Trichophyton interdigitale complex causing an ongoing alarming dermatophytosis outbreak in India: Genomic insights and resistance profile

Journal Pre-proofs A unique multidrug-resistant clonal Trichophyton population distinct from Trichophytonmentagrophytes / Trichophytoninterdigitale complex causing an ongoing alarming dermatophytosis outbreak in India: genomic insights and resistance profile Ashutosh Singh, Aradhana Masih, Juan Monroy-Nieto, Pradeep Kumar Singh, Jolene Bowers, Jason Travis, Ananta Khurana, David M. Engelthaler, Jacques F. Meis, Anuradha Chowdhary PII: DOI: Reference:

S1087-1845(19)30198-7 https://doi.org/10.1016/j.fgb.2019.103266 YFGBI 103266

To appear in:

Fungal Genetics and Biology

Received Date: Revised Date: Accepted Date:

14 June 2019 29 August 2019 29 August 2019

Please cite this article as: Singh, A., Masih, A., Monroy-Nieto, J., Kumar Singh, P., Bowers, J., Travis, J., Khurana, A., Engelthaler, D.M., Meis, J.F., Chowdhary, A., A unique multidrug-resistant clonal Trichophyton population distinct from Trichophytonmentagrophytes / Trichophytoninterdigitale complex causing an ongoing alarming dermatophytosis outbreak in India: genomic insights and resistance profile, Fungal Genetics and Biology (2019), doi: https://doi.org/10.1016/j.fgb.2019.103266

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A

unique

multidrug-resistant

clonal

Trichophyton

population

distinct

from

Trichophyton mentagrophytes / Trichophyton interdigitale complex causing an ongoing alarming dermatophytosis outbreak in India: genomic insights and resistance profile. Ashutosh Singh1, Aradhana Masih1, Juan Monroy-Nieto2, Pradeep Kumar Singh1, Jolene Bowers2, Jason Travis2, Ananta Khurana3, David M. Engelthaler2, Jacques F. Meis4,5, Anuradha Chowdhary1*

1Department

of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi,

Delhi, India;

2Translational

Genomics Research Institute, Flagstaff, Arizona, USA;

3Department of Dermatology, Dr. RML Hospital and PGIMER, New Delhi, India; 4Department

of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital (CWZ), Nijmegen; 5Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands.

Keywords: Trichophyton, outbreak, ITS, multi-locus, MDR, DermaGenius® 2.0 multiplex real-time PCR assays , whole-genome sequencing, India,

*Corresponding author Prof. Anuradha Chowdhary Department of Medical Mycology V. P. Chest Institute, University of Delhi Delhi-110007, India Email: [email protected]

Abstract

There has been a considerable upsurge of extensive, treatment recalcitrant, dermatophytosis presenting as tinea corporis and tinea cruris in India since the past few years. Genome analysis of Trichophyton species causing severe superficial dermatophytosis in North India confirmed a unique clade most recently related to the T. mentagrophytes/interdigitale complex, seeming to belong to an early diverging clade of the complex. The Indian Trichophyton species genomes were highly related showing only up to 42 SNPs between any two isolates confirming their clonal origin. Other genetic approaches such as ITS sequencing and multigene phylogeny used in this study were contradictory or inconclusive to show the differentiation of these isolates from T. mentagrophytes/T. interdigitale. Remarkably, high rates of resistance to all three commonly used oral antifungals, i.e., 36% for terbinafine (MICs 4 to ≥32 mg/L), 39.5% for fluconazole (MIC range 32 to ≥64 mg/L) and griseofulvin (Geometric mean MIC ≥4 mg/L) were observed. Two important amino acid substitutions (Leu393Phe or Phe397Leu) leading to a terbinafine resistant phenotype were found in the squalene epoxidase protein of all tested terbinafine resistant isolates. All 20 examined genomes presented a high mobility group (HMG) domain transcription factor gene corresponding to mating type (+). Of these, three isolates also showed positivity for both alpha-box and HMG in the genome which might indicate hybridization or an incomplete sexual cycle. Therefore, we highlight the potential of this organism to rapidly spread alleles that might be driving antifungal resistance among its population. This new population of Trichophyton with high rates of in vitro antifungal resistance seems to be driving an ongoing outbreak of dermatophytosis in India. Our study highlights

difficulties

in

identifying

isolates

from

the

Trichophyton

mentagrophytes/interdigitale clade of the genus using currently available molecular tools. High resistance rates of terbinafine warrant further clinical studies to assess its utility in the treatment of dermatophytosis caused by this strain.

1. Introduction Recalcitrant superficial dermatophytosis primarily due to Trichophyton mentagrophytes species complex has caused an alarming situation in India with extensive presentations, frequent relapses, and treatment failures. The cause of this increase in incidence of dermatophytosis is not clearly understood but multiple factors likely contribute. These include erratic over-the counter use of topical and oral antifungal agents and topical steroids, increased prevalence of Trichophyton mentagrophytes species complex (primarily T. interdigitale and T. mentagrophytes) infections, more humid and warmer climatic factors, and the recently recognised increasing burden of resistance to antifungal agents (Bishnoi et al., 2018; Singh et al., 2018; Rudramurthy et al., 2018). The unchecked availability of fixed drug combination creams containing steroid, antifungal and antibacterial compounds opens the way for irrational, inconsistent treatment possibly driving resistance (Bishnoi et al., 2018). Extensive dermatophytosis of the glabrous skin i.e. tinea cruris (involving groins)

and corporis

(involvement of rest of the skin surface excluding palms, soles, scalp and groins) is the commonest clinical presentation being seen in India at present. Recent studies of tinea cruris and tinea corporis in India, identifying the etiologic agent via internal transcribed spacer (ITS) region sequencing, have shown that Trichophyton interdigitale, a sibling species of T. mentagrophytes, is the most frequently encountered species (Rudramurthy et al., 2018; Singh et al., 2018). Although the rDNA ITS region is informative, the dermatophytes are a closely related group, and it remains challenging to use this genetic marker to differentiate isolates among species complexes such as Trichophyton rubrum and T. mentagrophytes. While T. mentagrophytes is a polymorphic sexual species, T. interdigitale is recognized as its clonal anthropophilic derivative (Metin and Heitman, 2017; Symoens et al., 2011). Further, phylogeny studies demonstrated trees with a paraphyletic or even polyphyletic T. mentagrophytes/T. interdigitale species complex, with T. tonsurans and T. equinum isolates

branching within their clade (Garcia Garces et al., 2016; Kawasaki et al., 2011; RezaeiMatehkolaei et al., 2014; Suh et al., 2018). All four species are being placed in the T. mentagrophytes series (Gräser et al., 2018). However, a recent study using whole genome sequencing (WGS) suggest that T. mentagrophytes and T. interdigitale are conspecific (Pchelin et al., 2018). Furthermore, accurate identification of Trichophyton spp. by ITS sequence comparisons with public databases (mainly GenBank, NCBI and Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands) have limitations as at present, these databases have not been updated and contain numerous sequences deposited under different names for strains of several taxa in the genus (Chowdhary et al., 2019). Here we investigated the ongoing epidemic of tinea corporis/tinea cruris using multigene phylogeny, ITS sequencing, WGS and antifungal resistance assays and characterized the terbinafine SQLE gene target in Trichophyton isolates originating from patients in North India. 2.

Materials and methods

2.1 Clinical specimens and fungal isolates A total of 166 Trichophyton isolates were obtained over a period of 5 years (2014-18) from individual patients in five hospitals in North India. The patients had predominantly tinea corporis/cruris (94%, n=156) followed by tinea capitis (6%, n=10). Skin scrapings (n=156) and hair (n=10) were processed for direct microscopic examination with 10% potassium hydroxide (KOH)/Blankophor. The specimens were inoculated on two plates each of Sabouraud’s dextrose agar (SDA) containing gentamicin and chloramphenicol and the other containing cycloheximide (0.05%) and were incubated at 28°C for 2 weeks. 2.2.

Morphological examination and phenotypic identification

The preliminary morphological identification was made on potato dextrose agar (PDA). Briefly, the primary growth on SDA was transferred to PDA and incubated at 28°C for 14 days.

For microscopic morphological details, slide cultures were prepared on PDA and mounted in lactophenol cotton blue. 2.3.

Molecular identification using the PCR DermaGenius® 2.0 kit

A total of 166 morphologically identified Trichophyton isolates were subjected to the DermaGenius® 2.0 (PathoNostics, Maastricht, The Netherlands) multiplex real-time PCR for identification of Trichophyton isolates. DNA extraction was done as per manufacturer’s instruction. Briefly, small pieces of mycelium were collected with a sterile scalpel from the SDA plates and transferred to green bead tubes (Roche Diagnostics, Indianapolis, IN, USA) and 500 µl of NucliSENS lysis buffer (bioMérieux, Amersfoort, the Netherlands) was added together with 5 µl of internal control. The mycelium suspension was subsequently bead-beaten in a MagNALyser instrument (Roche Diagnostics) for 45s at 6,500rpm. Proteinase K (Roche Diagnostics) was added, incubated for 10 min at 65°C, and subsequently inactivated for 10 min at 95°C. After centrifugation, the supernatant of the lysed mycelium suspension was transferred to a new tube. DNA of the mycelium suspension was extracted using the NucliSENSeasyMAG Extraction system (bioMérieux), according to the manufacturer’s instructions. DNA was stored at -20°C until testing with the real-time PCR DermaGenius® 2.0 complete multiplex kit for identification of Trichophyton isolates. For the real-time PCR, DNA samples were processed according to manufacturer’s instructions: 5 μl of DNA extract was added to the PCR mix 1 (Trichophyton spp.), and a Mic qPCR cycler (BMS, Australia) was used for amplification and melting curve analysis. Melting curve analysis is needed to differentiate between the dermatophyte species. The identification kit includes all clinically important Trichophyton species and Candida albicans. The species of Trichophyton that can be identified with the kit are T. mentagrophytes, T. interdigitale, T. rubrum, T. tonsurans, T. soudanense, T. violaceum, T.

benhamiae,

T. verrucosum,

Microsporum canis,

M.

audouinii,

and

Epidermophyton floccosum. A melting peak at specific temperatures (Tm-value) allows species

identification with the DermaGenius® 2.0 PCR assay. Positive and negative template controls were included in each PCR run. Data analysis was performed using the Mic qPCR software (v 2.6.4), and automatic Ct analysis/ Tm-range calculations were enabled. Data interpretation was done automatically using the parameter ‘Genotype’ in the Mic qPCR software, which resulted in dermatophyte species identification based on specific predefined Tm-values. Additionally, three other Trichophyton species confirmed by sequencing as T. rubrum (n=5), T. tonsurans (n=3) and T. violaceum (n=1) were also included. 2.4.

Confirmation of identification by ITS sequencing of Trichophyton isolates

To confirm the results obtained by DermaGenius Kit, we selected 73 Trichophyton isolates randomly and subjected them to ITS sequencing using ITS1 and ITS4 primers detailed in the following section. 2.5.

Multigene sequence analysis of Trichophyton isolates using ITS, β-tubulin (BT2), translation elongation factor 1-α (Tef 1-α ) and calmodulin genes

To study the population structure of Trichophyton, we performed multigene phylogeny of 36 isolates using previously published markers (de Hoog et al., 2017). Genomic DNA was extracted using phenol-chloroform-isoamyl alcohol method as described previously (Masih et al., 2016; Singh et al., 2017). Then, the extracted DNA was amplified using the ITS-1 (5′TCCGTAGGTGAACCTTGCGG-3′) and ITS-4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al., 1990), β-tubulin gene; Btub2Fd (5’-GTBCACCTYCARACCGGYCARTG-3’) and Btub4Rd (5’-CCRGAYTGRCCRAARACRAAGTTGTC-3’) (Groenewald et al., 2013), translation elongation factor 1-α gene; EF-DermF (5’-CACATTAACTTGGTCGTTATCG-3’) and EF-DermR (5’-CATCCTTGGAGATACCAGC-3’) (Mirhendi et al., 2015), calmodulin gene;

CF1

(5’-GCCGACTCTTTGACYGARGAR-3’)

and

CF4

(5’-

TTTYTGCATCATRAGYTGGAC-3’) (Ahmadi et al., 2016) primers. DNA sequencing was performed using the respective primers for PCR at 0.5 μmol/L concentration. All sequencing

reactions were carried out in a 10-μL reaction volume using the BigDye Terminator kit version 3.1 (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s recommendations, and analysed on an ABI 3130XL Genetic Analyzer (Applied Biosystems). The

sequences

were

queried

with

BLAST

searching

of

GenBank

records

(http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi). Sequence-based species identification was defined as ≥99% sequence similarity with ≥99% query coverage. Further, a neighbour-joining (NJ) tree based on aligned ITS gene sequences with 2,000 bootstrap replications was constructed using MEGA version 7 (Tamura et al., 2013). The sequences of the type/reference strains of various Trichophyton spp. were retrieved from GenBank and included for the phylogenetic analysis (de Hoog et al., 2017). ITS sequences of Indian T. mentagrophytes deposited as Type VIII ( DSM 107595-610, WM10.87, 490, 05-297-2873, Ahv-18, SRMC-K, 800036, 30816, NCCPF: 800062) and other types, i.e., Type II, III, IV, V and VII of T. mentagrophytes were retrieved from the NCBI database for comparison (Nenoff et al., 2019). The neotype strain of T. mentagrophytes (IHEM4268), T. interdigitale (CBS428.63) and reference strain of T. quinckeanum (216686/15, SU2013746, ATCC 32457,) and T. rubrum (AB430483.1) were also used for comparison. 2.6.

Antifungal susceptibility testing

A total of 129 Trichophyton isolates were available for antifungal susceptibility testing (AFST). This procedure was done using the Clinical and Laboratory Standards Institute broth microdilution (CLSI-BMD) (Clinical Laboratory Standards Institute, 2017). It is highly relevant to emphasize that in spite of using primary SDA culture media supplemented with chloramphenicol and gentamicin, bacterial contamination was a hurdle in primary cultures. Despite repeated subcultures on SDA media containing the above-mentioned antibiotics, bacterial contamination was evident in some cultures. To tackle this issue, the samples growing on SDA containing chloramphenicol and gentamicin were transferred to 3ml PBS containing

penicillin G (6g/L), streptomycin (0.5 mg/L) and gentamicin (0.25 mg/L) and were incubated at room temperature for 12 hours. A loopful of suspension was sub-cultured on PDA plates and incubated at 28°C for 5-7 days. The growth obtained after 5-7 days was free of bacteria and was then sub-cultured on PDA for 7 days at 28°C for sporulation. These axenic cultures were used for AFST. The conidial inocula of the pure cultures were prepared in 0.09% saline containing Tween 80 by gently scraping the surface of mature colonies with a sterile cotton swab moistened with sterile physiological saline and allowed to settle. The final inoculum concentration was adjusted to 1×103 to 3×103 CFU/mL counted by haemocytometer (CLSI, 2017). Antifungals tested included terbinafine (TRB; R-1012/16; Synergene India, Hyderabad, India), triazoles viz. itraconazole (ITC; ITFP07008; Lee Pharma, Hyderabad, India), voriconazole (VRC; 030M7505V; Sigma-Aldrich, Steinheim, Germany), and fluconazole (FLU;

036M4709V;

Sigma-Aldrich);

Imidazoles

viz.

sertaconazole

(SER;

OP-

SANP/05/16/011; Optimus, Hyderabad, India), luliconazole (LUZ, 2823343; Sun, Baddi, HP India), clotrimazole (CLT; 075K1032; Sigma-Aldrich), miconazole (MCZ; BCBD5966V; Sigma-Aldrich), and ketoconazole (KTC; SLBR1290V; Sigma-Aldrich) and griseofulvin (GRE; MKBQ4861V; Sigma-Aldrich). AFST of the isolates against TRB was performed for 20 dilutions ranging from 0.00006-32 mg/L. For the remaining tested drugs 10 dilutions were tested and the drug concentration ranges were: FLU, 0.25-64 mg/L; ITC, VRC, SER, and MCZ, 0.03-16 mg/L; CLT and KTC, 0.06-32 mg/L; GRE, 0.015-8 mg/L and LUZ, 0.0035-2 mg/L. Drug-free and mould-free controls were included, and microtiter plates were incubated at 35°C. The CLSI recommended control strains Candida krusei ATCC6258, Candida parapsilosis ATCC22019, and reference strain T. mentagrophytes (ATCC MYA-4439) were also included for susceptibility testing. Minimum inhibitory concentration endpoints for all the drugs were defined as the lowest concentration that inhibited 80% of the growth as read visually at 72 hrs. The reference CLSI-BMD method (CLSI, 2017) suggests reading MICs of dermatophytes at 4

days (96 hrs). However, in the present study confluent growth was observed after 3-days (72 hours) of incubation for all Trichophyton isolates. Therefore, MICs of all isolates were read visually after incubation at 35o C for 72 hours. The MIC range of the reference strain T. mentagrophytes (ATCC MYA-4439) against GRE, ITC, VRC and TRB were interpreted following guidelines of M61 (CLSI, 2017). 2.7.

Preliminary screening of Trichophyton isolates resistant to terbinafine

In order to validate a previously described agar screening method for detecting TRB resistance among Trichophyton spp., we screened 20 Trichophyton isolates (n=10, MIC 0.125-1 mg/L; n=10, MIC 4-≥32 mg/L) on SDA plates containing 0.2 mg/L TRB (Yamada et al., 2017). Briefly, a cotton swab dipped in sterile normal saline was rotated on the plate containing a pure growth of isolates to collect the spores. These spores were then inoculated at the centre of the SDA plates containing TRB and incubated at 28o C. Examination of fungal growth of susceptible and resistant isolates was performed after 7, 10, and 14 days intervals. 2.8.

Squalene epoxidase (SQLE) gene amplification and sequencing of terbinafine resistant and susceptible Trichophyton isolates

A total of 61 Trichophyton isolates including all isolates with high MICs of TRB (n=47, MIC2≥32 mg/L) and 16 randomly selected isolates with a low to moderate MICs range (0.25-1mg/L) were subjected to SQLE gene sequencing. The DNA was extracted as described above under the section multigene phylogeny (2.5). The extracted DNA was amplified using primers TrSQLE-F1

(5’-ATGGTTGTAGAGGCTCCTCCC-3’)

and

TrSQLE-R1

(5’-

CTAGCTTTGAAGTTCGGCAAA-3’) (Yamada et al., 2017). PCR was carried out in a 50µL reaction volume, and the conditions included initial denaturation for 5 min at 95ºC followed by 34 cycles of 30 s at 95ºC, 30 s at 60ºC and 180 s at 72ºC. DNA sequencing was performed using the PCR primers at 2.5 mM concentration. All sequencing reactions were carried out in a 10 µL reaction volume using BigDye Terminator Kit v3.1 (Applied Biosystems, Foster City,

CA, USA) according to the manufacturer’s recommendations and analysed on an ABI3130xL Genetic Analyzer (Applied Biosystems). 2.9.

Genome sequencing and phylogenetic analysis of Trichophyton isolates

For genomic sequencing, DNA was extracted using a column-based method with a QIAamp DNA minikit (Qiagen, Hilden, Germany) and was quantified by QUBIT 3 Fluorometer using dS DNA HS Dye. WGS libraries were prepared using NEBNext ultra II DNA FS kit (New England Biolabs, Ipswich, MA, USA). In brief, 200 nanograms of intact DNA was enzymatically fragmented by targeting 200-300 bp fragments sizes followed by purification using AMPure beads (Beckman Coulter life Sciences, Indianapolis, IN, USA). For sequencing, the libraries were normalized to 10 nMol/L concentration and pooled together at equal volumes. Further, the library pools were denatured using freshly prepared 0.2N NaOH for cluster generation on cBOT and sequenced on Illumina HiSeq 4000. To ascertain the relatedness of Indian Trichophyton isolates, we compared the WGS of 20 isolates with publicly available Sequence Read Archive (SRA) records and NCBI assemblies sampled on 02/22/2019 (Supplementary File 1). Relatedness was investigated using three methods: High-confidence genomic single nucleotide polymorphisms (SNPs) using NASP (Sahl et al., 2016), kmer content comparison using MASH (Ondov et al., 2016) , and taxonomyguided kmer content assignment via kraken2 (Wood and Salzberg, 2014) . Sequence read data were initially trimmed of adapter remnants, artefacts, and low-quality base calls using BBDuk from BBTools (https://github.com/kbaseapps/BBTools). We identified high-certainty SNPs in the samples using the NASP pipeline (v. 1.1.2) against a public assembly from isolate D15P135 (Accession: GCA_003664455.1). The pipeline was set to use BWA (v 0.7.15) (Li and Durbin, 2010) as the read aligner and GATK (v3.7) (McKenna et al., 2010) as the SNP caller. The pipeline also filtered out positions with depth of coverage below 10X, those with base concordance below 90% among the aligned reads, and any positions that

weren’t present in all samples of the set. SNP data were processed in MEGA7 (Kumar et al., 2016). Phylogenies were calculated using the consensus of most parsimonious trees at 95% agreement; splits below this threshold are shown as polytomies. For kmer comparisons, each sample’s read-data was parsed or “sketched” for MASH analysis; the resulting databases contained at most 10,000 non-redundant min-hashes. The comparisons were averaged based on species identification of the target sketches. Some samples, those that were learned to be misidentified according to our SNP analysis, were corrected for measurements in this analysis: M8436 (T. interdigitale -> T. mentagrophytes), D15P152 (T. mentagrophytes -> T. interdigitale), MR1463 (T. rubrum -> T. interdigitale). The taxonomy-supported kmer content assignment was executed running kraken2 over a custom database created with all GenBank records for fungi (TaxID 4751) using all default settings. 2.10.

Sex identity gene identification from whole genome data

Assemblies from the sequence reads were created using Perga (V. 0.5.03.02) (Zhu et al., 2014) and SPAdes (V. 3.10.1) (Nurk et al., 2013). Sex genes encoding, a high mobility group (HMG) domain transcription factor gene and alpha-box were identified from assembled genomic data using the in silico PCR software VIPR (https://github.com/TGenNorth/vipr), searching for hits as long as 1500 bp with all permutations of primers for a given target. The primers used are available in the informatics resources (Supplemental file 1) 3. Results 3.1.

Identification of isolates by DermaGenius® 2.0 multiplex real-time PCR and ITS sequencing

The isolates microscopically identified as Trichophyton spp. were further characterized as T. interdigitale (99%; n=164) and the remaining two isolates as T. mentagrophytes by DermaGenius (DG) real-time PCR. All positive controls and no-template controls (NTCs) were

in the acceptable range. All DNA extracts contained an IC signal, which indicated no inhibition. Contrasting the results of the kit, BLAST results of ITS region sequencing of 73 Trichophyton isolates yielded identification as T. mentagrophytes and T. interdigitale both with identity score 100% and 100% query coverage in GenBank, NCBI and Westerdijk Fungal Biodiversity Institute, databases. Conversely, in the phylogenetic analysis of ITS sequences (Figure 1) all Trichophyton isolates investigated in the present study and T. mentagrophytes type VIII isolates retrieved from GenBank clustered together comprising a single clade. Furthermore, other T. mentagrophytes types (II, III, IV, V, VII) fell in their respective distinct clades. T. mentagrophytes neotype (IHEM 4268) strain clustered within Type III T. mentagrophytes clade. GenBank numbers (MH990799-MH990859) of the ITS sequences of Trichophyton isolates examined in this study have been submitted as T. interdigitale in previous publication (Singh et al., 2018). Interestingly, neotype T. interdigitale (CBS 428.63) and all other T. interdigitale strains retrieved from GenBank were resolved into another clade that was distinct from the clade housing the present study isolates, suggesting that all Indian isolates comprise a unique genotype of T. mentagrophytes. It is pertinent to mention here that the majority of type VIII strains deposited in the NCBI database have been isolated from Indian patients in a recent study and four isolates outside India were from Iran ( n=2), Oman (n=1) and Australia ( n=1). 3.2.

Multigene comparison

β-tubulin gene sequences of 36 T. mentagrophytes/interdigitale complex isolates generated in this study showed a 100% query coverage and identity with the neotype strain of T. interdigitale (CBS 428.63), whereas they differed at five positions from the neotype strain of T. mentagrophytes (strain IHEM 4268) and sequences of the Tef-1α and partial calmodulin gene showed 99%-100% identity with T. interdigitale and T. mentagrophytes strains with equal query coverage.

3.3.

In vitro susceptibility testing

The results of in vitro antifungal susceptibility profiles of 129 Trichophyton isolates are summarised in Table 1. It is noteworthy that 36% and 39.5% of isolates displayed high resistance for TRB (MICs: 4 to ≥32 mg/L) and FLU (MIC range: 32 to ≥64 mg/L). In addition, they also showed reduced susceptibility to GRE (GM: 3 mg/L) as 95% isolates had MICs >2 mg/L. However, as compared to FLU, all other azoles tested, i.e., VRC (GM MIC: 0.18mg/L) and ITC (GM MIC: 0.24 mg/ L) had low MICs. Overall, 3% (n = 16) of isolates were resistant to all three orally available antifungals, i.e., TRB, FLU, and GRE. Moreover, 24% and 7% of isolates were resistant to TRB+GRE (TRB, MICs: 4 to ≥32 mg/L; GRE, MICs: ≥4) and TRB+FLU, (TRB, MICs: 4 to ≥32 mg/L; FLU, MICs: 32 to ≥64 mg/L) respectively. For KTC, MCZ, LUZ, and CLT no ranges have been given by CLSI, so we were unable to interpret MICs of

reference

strains.

For

these

drugs,

we

compiled

data

of

clinical

T. mentagrophytes/interdigitale spp. complex strains based on previously published studies (Badali et al., 2015; Baghi et al., 2016; Rezaei-Matehkolaei et al., 2018; Wiederhold et al., 2014) and observed wide MIC ranges for LUZ, (0.0002–0.025 mg/L), SER (0.03-16 mg/L), KTC (0.03-16 mg/L), MCZ (0.03-8 mg/L), and CLT (0.01-2 mg/L). Overall, in the present study, LUZ (GM MIC: 0.007 mg/L) showed excellent activity whereas CLT (GM: 2.2 mg/L), MCZ (GM: 1.8 mg/L), and SER (GM: 1.12 mg/L) showed reduced susceptibility against all Trichophyton isolates. 3.4.

SQLE gene analysis

Two important previously recognised amino acid substitutions (Leu393Phe or Phe397Leu) leading to a TRB resistant phenotype in T. mentagrophytes isolates were found in SQLEp of all tested TRB resistant (MIC: 4 to ≥32 mg/L) isolates. The wild-type genotype was noted in isolates with the MIC range of 0.125-2 mg/L. In 85 % (n=39) of isolates, amino acid

substitution Phe397Leu was observed, whereas in the remaining 15% (n=7) isolates amino acid substitution Leu393Phe was noted (Table 2). 3.5.

Screening of Trichophyton isolates resistant to terbinafine

The screening of 20 Trichophyton (n=10, MIC 0.125-1 mg/L; n=10, MIC 4-≥32 mg/L) isolates on SDA plates containing 0.2 mg/L TRB showed that all Trichophyton isolates with high MICs against TRB (4-≥32 mg/L) showed rich confluent growth within 7 days of incubation at 28°C. However, isolates with MICs of ≥ 0.25 mg/L showed poor growth after 7-10 days of incubation. In contrast, isolates with MICs of 0.125 mg/L presented no growth at day 14 under the same conditions. 3.6.

Genome sequencing and phylogenetic analysis of Trichophyton isolates

The whole genome phylogenetic and kmer content analyses were conducted using 20 Indian Trichophyton isolates, including both TRB resistant and susceptible strains. MinHash distances between our samples and public genomic data for the genus, calculated using MASH, were an average of 0.031, 0.071, and 0.072 for T. mentagrophytes, T. tonsurans, and T. equinum respectively. Kmer content in T. interdigitale data varied greatly, rendering this distance measurement inconclusive and it is therefore not reported. For the kmer taxonomy-guided recognition, 89.26% of our data in the form of reads belonged to the superset Fungi, 82.42% of the total was exclusive for Trichophyton. The average kmer content assigned to speciesspecific clades was 44.37%, 15.30%, and 15.22% for T. rubrum, T. verrucosum, and T. benhaminae. For phylogenetic SNP reconstruction, the reference genome D15-P135 (GCA_003664455.1) is a T. mentagrophytes isolate associated with human infection and isolated in Mumbai, India in 2016 (Pchelin et al., 2018). It is the closest genome relating to the set studied here. Phylogenetic analysis between our samples and other Trichophyton isolates available in the NCBI database shows that these genomes are closely related to one another and most recently

related to the T. mentagrophytes/interdigitale complex, seeming to belong to an early diverging clade of the complex (Figure 2A). Among themselves, the Indian genomes have up to 42 SNPs difference between any two isolates suggesting highly related isolates (Figure 2B).

4. Discussion This study reports that isolates of Trichophyton sp. causing severe superficial dermatophytosis (tinea cruris and tinea corporis) in North India belong to a unique clade of the genus, as confirmed by whole genome sequencing. At the same time, other molecular approaches used in this study were contradictory or inconclusive to show the distinction of these isolates from T. mentagrophytes/ T. interdigitale species of the genus. This finding uncovers a formidable challenge for diagnostic and therapeutic efforts. The multidrug resistance observed in Indian Trichophyton isolates may translate to treatment failure and relapses in dermatophytosis in India. The ineffective response to TRB therapy has recently been documented in a study from India investigating the clinical correlation of MICs of TRB and SQLE mutations in 30 patients with tinea corporis/cruris (Khurana et al., 2018). The authors reported that an infection with Trichophyton isolates exhibiting a TRB MIC <1µg/ml is 2.5 times more likely to respond to TRB therapy than those with higher MICs (Khurana et al., 2018). In the present study, 36% of patients harboured isolates that exhibited high TRB MICs 4 to ≥32 mg/L, indicating that TRB therapy might be inadequate in these patients. Remarkably, we also found high rates of resistance among 129 T. mentagrophytes isolates to all three commonly used oral antifungals in dermatophytosis treatment, i.e., TRB (36%), FLU (39.5%) and GRE (56%). It is pertinent to mention here that dermatophyte susceptibility testing remains difficult to perform as contamination of the susceptibility plate invariably occurs despite primary isolation on selective agars (Saunte et al., 2019).

A recently proposed modified EUCAST method

with/without cycloheximide and chloramphenicol supplementation to the growth medium may

contribute to making terbinafine susceptibility testing more accessible by preventing overgrowth of non-dermatophyte moulds and bacteria (Saunte et al., 2019) Although global studies report TRB as the most effective antifungal agent for treating dermatophytosis in India, high resistance rates of TRB warrant a reappraisal of its utility in the treatment of dermatophytosis. Interestingly, prior to 2017, only two documented cases of TRB resistance in patients with onychomycosis were recorded in Swiss patients (Osborne et al., 2005, 2003). Alarmingly a recent upsurge of reports documenting TRB resistance in T. mentagrophytes and T. interdigitale from Switzerland (Yamada et al., 2017) and India (Rudramurthy et al., 2018; Singh et al., 2018) and T. rubrum and T. tonsurans from Denmark (Digby et al., 2017; Schøsler et al., 2018), Iran (Salehi et al., 2018) and Japan (Suh et al., 2018) have been a matter of concern. Recently, 14 cases of terbinafine treatment failure in Trichophyton infected Danish patients due to acquired resistance in patients with T. rubrum (n=12) or T. interdigitale (n=2) infections with elevated TRB MICs were reported. Overall, nine isolates (64%) displayed high (MIC 4->8 mg/L), two (14%) moderate (MIC 1-2 mg/L) and three (21%) low terbinafine resistance (MIC 0.125-0.25 mg/L). Further, all resistant isolates harboured known and novel SQLEp amino acid substitutions (F397L, L393F, L393S, F415S, H440Y/F484Y and I121M/V237I). Similarly, all isolates with high MICs (4->8 mg/L) in the present study had two significant substitutions i.e. F397L and L393F. These two substitutions (L393F and F397L) cause conformational changes in the enzyme that result in reduced drug affinity (Nowosielski et al., 2011) and have been associated with high resistance while other variants result in lower MIC elevation (Saunte et al., 2019). However, we did not observe other amino acid substitutions such as F415S, H440Y/F484Y and I121M/V237I in isolates with moderately high terbinafine MICs (1-2 mg/L) suggesting other possible mechanisms may confer resistance in clinical settings. In contrast to high resistance rates of TRB noted in Indian Trichophyton isolates, data from other countries show low (1-2%) TRB

resistance rates (Salehi et al., 2018; Yamada et al., 2017). In addition, high GRE MICs (modal MIC 4mg/L) with 56% of the isolates exhibiting MICs of ≥4mg/L were found. Artis et al. (1981) compared clinical outcomes with the respective GRE MIC values and found that a MIC of ≥3 mg/L indicates relative GRE resistance. GRE had largely been superseded by TRB and ITR for treatment of tinea corporis/ cruris since the introduction of these drugs in 1990s, and its clinical utility at present mainly lies in treatment of tinea capitis. However, TRB has been a frontline drug for tinea corporis/cruris worldwide and the rising resistance to it is worrisome. Increasing drug exposure by means of longer durations and higher dosages of TRB has been shown to surmount the in vitro resistance to some extent, but the high failure rates still are a cause of concern (Khurana et al., 2018). The DermaGenius® 2.0 multiplex real-time PCR assays is a rapid molecular diagnostic method that identifies the most common fungal infections in nail, hair, and skin samples within 3 hours. We used this assay for confirming the identity of Trichophyton isolates cultured from skin scrapings. All the Trichophyton isolates in this present study were identified as T. interdigitale by DermaGenius® 2.0 which is in concordance with the results of the BLAST searches of ITS sequences. However, both ITS and whole-genome phylogenies cluster our samples away from T. interdigitale. This indicates that the BLAST searches showed equivocal identification of all the Trichophyton isolates analysed in the present study. Similarly, although control identifications of T. rubrum, T tonsurans and T. violaceum were accurate, the DermaGenius® 2.0 multiplex real-time PCR assay was unable to differentiate species in the T. mentagrophytes complex. Regarding our BLAST results, we attribute this misidentification to likely incorrect labelling of sequences deposited in the public databases mainly in GenBank, NCBI and Westerdijk Fungal Biodiversity Institute (Chowdhary et al., 2019). The identities of public data were accounted for only after genomic analysis demonstrated the correct identity of the samples.

A cautious phylogenetic analysis of sequences is required to resolve these labelling issues specifically in the T. mentagrophytes complex. We expect that genomic and multigene approaches help resolve these issues. Initial approaches using multigene phylogenies by de Hoog et al. (2017) and genome wide analyses (Pchelin et al., 2018) seem to be in topological concordance with our phylogenomic observations of the genus members, making the potential of these techniques evident. Even so, increasing the number of public sequences for gene markers and genomes is required for these methods to take a central stage in taxonomic designation. Phylogenetic analysis of the concatenated loci can resolve species boundaries between T. mentagrophytes and T. interdigitale provided enough sequences of all the loci from several geographical regions are available in the database. Baert et al. (2019) analysed 688 β-tubulin and ITS sequences of several genera of dermatophytes and suggested that combined analysis of ITS and β-tubulin gene provides strong support for the major clades of the Arthrodermatacae family. Combining these two genes provided a phylogenetic tree with more significant support than using any one gene alone. The phylogenetic analysis using ITS sequences and ITS and Tef 1-α in the current study placed the Indian Trichophyton isolates closely to T. mentagrophytes type VIII; many of the samples from this type have been added recently to GenBank and have been isolated from tinea corporis and cruris patients from India and probably represent the same background as the isolates investigated here. We also generated whole-genome Illumina sequences for a subset of 20 of our isolates and found the distance from Indian Trichophyton spp. isolates to the public T. mentagrophytes and T. interdigitale genomes was greater than the distance between the public T. mentagrophytes and T. interdigitale. Indeed, the clade we study here shares a recent common ancestor with T. mentagrophytes/interdigitale complex. Therefore, we refrain from calling these isolates T. mentagrophytes since this would create a paraphyletic group. The

unique Indian Trichophyton clade among T. mentagrophytes and T. interdigitale raises an interesting question: whether an independent Indian Trichophyton clade exists which warrants further in depth phylogeographic studies. We examined these 20 genomes for mating type markers. In dermatophytes this is specified by the presence of one of two idiomorphs at a single mating type (MAT) locus; each idiomorph includes either an alpha-box domain or a high mobility group (HMG) domain transcription factor gene (Persinoti et al., 2018; Li et al., 2010). Across the 20 examined genomes, all had HMG domains and therefore correspond to mating type (+). Particularly, three isolates P161161, P16-1073, and P16-2386 show positivity for both alpha-box and HMG which might indicate hybridization or an incomplete sexual cycle. Further, the contigs housing the HMG and alpha-box domains are distinct in the three isolates displaying both loci. The large size of these contigs suggests that the domains represent different loci. Figure 3 represents the diagram of the findings using the in silico PCR, Vipr (https://github.com/TGenNorth/vipr), with the primers shown in the figure The results concordantly reported one locus for each domain for all working permutations of the primers in every assembly displaying both (Lee et al., 2010). MAT loci are usually thousands of base pairs long; this is much lower than the distances flanking the domains, which indicates it is unlikely that the alpha-box and HMG domains reside in the same genomic loci. Most likely they are housed in the same locus in different chromosomes. Isolates of both mating types present in this clonal outbreak, and those of opposite mating type could undergo mating. This highlights the potential of these dermatophytes to rapidly spread alleles that might be driving antifungal resistance among its population (Martinez et al., 2012; Burmester et al., 2011).

Conclusion We have identified a new population of Trichophyton with high rates of in vitro antifungal resistance. This population seems to be driving an ongoing outbreak of dermatophytosis in India. Our study highlights complications in identifying isolates from the Trichophyton mentagrophytes/interdigitale clade of the genus using currently available molecular tools. Further investigation of these isolates is required to understand this previously unrecognised potential public-health threat.

Acknowledgments This work was carried out in part with financial assistance from the Council of Scientific & Industrial Research (F. No. 09/174(0068)/2014-EMR-I) to A.S. We thank Gijs Dingemans from Pathonostics, Maastricht, The Netherlands with help with the Dermagenius assay.

Conflict of Interest J.F.M. received grants from F2G, Basilea, and Pulmozyme. He has been a consultant to Astellas, Basilea, and Scynexis and has received speaker’s fees from United Medical, TEVA and Gilead. All other authors declare no potential conflicts of interest. We alone are responsible for the content and writing of the paper.

References: Ahmadi, B., Mirhendi, H., Makimura, K., de Hoog, G.S., Shidfar, M.R., Nouripour-Sisakht, S., Jalalizand, N., 2016. Phylogenetic analysis of dermatophyte species using DNA sequence

polymorphism

in

calmodulin

gene.

Med.

Mycol.

54,

500–514.

https://doi.org/10.1093/mmy/myw004 Artis, W.M., Odle, B.M., Jones, H.E., 1981. Griseofulvin-resistant dermatophytosis correlates with

in

vitro

resistance.

Arch.

Dermatol.

117,

16.

https://doi.org/10.1001/archderm.1981.01650010022016 Badali, H., Mohammadi, R., Mashedi, O., de Hoog, GS., Meis JF., 2015. In vitro susceptibility patterns of clinically important Trichophyton and Epidermophyton species against nine antifungal drugs. Mycoses. 58, 303-7. https://doi.org/10.1111/myc.12315. Baert, F., Stubbe, D., D’hooge, E., Packeu, A., Hendrickx, M., 2019. Updating the taxonomy of dermatophytes of the BCCM/IHEM collection according to the new standard: A phylogenetical approach. Mycopathologia. https://doi.org/10.1007/s11046-019-00338-7 Baghi, N., Shokohi, T., Badali, H., Makimura, K., Rezaei-Matehkolaei, A., Abdollahi, M., Didehdar, M., Haghani, I., Abastabar, M., 2016. In vitro activity of new azoles luliconazole and lanoconazole compared with ten other antifungal drugs against clinical dermatophyte isolates. Med. Mycol. 54, 757–763. https://doi.org/10.1093/mmy/myw016 Bishnoi, A., Vinay, K., Dogra, S., 2018. Emergence of recalcitrant dermatophytosis in India. Lancet Infect. Dis. 18, 250–251. https://doi.org/10.1016/S1473-3099(18)30079-3 Burmester, A., Sheles,t E., Glöckner, G., Heddergott, C., Schindler, S., Staib, P., Heidel, A., Felder, M., Petzold, A., Szafranski, K., Feuermann, M., Pedruzzi, I., Priebe, S., Groth, M., Winkler, R., Li, W., Kniemeyer, O., Schroeckh, V., Hertweck, C., Hube, B., White, TC., Platzer, M., Guthke, R., Heitman, J., Wöstemeyer, J., Zipfel, PF., Monod, M., Brakhage,

AA., 2011. Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi. Genome Biol. 12: R7. https://doi.org/10.1186/gb-2011-12-1-r7. Chowdhary, A., Singh, A., Singh, P.K., Khurana, A., Meis, J.F., 2019. Perspectives on misidentification of Trichophyton interdigitale / Trichophyton mentagrophytes using internal transcribed spacer region sequencing: Urgent need to update the sequence database. Mycoses 62, 11–15. https://doi.org/10.1111/myc.12865 de Hoog, G.S., Dukik, K., Monod, M., Packeu, A., Stubbe, D., Hendrickx, M., Kupsch, C., Stielow, J.B., Freeke, J., Göker, M., Rezaei-Matehkolaei, A., Mirhendi, H., Gräser, Y., 2017. Toward a novel multilocus phylogenetic taxonomy for the dermatophytes. Mycopathologia 182, 5–31. https://doi.org/10.1007/s11046-016-0073-9 Digby, S., Hald, M., Arendrup, M. C, Hjort, S., Kofoed, K., 2017. Darier disease complicated by terbinafine-resistant Trichophyton rubrum: A case report. Acta Derm. Venereol. 97, 139–140. https://doi.org/10.2340/00015555-2455 Garcia Garces, H., Hrycyk, M.F., Giacobino, J., Capela Machado, G., Domingos Arantes, T., Theodoro, R.C., Bosco, S. de M.G., Bagagli, E., 2016. Molecular identification and phylogenetical analysis of dermatophyte fungi from Latin America. Mycoses 59, 787– 797. https://doi.org/10.1111/myc.12532 Gräser, Y., Monod, M., Bouchara, J.-P., Dukik, K., Nenoff, P., Kargl, A., Kupsch, C., Zhan, P., Packeu, A., Chaturvedi, V., de Hoog, S., 2018. New insights in dermatophyte research. Med. Mycol. 56, S2–S9. https://doi.org/10.1093/mmy/myx141 Groenewald, J.Z., Nakashima, C., Nishikawa, J., Shin, H.-D., Park, J.-H., Jama, A.N., Groenewald, M., Braun, U., Crous, P.W., 2013. Species concepts in Cercospora: spotting the weeds among the roses. Stud. Mycol. 75, 115–170. https://doi.org/10.3114/sim0012 Kawasaki, M., Anzawa, K., Ushigami, T., Kawanishi, J., Mochizuki, T., 2011. Multiple gene analyses are necessary to understand accurate phylogenetic relationships among

Trichophyton species. Med. Mycol. J. 52, 245–254. https://doi.org/10.3314/mmj.52.245 Khurana, A., Masih, A., Chowdhary, A., Sardana, K., Borker, S., Gupta, A., Gautam, R.K., Sharma, P.K., Jain, D., 2018. Correlation of in vitro susceptibility based on MICs and squalene epoxidase mutations with clinical response to terbinafine in patients with tinea corporis/cruris.

Antimicrob.

Agents

Chemother.

62,

pii:

e01038-18.

https://doi.org/10.1128/AAC.01038-18 Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular evolutionary genetics analysis version

7.0

for

bigger

datasets.

Mol.

Biol.

Evol.

33,

1870–1874.

https://doi.org/10.1093/molbev/msw054 Lee, S.C., Ni, M., Li, W., Shertz, C., Heitman, J., 2010. The evolution of sex: a perspective from the fungal kingdom. Microbiol. Mol. Biol. Rev. 74, 298-340. https://doi.org/ 10.1128/MMBR.00005-10. Li, H., Durbin, R., 2010. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595. https://doi.org/10.1093/bioinformatics/btp698 Li, W., Metin, B., White, TC., Heitman, J., 2010. Organization and evolutionary trajectory of the mating type (MAT) locus in dermatophyte and dimorphic fungal pathogens. Eukaryot. Cell. 9, 46-58. https://doi.org/10.1128/EC.00259-09 Masih, A., Singh, P.K., Kathuria, S., Agarwal, K., Meis, J.F., Chowdhary, A., 2016. Identification by molecular methods and matrix-assisted laser desorption ionization–time of flight mass spectrometry and antifungal susceptibility profiles of clinically significant rare Aspergillus species in a referral chest hospital in Delhi, India. J. Clin. Microbiol. 54, 2354–2364. https://doi.org/10.1128/JCM.00962-16 Martinez, DA., Oliver, BG., Gräser, Y., Goldberg, JM., Li, W., Martinez-Rossi, NM., Monod, M., Shelest, E., Barton, RC., Birch, E., Brakhage, AA., Chen, Z., Gurr, SJ., Heiman, D., Heitman, J., Kosti, I., Rossi, A., Saif, S., Samalova, M., Saunders, CW., Shea, T.,

Summerbell, RC., Xu, J., Young, S., Zeng, Q., Birren, BW., Cuomo, CA., White, TC., 2012. Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals

candidate

genes

involved

in

infection.

MBio.

3,

e00259-12.

https://doi.org/10.1128/mBio.00259-12 McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M., DePristo, M.A., 2010. The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303. https://doi.org/10.1101/gr.107524.110 Metin, B., Heitman, J., 2017. Sexual reproduction in dermatophytes. Mycopathologia 182, 45– 55. https://doi.org/10.1007/s11046-016-0072-x Mirhendi, H., Makimura, K., de Hoog, G.S., Rezaei-Matehkolaei, A., Najafzadeh, M.J., Umeda, Y., Ahmadi, B., 2015. Translation elongation factor 1-α gene as a potential taxonomic and identification marker in dermatophytes. Med. Mycol. 53, 215–224. https://doi.org/10.1093/mmy/myu088 Nenoff, P., Verma, S.B., Vasani, R., Burmester, A., Hipler, U.-C., Wittig, F., Krüger, C., Nenoff, K., Wiegand, C., Saraswat, A., Madhu, R., Panda, S., Das, A., Kura, M., Jain, A., Koch, D., Gräser, Y., Uhrlaß, S., 2019. The current Indian epidemic of superficial dermatophytosis due to Trichophyton mentagrophytes-A molecular study. Mycoses 62, 336–356. https://doi.org/10.1111/myc.12878 Nowosielski, M., Hoffmann, M., Wyrwicz, L.S., Stepniak, P., Plewczynski, D.M., Lazniewski, M., Ginalski, K., Rychlewski, L., 2011. Detailed mechanism of squalene epoxidase inhibition

by

terbinafine.

J.

Chem.

Inf.

Model.

51,

455–462.

https://doi.org/10.1021/ci100403b Nurk, S., Bankevich, A., Antipov, D., Gurevich, A., Korobeynikov, A., Lapidus, A., Prjibelsky, A., Pyshkin, A., Sirotkin, A., Sirotkin, Y., Stepanauskas, R., McLean, J., Lasken, R.,

Clingenpeel, S.R., Woyke, T., Tesler, G., Alekseyev, M.A., Pevzner, P.A., 2013. Assembling genomes and mini-metagenomes from highly chimeric reads, in: Lecture notes in computer science (Including subseries lecture notes in artificial intelligence and lecture notes in bioinformatics). pp. 158–170. https://doi.org/10.1007/978-3-642-371950_13 Ondov, B.D., Treangen, T.J., Melsted, P., Mallonee, A.B., Bergman, N.H., Koren, S., Phillippy, A.M., 2016. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 17, 132. https://doi.org/10.1186/s13059-016-0997-x Osborne, C.S., Hofbauer, B., Favre, B., Ryder, N.S., 2003. In vitro analysis of the ability of Trichophyton rubrum to become resistant to terbinafine. Antimicrob. Agents Chemother. 47, 3634–3636. https://doi.org/10.1128/AAC.47.11.3634 Osborne, C.S., Leitner, I., Favre, B., Ryder, N.S., 2005. Amino acid substitution in Trichophyton rubrum squalene epoxidase associated with resistance to terbinafine. Antimicrob. Agents Chemother. 49, 2840–2844. https://doi.org/10.1128/AAC.49.7.28402844.2005 Pchelin, I.M., Azarov, D. V, Churina, M.A., Scherbak, S.G., Apalko, S. V, Vasilyeva, N. V, Taraskina, A.E., 2019. Species boundaries in the Trichophyton mentagrophytes / T. interdigitale

species

complex.

Med.

Mycol.

57,

781–789.

https://doi.org/10.1093/mmy/myy115 Performance standards for antifungal susceptibility testing of filamentous fungi. CLSI M61, 1st Ed., 2017. Clinical and Laboratory Standards Institute , Wayne, PA. Persinoti, GF., Martinez, DA., Li W, Döğen A., Billmyre, RB., Averette, A., Goldberg, JM., Shea, T., Young, S., Zeng, Q., Oliver, BG., Barton, R., Metin, B., Hilmioğlu-Polat, S., Ilkit, M., Gräser, Y., Martinez-Rossi, NM., White, TC., Heitman, J., Cuomo, CA., 2018. Whole-Genome Analysis Illustrates Global Clonal Population Structure of the Ubiquitous

Dermatophyte

Pathogen

Trichophyton

rubrum.

Genetics.

208,

1657-1669.

https://doi.org/10.1534/genetics.117.300573 Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. CLSI standard M38, 3rd Ed., 2017. Clinical and Laboratory Standards Institute , Wayne, PA. Rezaei-Matehkolaei, A., Khodavaisy, S., Alshahni, M.M., Tamura, T., Satoh, K., Abastabar, M., Shokoohi, G.R., Ahmadi, B., Kord, M., Taghipour, S., Makimura, K., Badali, H., 2018. In vitro antifungal activity of novel triazole efinaconazole and five comparators against dermatophyte isolates. Antimicrob. Agents Chemother. 62, pii: e02423-17. https://doi.org/10.1128/AAC.02423-17 Rezaei-Matehkolaei, A., Mirhendi, H., Makimura, K., de Hoog, G.S., Satoh, K., Najafzadeh, M.J., Shidfar, M.R., 2014. Nucleotide sequence analysis of beta tubulin gene in a wide range of dermatophytes. Med. Mycol. 52, 674–688. https://doi.org/10.1093/mmy/myu033 Rudramurthy, S.M., Shankarnarayan, S.A., Dogra, S., Shaw, D., Mushtaq, K., Paul, R.A., Narang, T., Chakrabarti, A., 2018. Mutation in the squalene epoxidase gene of Trichophyton interdigitale and Trichophyton rubrum associated with allylamine resistance.

Antimicrob.

Agents

Chemother.

62,

pii:e02522-17.

https://doi.org/10.1128/AAC.02522-17 Sahl, J.W., Lemmer, D., Travis, J., Schupp, J.M., Gillece, J.D., Aziz, M., Driebe, E.M., Drees, K.P., Hicks, N.D., Williamson, C.H.D., Hepp, C.M., Smith, D.E., Roe, C., Engelthaler, D.M., Wagner, D.M., Keim, P., 2016. NASP: an accurate, rapid method for the identification of SNPs in WGS datasets that supports flexible input and output formats. Microb. Genomics 2, 1–12. https://doi.org/10.1099/mgen.0.000074 Salehi, Z., Shams-Ghahfarokhi, M., Razzaghi-Abyaneh, M., 2018. Antifungal drug susceptibility profile of clinically important dermatophytes and determination of point

mutations in terbinafine-resistant isolates. Eur. J. Clin. Microbiol. Infect. Dis. 37, 1841– 1846. https://doi.org/10.1007/s10096-018-3317-4 Saunte, D.M.L., Hare, R.K., Jørgensen, K.M., Jørgensen, R., Deleurand, M.,. Zachariaee, C. O., Thomsen, S.F., Bjørnskov-Halkierh, L., Kofoede, K., Arendrup, M.C., 2019. Emerging terbinafine resistance in Trichophyton: clinical characteristics, squalene epoxidase gene mutations and a reliable EUCAST method for detection. Antimicrob. Agents Chemother. https://doi.org/10.1128/AAC.01126-19 Schøsler, L., Andersen, L.K., Arendrup, M.C., Sommerlund, M., 2018. Recurrent terbinafine resistant Trichophyton rubrum infection in a child with congenital ichthyosis. Pediatr. Dermatol. 35, 259–260. https://doi.org/10.1111/pde.13411 Singh, A., Masih, A., Khurana, A., Singh, P.K., Gupta, M., Hagen, F., Meis, J.F., Chowdhary, A., 2018. High terbinafine resistance in Trichophyton interdigitale isolates in Delhi, India harbouring mutations in the squalene epoxidase gene. Mycoses 61, 477–484. https://doi.org/10.1111/myc.12772 Singh, A., Singh, P.K., Kumar, A., Chander, J., Khanna, G., Roy, P., Meis, J.F., Chowdhary, A., 2017. Molecular and matrix-assisted laser desorption ionization–time of flight mass spectrometry-based characterization of clinically significant melanized fungi in India. J. Clin. Microbiol. 55, 1090–1103. https://doi.org/10.1128/JCM.02413-16 Suh, S.-O., Grosso, K.M., Carrion, M.E., 2018. Multilocus phylogeny of the Trichophyton mentagrophytes species complex and the application of matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry for the rapid identification

of

dermatophytes.

Mycologia

110,

118–130.

https://doi.org/10.1080/00275514.2018.1443653 Symoens, F., Jousson, O., Planard, C., Fratti, M., Staib, P., Mignon, B., Monod, M., 2011. Molecular analysis and mating behaviour of the Trichophyton mentagrophytes species

complex.

Int.

J.

Med.

Microbiol.

301,

260–266.

https://doi.org/10.1016/j.ijmm.2010.06.001 Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular evolutionary

genetics

analysis

version

6.0.

Mol.

Biol.

Evol.

https://doi.org/10.1093/molbev/mst197 White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics: PCR - Protocols and applications - A laboratory manual,

PCR

protocols:

A

Guide

to

methods

and

applications.

https://doi.org/10.1016/B978-0-12-372180-8.50042-1 Wiederhold, N.P., Fothergill, A.W., McCarthy, D.I., Tavakkol, A., 2014. Luliconazole demonstrates potent in vitro activity against dermatophytes recovered from patients with onychomycosis.

Antimicrob.

Agents

Chemother.

58,

3553–3555.

https://doi.org/10.1128/AAC.02706-13 Wood, D.E., Salzberg, S.L., 2014. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 15, R46. https://doi.org/10.1186/gb-2014-15-3-r46 Yamada, T., Maeda, M., Alshahni, M.M., Tanaka, R., Yaguchi, T., Bontems, O., Salamin, K., Fratti, M., Monod, M., 2017. Terbinafine resistance of Trichophyton clinical isolates caused by specific point mutations in the squalene epoxidase gene. Antimicrob. Agents Chemother. 61, pii: e00115-17 . https://doi.org/10.1128/aac.00115-17 Zhu, X., Leung, H.C.M., Chin, F.Y.L., Yiu, S.M., Quan, G., Liu, B., Wang, Y., 2014. PERGA: A paired-end read guided de novo assembler for extending contigs using SVM and look ahead approach. PLoS One 9, e114253. https://doi.org/10.1371/journal.pone.0114253

Table 1: MIC distribution of Trichophyton spp. (n= 129) against 10 antifungals drugs tested using CLSI-BMD method. No. of isolates with MIC (mg/L) of: Drugsa ≤0.03

0.06

2.881

1

32

51

0.5->32

12.099

16

32

2

0.03->16

0.254

0.25

1

1

0.03->16

0.181

0.125

0.5

7

0.03->16

1.155

1

4

3

0.125->16

1.816

2

4

0.25->8

2.977

4

4

0.125-16

2.215

2

4

0.06->32

0.689

0.5

2

≤0.03-0.5

0.006

0.0035

0.03

8

16

32

2

5

17

53e

4

1

3

3

4

5

15

10

7

37

8

1

21

26

35

7

VRC

6

20

49

22

25

6

SER

1

1

4

13

32

17

30

24

1

1

6

23

81

13

1

1

1

4

51

60

12

3

20

63

36

5

57

29

7

4

1

MCZ GRE CLT

1 1 6

1

24

1

1 5

terbinafine; FLU, fluconazole; ITC, itraconazole; VRC, voriconazole; SER, sertaconazole; MCZ, miconazole; GRE, griseofulvin; CLT, clotrimazole; KTC,

ketoconazole; LUZ, luliconazole. geometric mean MICs.

cMIC50,MIC

at which 50 % of test isolates were inhibited.

dMIC90,MIC

at which 90 % of test isolates were inhibited.

eModal

0.125->32

4

25

bGM,

41

2

4

aTRB,

MIC90d

1

ITC

122

MIC50c

0.5

FLU

LUZ

GMb

0.25

TRB

KTC

Range 0.125

MICs are indicated with underlined numbers.

Table 2: In vitro susceptibility and amino acid substitutions in SQLEp of Trichophyton spp. (n=61). Amino acid Isolate No.

Hospital

MIC (mg/L)

substitution/TRB MIC (mg/L)

FLU

ITC

VRC

SER

MCZ

GRE

CLT

KTC

LUZ

1/P/16

A

WT/ 0.125

32

0.5

0.255

2

1

2

4

1

0.015

2/P/16

A

WT/ 0.25

2

4

0.5

4

2

2

4

2

0.06

3/P/14

B

WT/ 0.5

32

0.5

0.25

2

1

2

2

0.5

0.015

4/P/15

C

WT/ 1

2

0.06

0.06

1

2

1

2

0.25

0.0035

5/P/15

C

WT/ 1

1

0.03

0.06

1

2

4

1

0.125

0.0035

6/P/16

C

WT/ 1

2

0.06

0.06

1

2

2

2

0.25

0.0035

7/P/16

A

WT/ 1

32

0.25

0.125

0.25

2

2

1

1

0.0035

8/P/16

A

WT/ 1

32

0.5

0.125

4

2

>8

4

>32

0.015

9/P/16

D

WT/ 1

32

>16

0.25

>16

8

>8

2

4

0.015

10/P/16

D

WT/ 1

64

2

0.5

4

2

4

4

1

0.06

11/P/16

C

WT/ 1

8

0.25

0.5

2

4

2

4

0.5

0.007

12/P/16

D

WT/ 1

64

2

0.5

4

2

4

4

1

0.06

13/P/16

C

WT/ 1

4

0.125

0.5

0.25

0.5

2

0.5

0.25

0.0035

14/P/16

D

WT/ 1

>64

>16

>16

>16

>16

>8

16

>32

0.5

15/P/15

C

WT/ 2

16

0.5

1

>16

>16

2

4

1

0.015

16/P/15

C

F397L/ 4

16

0.06

0.06

0.5

2

2

2

0.5

0.0035

17/P/15

A

F397L/ 8

16

0.125

0.125

0.25

2

4

4

0.5

0.0035

18/P/16

A

F397L/ 8

16

0.5

0.5

≥16

2

≥8

4

4

0.007

19/P/15

C

L393F/ 16

16

0.06

0.06

0.5

2

4

2

0.5

0.0035

20/P/16

A

F397L/ 16

16

0.25

0.125

0.5

1

4

1

0.5

0.0035

21/P/17

E

F397L/ 16

16

0.25

0.06

0.5

1

2

1

0.5

0.0035

22/P/16

C

F397L/ 32

16

0.125

0.06

0.5

2

4

2

0.5

0.0035

23/P/16

C

F397L/ 32

16

0.125

0.06

0.5

2

2

2

0.5

0.0035

24/P/16

A

F397L/ 32

16

0.125

0.03

0.5

2

4

2

0.5

0.0035

25/P/16

A

F397L/ 32

16

0.25

0.06

0.5

2

4

2

0.5

0.0035

26/P/16

C

F397L/ 32

32

0.5

0.25

2

4

≥8

2

8

0.0035

27/P/15

C

F397L/ 32

16

0.06

0.06

0.25

2

4

2

0.5

0.015

28/P/16

C

F397L/ 32

0.5

0.125

0.125

2

1

4

2

0.5

0.007

29/P/16

C

F397L/ 32

4

0.06

0.06

2

2

2

2

0.5

0.0035

30/P/16

C

F397L/ 32

16

0.25

0.125

0.5

4

4

2

0.5

0.0035

31/P/17

D

F397L/ 32

2

0.06

0.125

4

2

4

2

0.25

0.0035

32/P/15

C

F397L/ ≥32

32

0.06

0.06

0.5

1

2

1

0.5

0.0035

33/P/16

A

F397L/ ≥32

32

0.25

0.06

0.5

2

4

2

0.5

0.0035

34/P/16

A

F397L/ ≥32

16

0.25

0.03

0.25

2

8

4

1

0.015

35/P/16

D

F397L/ ≥32

0.5

0.125

0.125

2

2

8

4

1

0.03

36/P/16

D

F397L/ ≥32

16

0.25

0.06

0.5

2

4

2

0.5

0.0035

37/P/15

C

F397L/ ≥32

16

0.125

0.125

0.5

1

2

1

0.5

0.0035

38/P/15

C

F397L/ ≥32

16

0.125

0.125

0.5

1

2

1

0.5

0.0035

39/P/17

D

F397L/ ≥32

16

0.125

0.125

0.5

2

4

2

0.5

0.0035

40/P/16

C

F397L/ ≥32

1

0.25

0.5

2

2

4

4

0.5

0.007

41/P/15

C

F397L/ ≥32

16

0.125

0.125

0.5

2

4

4

0.5

0.007

42/P/16

C

F397L/ ≥32

16

0.5

0.125

1

2

2

2

0.5

0.0035

43/P/16

C

F397L/ ≥32

32

0.25

0.125

0.5

2

2

1

0.5

0.0035

44/P/15

B

F397L/ ≥32

32

0.5

0.5

≥16

2

4

8

2

0.03

45/P/16

A

L393F/ ≥32

1

0.25

0.5

2

2

4

4

0.5

0.007

46/P/16

A

L393F/ ≥32

8

0.25

0.03

0.25

0.5

4

1

0.25

0.0035

47/P/14

B

L393F/ ≥32

16

0.25

0.25

1

2

4

2

0.5

0.0035

48/P/15

B

F397L/ ≥32

16

0.5

0.5

2

1

4

4

1

0.015

49/P/16

D

L393F/ ≥32

32

0.25

0.125

4

2

4

8

0.5

0.015

50/P/16

C

F397L/ 32

4

0.125

0.125

2

2

2

2

0.25

0.0035

51/P/17

E

F397L/ ≥32

32

0.25

0.125

0.5

2

2

2

0.5

0.0035

52/P/17

E

F397L/ ≥32

32

0.5

0.125

1

2

2

2

0.5

0.0035

53/P/16

A

F397L/ ≥32

1

0.5

0.5

2

2

4

4

0.5

0.007

54/P/16

C

F397L/ ≥32

16

0.125

0.125

2

4

4

4

1

0.007

55/P/16

C

F397L/ ≥32

16

0.25

0.06

0.125

2

2

1

0.25

0.0035

56/P/17

E

F397L/ ≥32

16

0.125

0.06

0.25

1

2

1

0.5

0.0035

57/P/16

D

F397L/ ≥32

16

0.5

0.25

1

2

4

2

0.5

0.0035

58/P/15

C

L393F/ ≥32

16

0.5

0.25

1

2

4

2

0.5

0.0035

59/P/16

D

L393F/ ≥32

64

2

0.125

4

4

2

2

4

0.007

60/P/16

D

F397L/ ≥32

0.5

0.25

0.25

2

2

8

8

1

0.03

61/P/15

C

F397L/ ≥32

16

0.06

0.125

0.5

2

4

1

0.5

0.0035

Bold faces denote high MICs for the respective antifungals. TRB, terbinafine; FLU, fluconazole; ITC, itraconazole; VRC, voriconazole; SER, sertaconazole; MCZ, miconazole; GRE, griseofulvin; CLT, clotrimazole; KTC, ketoconazole; LUZ, luliconazole.

Legend: Figure 1: Phylogenetic tree based on ITS sequences using neighbour-joining analysis, MEGA version 6, with 2,000 bootstrap replications of Trichophyton isolates (n=73). Sequences of neotype/type strains were retrieved from GenBank for the analysis. Bootstrap values are shown above the branches.

Figure 2. Genome analysis of selected samples with low and high MICs. (A) Consensus maximum parsimony phylogeny of distantly related Trichophyton genomes. This analysis included 50 most-parsimonious trees and shows splits with <95% confidence as polytomies. This included 1’356.314 high-confidence SNP positions that covered ~59.8% of the genome used as reference (D15P135). SNP counts below 200 are not shown, this tree was rooted using the genome assembly GCA_000151145.1 of Microsporum canis CBS 113480, samples sequenced for this study are marked with green circles, consistency index was 0.921. (B) Maximum parsimony consensus tree of the 40 most parsimonious trees showing phylogenomic relations of the isolates sequenced for this study and their related genome size, sex loci, and MICs information. Splits with confidence below 95% are shown as polytomies, 96.32% of the reference genome D15P135 (used as root standing 98 SNPs away from common ancestor of this clade, not shown) is covered. 204 high-confidence SNP positions were analysed and the consistency indexed was 0.995.

Figure 3. Genomic position of HMG domain and alpha-box domain with reference to the isolate number P16-1073, P16-1161and P16-2386.

Highlights (3-5 points, each with a limit of maximum 85 characters) 1. WGS confirmed a unique population of Trichophyton, causing dermatophytosis in India. 2. High rates of resistance to terbinafine observed due to mutation in SQLE gene. 3. Available

molecular

tools

obfuscate

the

identification

of

T.mentagrophytes/interdigitale. 4. HMG and alpha-box gene in this population indicates hybridization or an incomplete sexual cycle driving resistant alleles.