single group in which all the T. interdigitale genotypes I and II are present (Supplementary Figure S1) [13]. The confusion in identification is due to a need for more upgradation in strain details deposited in the NCBI and CBS databases. Few of the Trichophyton spp. sequence details are incorrect (Supplementary Table S2), which has also been emphasized by Chowdhary et al. [7] Two isolates (IHEM 10162 and JCM 1891), which were previously described as T. mentagrophytes genotype IV based on ITS, form a separate cluster (cluster 3-Supplementary Figure S2) as per the TEF 1-α gene analysis in the present study [12]. Further, due to the unavailability of gene bank accession numbers for the type and neotype strain of T. mentagrophytes and T. interdigitale, we could not assess whether this gene could differentiate them. Only the neotype strain of T. mentagrophytes (IHEM 4268 NT ) sequence was available (ITS genotype III* based on ITS gene), which formed a cluster with isolates of T. mentagrophytes ITS genotype III, III*, V, and T. interdigitale (CBS 130806) strain (Supplementary Figure S2).
Recently, Tang et al. suggested that the presence of HMG transcription factor without α-box may provide the answer to identify the etiological agent responsible for the ongoing epidemic due to T. indotineae. HMG and α box are the drivers of evolution among dermatophytes [21]. T. mentagrophytes is the ancestral species from which 'clonal offshoots' T. interdigitale and T. indotineae were derived [11]. The occurrence of α box indicates the presence of MAT1-1 mating type, whereas the presence of MAT1-2 mating is indicative of the HMG domain [22]. Previously, Persinoti et al. reported that all the T. rubrum and T. interdigitale isolates used in their study showed the presence of a single mating type and all the population are clonally similar with a low level of diversity [22]. Further, based on the HMG gene, they described T. rubrum and T. interdigitale (causing an ongoing epidemic in India or T. indotineae) as anthropophilic. Anthropophilic dermatophytes have lost their sexuality [23], as seen among T. interdigitale. Similar to Nenoff et al. and Tang et al., all our human isolates (n = 11) belong to the "plus mating type" and carry only the HMG transcription factor at the MAT locus suggesting that the ongoing epidemic in India was due to a single type of mating strain (i.e., HMG transcription factor). Further, this finding shows agreement with the phylogenetic data of ITS and TEF1-α in which all the ongoing Indian epidemic isolates cluster together [11,19].
Chowdhary et al. reinforced that all the isolates used in the study by Singh et al. were T. interdigitale based on concatenated phylogeny of ITS and beta-tubulin sequences [7]. Further, they also reported that the patient presented with an atypical clinical appearance suggesting more like zoophilic infection. Thus, the question is whether the disease in India is due to the zoophilic T. mentagrophytes or anthropophilic T. interdigitale species. In our experience, of the 195 patients included in our previous study, only 28 (14.3%) gave a history of possible animal contact [4]. This is in full agreement with the study by Khurana et al. in which among 64 (75.3%) culture positive cases, only 2 (3.12%) patients had a history of animal contact [24]. We also agree that rearing a pet in India is relatively rare [7].
We did not find any nucleotide variation in the ITS and TEF sequences of human and animal origin (five isolates) T. mentagrophytes type VIII isolates. The animal-origin isolates of T. mentagrophytes type VIII only had plus mating-type of HMG transcription factor and did not show the presence of αbox, suggesting its nature like human-origin isolates. Till date, all the reported T. mentagrophytes ITS genotype VIII in the literature isolated from a human source, but in the present study, for the first time, we isolated T. mentagrophytes ITS genotype VIII from animal origin (pet dogs). Furthermore, several isolates are required to support the validity of the circulated anthropophilic T. indotineae among the animals.
A possible solution to resolve the confusion is to curate the sequences deposited in the public databases. There is also a need to define barcode sequence, positions (trimmed ends) of ITS to designate the Trichophyton species. An international consortium to discuss the taxonomy and designating neotype or type strain for any new species or subspecies (based on housekeeping gene sequence or multiple gene sequences) may help resolve the taxonomy issues. Further, the full genome sequencing project on dermatophytes might help to resolve the current taxonomy issues.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof9050577/s1, Figure S1: Phylogenetic tree based on ITS sequences Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the Kimura 2-parameter method. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA X., Figure S2: Phylogenetic tree based on TEF sequences Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the Kimura 2-parameter method. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA X., Figure S3: Phylogenetic tree based on BT sequences Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the Kimura 2-parameter method. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA X., Figure S4: Phylogenetic tree based on LSU sequences Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the Kimura 2-parameter method. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA X., Figure S5: Phylogenetic tree based on CAL sequences Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the Kimura 2-parameter method. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA X., Figure S6: Phylogenetic tree based on HMG sequences Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the Kimura 2-parameter method. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA X; Table S1: Primers used in the present study, Table S2: The discrepancy in the naming of Trichophyton mentagrophytes complex in the NCBI database. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: All data were available in NCBI database accession number provided in manuscript (Url: https://www.ncbi.nlm.nih.gov/ accessed on 21 September 2021).

Conflicts of Interest:
The authors declare no conflict of interest.