Genomic Analysis of Antifungal Drug Resistance Induced in Trichophyton rubrum After Prolonged Culture with Terbinafine

In this study, we induced terbinafine (TRF) resistance in a T. rubrum strain in vitro for 18 months then compared the genomes of the TRF-resistant (N42-3) and TRF-susceptible wild-type (N42WT) strains to identify mutations. In the SQLE gene, N42WT had no mutation while N42-3 had a F397L mutation. We sequenced approximately 22.53 Mb of the genomes of the N43WT and N42-3 strains. Other than the F397L mutation in SQLE, there were three other genetic mutations in three different genes that were found in N42-3, but not in N43WT; however, these three mutations were not detected in other TRF-resistant T. rubrum strains. From this genome sequencing analysis, the only variation that was confirmed to be associated with drug resistance in the genome of the TRF-resistant T. rubrum was a hotspot mutation in SQLE.

Dermatophytoses are common fungal skin infections in humans.More than 60% of the fungal infections worldwide are caused by dermatophytes, predominantly Trichophyton rubrum (50%) [1].The antifungal drug terbinafine (TRF) inhibits squalene epoxidase (SQLE), which is involved in an early step of ergosterol biosynthesis; ergosterol is a compound essential for the structure and function of the plasma membrane in fungi [2].Although TRF his often used as a treatment for dermatophytosis, TRF-resistant strains have recently been isolated from human patients around the world [2,3].In 2020, we investigated the antifungal susceptibility of clinical isolates from Japanese patients to assess the TRFresistance patterns in T. rubrum strains; among 128 T. rubrum infections, we isolated 5 (3.9%)TRF-resistant strains of T. rubrum [4].There is a risk that the prevalence of dermatophytosis due to antifungalresistant strains may increase worldwide.Almost all TRF-resistant strains of dermatophytes have the L393F or F397L amino acid substitution in the SQLE gene [2][3][4].However, the intracellular molecular mechanisms that lead to the acquisition of TRF resistance remain poorly understood, and therefore, effective methods to control TRF resistance have not yet been developed.We are also interested in determining whether fungi acquire drug resistance through genome-wide changes or through mutations in a single gene.
To better understand how the genome changes when dermatophytes acquire drug resistance, we induced TRF resistance in a T. rubrum strain in vitro, then compared the genomes of the TRFresistant and wild-type (WT) strains to identify mutations.
The N42WT strain of T. rubrum was isolated from a tinea cruris case in 2020.This strain is susceptible to TRF, itraconazole (ITZ), ravuconazole (RVZ), and luliconazole (LUZ); the minimum inhibitory concentrations (MICs) of these drugs were all \ 0.03 lg/mL as determined according to the Clinical & Laboratory Standards Institute (CLSI) M38-A2 guidelines with modifications as previously described (Table 1) [5,6].The internal transcribed spacer (ITS) sequence homology of the isolated strain was 100% identical to that of the Trichophyton rubrum CBS 392.58 ITS region from TYPE material (GenBank accession number NR_131330.1).
Aliquots of N42WT (2.4 9 10 6 microconidia/plate) were cultured on Sabouraud's dextrose agar (SDA: 1% of peptone, 2% of dextrose and 2% of agar) containing 0.005 lg/mL of TRF in plates that were 9 cm in diameter, and incubated at room temperature for 3 weeks.Nine individual colonies developed on the primary selection plates; these colonies were subcultured on SDA containing 0.01 lg/mL of TRF, and incubated at room temperature for 2 weeks.The nine colonies grew on new plates containing 0.03 lg/mL of TRF after 2 weeks, and they were passaged on SDA containing 0.06 lg/mL of TRF.After 3 weeks of culture SDA containing 0.06 lg/mL of TRF, there were three colonies that grew on the SDA; these colonies were transferred onto SDA with a TRF concentration of 0.1 lg/mL.After 4 weeks of culture, one colony grew on the SDA, and it was transferred onto new SDA with a TRF concentration of 0.1 lg/mL for 2 weeks.Then the colony was maintained for 12 months by passaging the cells every 4 weeks onto SDA with a TRF concentration of 0.1 lg/mL.During this period, attempts were made to grow the cells on SDA with a TRF concentration of 1 lg/mL every 1 month, but the strain did not proliferate.After 12 months by passaging on SDA with a TRF concentration of 0.1 lg/mL, the strain was passaged grow on SDA with a TRF concentration of 1 lg/mL.After 4 weeks of incubation on SDA with a TRF concentration of 1 lg/mL, colony growth was observed, and the culture was transferred onto SDA with a TRF concentration of 5 lg/mL.After 4 weeks of incubation, colony growth was observed, and this strain was used for further investigations.In total, it took approximately 18 months to establish this TRF-resistant strain.This strain was designated as N42-3, and was resistant to TRF (MIC [ 32 lg/mL), but susceptible to ITZ, RVZ, and LUZ (MICs \ 0.03 lg/mL for all) by the M38-A2 method with modifications (Table 1) [5,6].
Mycelial cells of the N42-3 and N42WT strains were collected and frozen in liquid nitrogen, then homogenized.The homogenates were subsequently lysed in lysis buffer containing 0.1 mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate, and 10 mM Tris hydrochloride (Tris-HCl, pH 8.0).High molecular weight DNA was treated with RNase, and extracted from the mycelial cells using the phenol method and ethanol preparation.The DNA samples dissolved in TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM ethylenediaminetetraacetic acid) were used for further analysis.
Whole-genome sequencing (PacBio Sequel IIe; https://www.pacb.com/technology/hifi-sequencing/sequel-system/) and data analysis of N42WT and N42-3 were performed by Bioengineering Lab.Co., Ltd.(Kanagawa, Japan).Genomic DNA samples of approximately 2.5 lg were extracted from growing mycelia according to a previously described method.After elimination of the short DNA fragments (\ 10 kb) using the Short Read Eliminator XS Kit (NIPPON Genetics Co., Ltd.Tokyo, Japan), the resulting DNA was sheared to 10-to 20-kb pieces using a g-TUBE device (Covaris, Osaka, Japan) prior to library preparation.High-fidelity sequencing libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA, USA), and bound to the sequencing polymerase enzyme using the Sequel II Binding Kit 2.0 (Pacific Biosciences) according to the manufacturer's protocol.Shotgun genomic DNA sequencing data were collected on the Sequel IIe system (Pacific Biosciences), and assembled using the IPA HiFi Genome Assembler (version 1.5.0;Pacific Biosciences).
For genome sequencing, we sequenced approximately 22.53 Mb of the whole genomes of the N43WT and TRF-resistant N42-3 strains.An average of 10,000 bp was sequenced per read for the genomes of both strains, and approximately 260,000 reads were analyzed, resulting in a total of approximately 2.3 billion bp sequenced in each strain.The mapping using Pbmm2 (ver.1.10.0)[7] was performed based on Trichophyton rubrum CBS 118892 (assembly ASM15142v1 in GenBank), which is registered in GenBank, and approximately 98.3% identity was confirmed between the genomes of T. rubrum CBS 118892 and the two strains.
The SMRTLink (v.11.0.0.146107; https://www.pacb.com/support/software-downloads/)software was used to remove the overhang adapters from the sequenced data to make subreads.After creating a consensus sequence (CCS) aligned.The unreliable reads from the CCS were removed and designated as HiFi reads, which were used for the detection of gene mutation analysis.
After these bioinformatics analyses, four genetic variants were identified only in N42-3, and not in N43WT, one of which was a mutation of F397L in SQLE (Table 2).Other than the SQLE gene mutation, three other mutations in three different genes were found in N42-3; these mutations were sequenced to examine whether they are also present in other TRF-resistant strains (strains N22-2, N99-2, and NUBS210011; TRF MICs: C 32 mg/mL in all strains; Table 1).
The primer sets are listed in Table 3.DNA samples (100 ng) were amplified by PCR in a reaction volume of 30 lL.The reaction mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.001% gelatin, 2.5 mM of each deoxynucleoside triphosphate, 1.0 unit of Taq polymerase (Takara, Kyoto, Japan), and 0.3 lM of the primer pairs.PCR amplification was carried out for 35 cycles, and consisted of template denaturation (30 s at 94 °C), primer annealing (30 s at 60 °C), and polymerization (30 s at 72 °C).The PCR products were detected by electrophoresis on a 2% agarose gel followed by staining with ethidium bromide, and visualization under ultraviolet light.The sequencing procedures were as reported previously [8].Sequencing results showed that the three genetic mutations in three different genes (TERG_00615, TERG_08545, and TERG_00315) other than F397L in SQLE that were found in N42-3 were not found in any of the three other TRF-resistant strains (N22-2, N99-2, and NUBS210011).
In this study, TRF resistance in T. rubrum was induced within 18 months by incubating the fungi with TRF.The sequencing of SQLE in the TRFresistant strain acquired resistance confirmed the F397L mutation, similar to the resistant strains that have been isolated from human dermatophytoses cases thus far.Therefore, the resistance mechanism in the strain generated in vitro in this study was assumed to reflect the resistance mechanism in strains isolated from clinical practice.
The whole genome sequencing analysis indicated that the genetic variations in both the N42-3 and N43WT strains in comparison to Trichophyton rubrum CBS 118892 consisted of 3293 gene substitutions, deletions, and insertions.These 3,293 genetic variations accounted for a 0.014% difference in the genomes, and the variations were taken to be mutations that represent the individuality of the N42WT strain, and that may have occurred randomly in the genome.In a previous report, the intraspecific variation in T. rubrum genomes was extremely low (99.99% match within the same species), which is indicative of the clonal nature of this species [9].However, the present results suggest that the intraspecific variation may be a little richer depending on the geographic distance between the areas from which the strains were isolated.
Comparison of the genomic mutations in the TRFresistant strain (N42-3) to those in N42WT revealed four mutations that developed over the 18 month period, including the F397L mutation in SQLE.However, the other three mutations were not found in other TRF-resistant strains that have been isolated in Japan, and were thus considered to be unrelated to TRF resistance.Presumably, these genetic mutations unrelated to resistance were random genetic mutations in the dermatophytes.
Interestingly, the SQLE hotspot mutation (L393F or F397L) was selected in 22.53-Mb bp of whole genome when the strain was grown in the presence of TRF within a period of only 18 months.Further studies will be conducted to elucidate the mechanism by which a specific gene locus in the genome is actively selected and mutated to acquire resistance.

Data Summary
The updated sequences have been submitted to GenBank with the following identification numbers.
For genomic analysis data of N42WT strain.

Table 2
Genetic variants only in N42-3 and not in N43WT

Table 3
Primer pairs used for amplification of TERG_00615, TERG_08545, and TERG_00315 genes