Drug-resistance genes and antifungal susceptibility of Trichophyton verrucosum variants isolated from bovine skin lesions and farm environments

doi:10.21203/rs.3.rs-3012124/v1

• Background

Trichophyton verrucosum is a zoophilic dermatophyte that causes a highly contagious disease in bovine, and can be occasionally transmitted to humans. Azoles are wildly used as antifungal drugs in bovine; they act by targeting the ergosterol biosynthesis pathway. However, recently, several cases of failure and relapse of dermatophyte infection have been reported due to gene mutations in the drug target site. In addition, subtilisin (SUB) genes play an important role in pathogenesis. However, there is limited information regarding T. verrucosum variants. This study aimed to classify T. verrucosum variants based on their antifungal susceptibility and the presence or absence of ergosterol biosynthesis (ERG) and SUB genes, isolated from animals with clinical symptoms and cattle environments (fence and water bowel) in the Republic of Korea.

• Results

Of 139 clinical samples and 39 environmental samples, 86 and 21 were found to be infected, respectively. The positive ratio of calves aged 1–6 months was 73.1%, which was significantly higher than that of calves aged > 6 months (55.2%). Twenty-seven T. verrucosum strains were identified and classified as T. album (n = 9), T. ochraceum (n = 6), and T. discoides (n = 12). Antifungal susceptibility testing showed that enilconazole had the lowest geometric mean antifungal activity, of 1.08, 1, and 0.94 µg/ml against T. album, T. ochraceum, and T. discoides, respectively. All strains harbored SUB6 and ERG11. The distribution of SUB5, ERG3, and ERG6 among the three variants was significantly different.

• Conclusions

To the best of our knowledge, this is the first report of the antifungal susceptibility and presence/absence of SUB and ERG genes in T. verrucosum variants isolated from bovine and farm environments in the Republic of Korea. This information regarding T. verrucosum variants may help prevent and manage dermatophytosis in cattle.

Trichophyton verrucosum variants

Antifungal susceptibility test

Virulence gene

Drug-resistance gene

Dermatophytosis, commonly known as ringworm or tinea, is caused by dermatophytes and involves superficial mycosis invading the coat and stratum corneum of the epidermis and keratinized tissues, such as hair and nails, in humans and animals (1). The causative agents of dermatophytosis include three genera: Microsporum, Trichophyton, and Epidermophyton. These can be divided based on the organisms infected: human (anthropophilic), animal and human (zoophilic), or soil, human, and animal (geophilic) (2). Trichophyton verrucosum is the predominant etiological agent for bovine dermatophytosis. Other causative agents, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton simii, and Microsporum gypseum, have also been reported (3). T. verrucosum has three variants: T. verrucosum var album (T. album), T. verrucosum var ochraceum (T. ochraceum), and T. verrucosum var discoides (T. discoides) (4). In cattle, ringworm causes economic losses attributed to hide damage, weight loss, decimated meat and milk, contagiousness among animals, and treatment costs (5). This is a cause for public health concern because T. verrucosum, the zoophilic dermatophyte responsible for dermatophytosis in cattle, can spread to humans through direct contact with cattle or indirect contact with infected fomites, causing inflammatory skin and hair dermatophytosis (6).

Topical therapy is the most practical and popular way to treat dermatophytosis and is usually sufficient to eliminate the pathogen. Enilconazole is the most widely used antifungal drug, and other drugs, such as thiabendazole, natamycin, resorcinol, and iodine, are used in veterinary medicine to treat bovine dermatophytosis (7).

Paradoxically, despite advances in modern medicine, there have been relapses and untreated cases of dermatophyte infections (8, 9). Several causes of tolerance or resistance to antifungal agents have been reported, such as mutations in ergosterol biosynthesis (ERG) genes encoding target enzymes, overexpression of genes involved in cellular detoxification, and ATP-binding cassette transporters for azole-resistant phenotypes of dermatophytes (10). Subtilisin (SUB) genes are major virulence genes that degrade proteins in keratinized skin tissues and play a role in the invasion of dermatophytes during infection (11). Screening for specific virulence genes can contribute to our understanding of the pathogenesis of dermatophytosis. Moreover, identifying the susceptibility of pathogenic fungi to antifungal drugs and the genomic characteristics of pathogenic factors may contribute to the prevention and treatment of dermatophytosis. However, to our knowledge, there is limited information regarding the classification of T. verrucosum variants and no information regarding their antifungal susceptibility and genetic characteristics. Therefore, in this study, we aimed to classify T. verrucosum variants isolated from animals with clinical symptoms and cattle environments in the Republic of Korea, determine their antifungal susceptibility, and evaluate the presence or absence of ERG and SUB.

Dermatophyte identification

In this study, of the 139 clinical samples and 39 environmental samples, 86 and 21 samples were found to be infected, respectively, after combining the results of direct microscopic examination and fungal culture (Table 1). The overall detection rate of dermatophytes was 60.1% (107/178). In the clinical samples, the positive ratio of calves aged 1–6 months was 73.1%, which was significantly higher (p < 0.001) than that of calves aged > 6 months (55.2%). However, no significant difference (p > 0.05) was observed for sample type and sex.

Table 1

Diagnosis of 178 dermatophytes based on sample type, age, and sex.
Variables		Positive ratio	P-value	F
Sample type	Animal	61.9% (86/139)	0.183	1.790
Sample type	Environment	53.8% (21/39)	0.183	1.790
Age	> 6 months	55.2% (48/87)	< 0.001	18.936
	≤ 6 months	73.1% (38/52)	< 0.001	18.936
Sex	Male	63.5% (40/63)	0.475	0.513
	Female	60.5% (46/76)	0.475	0.513
Total		60.1% (107/178)

Isolation and classification of T. verrucosum variants

The isolated fungi were initially classified according to the colony morphology on SDA (Table 2). A total of 178 samples were cultured in SDA at 37℃. Among these, 27 strains glowed slowly, and their shapes were rounded and grey to white or white to yellow. Twenty-seven strains were classified as T. album (n = 9), T. ochraceum (n = 6), and T. discoides (n = 12) based on their morphology (Table 2). In the NJ phylogenetic tree, the 27 T. verrucosum isolates clustered with T. verrucosum reference isolates from different geographical locations in Republic of Korea (Fig. 1). The constructed phylogenetic tree showed that all strains were on the same branch. All isolates were obtained from NCBI GenBank accession numbers (OR058550-OR058576).

Antifungal activity

The results of the antifungal susceptibility test of the 27 T. verrucosum variant isolates are shown in Table 3. Enilconazole had the lowest geometric mean antifungal activity, of 1.08, 1, and 0.94 µg/ml against T. album, T. ochraceum, and T. discoides, respectively. The Minimum inhibitory concentration 50 (MIC₅₀) of enilconazole was the same for all variants (1 µg/ml). However, T. ochraceum had a lower MIC₉₀ of enilconazole (1 µg/ml) than the other two variants (2 µg/ml). Moreover, just one T. discoides isolate had a high MIC (8 ppm). The MIC₉₀ of thiabendazole was similar for all variants (2 µg/ml). However, the MIC₅₀ of thiabendazole for T. ochraceum and T. discoides (1 µg/ml) was lower than that for T. album (2 µg/ml), but the difference between the values was not significant. The MIC of natamycin, resorcinol, and iodine was 128 µg/ml for all 27 strains. Although all T. verrucosum isolates had different MICs, most showed similar MICs for all five agents used in this study.

Table 3

*In vitro* activity susceptibility of 27 strains of *Trichophyton verrucosum* variants against five antifungals.
Species			Geometric means	MIC₅₀	MIC₉₀	Range
Trichophyton verrucosum	var album (n = 9)	TB	1.47	2	2	[1–2]
		ENL	1.08	1	2	[0.25-2]
		NAT	128	128	128	[> 64}
		RES	128	128	128	[> 64}
		I	128	128	128	[> 64}
	var ochraceum (n = 6)	TB	1.41	1	2	[1–2]
		ENL	1	1	1	[1]
		NAT	128	128	128	[> 64}
		RES	128	128	128	[> 64}
		I	128	128	128	[> 64}
	var discoides (n = 12)	TB	1.33	1	2	[1–2]
		ENL	0.94	1	2	[0.25-8]
		NAT	128	128	128	[> 64}
		RES	128	128	128	[> 64}
		I	128	128	128	[> 64}
^§Antifungal agents: TB: Thiabendazole, ENL: Enilconazole, NAT: Natamycin, RES: Resorcinol, I: Iodine

Virulence genes

Screening for the presence of three ERG and seven SUB genes in 27 T. verrucosum variants was conducted using PCR assays. The rates of positivity of the ERG3, ERG6, and ERG11 genes in T. album, T. ochraceum, and T. discoides isolates were 66.7–100%, 0.0–100%, and 0.0–100%, respectively, whereas those of the SUB1-7 genes in T. album, T. ochraceum, and T. discoides isolates were 55.6–100%, 16.7–100%, and 58.3–100%, respectively. The analysis revealed the presence of SUB6 and ERG11 in all strains. In addition, 100% of T. ochraceum isolates were positive for SUB2 compared to T. album (77.8%) and T. discoides (91.7%). ERG3 was not detected in T. ochraceum, and ERG6 was not detected in T. ochraceum and T. discoides (Fig. 2). According to the Pearson χ2 and cross-tabulation tests, the distributions of SUB5, ERG3, and ERG6 among the three variants were significantly different (p < 0.05; Fig. 2). No significant differences were observed in the distribution of SUB1-4, SUB6-7, and ERG6 and ERG11 (p > 0.05; Fig. 2).

To the best of our knowledge, this study is the first to investigate antifungal susceptibility and genetic characteristics of T. verrucosum variants in clinical and environmental samples. Moreover, three drug-resistance genes and seven virulence genes were analyzed to understand pathogenicity better.

In this study, 61.9% of the clinical and 53.8% of the farm environment samples were positive for T. verrucosum, as confirmed by direct microscopic examination and fungal culture. The present diagnostic results differ from those observed in symptomatic animal samples in India (55.3%) (12), Egypt (84.9%) (13), Italy (95.7%) (14), and Iran (92.6%) (15). This is because the difficulty in clinically differentiating dermatophytosis from other non-dermatophyte infections and the similar clinical manifestations of different dermatophyte species make clinical examination less reliable for diagnostic purposes (16). A comparison of the sample types revealed that T. verrucosum-positivity ratio between clinical and environmental samples was not significantly different. Trichophyton verrucosum is transmitted by direct contact with infected animals and environment. It separates from skin lesions in the environment and transmitted to other animals by contact (17). One of the most well-known risk factors for T. verrucosum infection is cattle age (13). In symptomatic animal samples, the T. verrucosum-positivity ratio in calves aged 1–6 months (73.1%) was significantly higher than that in calves aged > 6 months (55.2%). Young animals are more easily infected by ringworm because they are not immune to the effects of exposure to dermatophytes (14).

In this study, the 27 T. verrucosum isolates were classified as T. ochraceum (n = 9, 33%), T. album (n = 6, 22%), and T. discoides (n = 12, 44%). However, other studies have shown different results for separation rates among variants (18, 19). Classification of T. verrucosum into three variants was done by Sabouraud in 1910 (20). In 1950, Georg (4) found that these three fungi belonged to the same species, T. verrucosum. Morphologically, T. verrucosum is classified into three variants: T. ochraceum, flat and verrucose but with an ochraceous pigment; T. album, heaped and folded with a waxy appearance; T. discoides, disc-shaped with a small central knob. The sequencing of the ITS region of rDNA has been widely used for species identification (21). In previous studies (22, 23), T. verrucosum isolates showed 99% similarity in their ITS sequencing data, which is consistent with our results.

In this study, the MICs of five antifungal agents for T. verrucosum variant isolates were measured because of the importance of drug sensitivity test in the selection and dosage determination of drugs to effectively treat bovine dermatophytosis (24). Our results showed that enilconazole had the lowest geometric mean antifungal activity among the five antifungal agents tested against the three T. verrucosum variant isolates, followed by thiabendazole. Moreover, the MIC₉₀ of enilconazole was 1 or 2 µg/ml and that of thiabendazole was 2 µg/ml for the three variants. The MIC₉₀ of enilconazole was slightly higher than that reported previously (25). Our values were still lower than the commensal enilconazole drug dose (2 µg/ml) (7). Despite the wide application of enilconazole for bovine dermatophytosis, only one T. discoides isolate had a high MIC of enilconazole (8 ppm). However, there are no reports on T. verrucosum with a high MIC. Further research is needed to clarity resistance or tolerance of T. verrucosum to enilconazole. Thiabendazole is a well-known antifungal drug for bovine dermatophytosis, and our results are similar to those of a study from Mexico (26). Natamycin, resorcinol, and iodine are not widely used to treat dermatophytosis (7). The present study showed that the three antifungal agents were ineffective against all 27 isolates. No significantly different geometric means of MICs were observed for all antifungal agents. We recommend that antifungal susceptibility test be performed before choosing appropriate antifungal drugs to treat bovine dermatophytosis.

Ergosterol is a major sterol in fungi and maintains membrane fluidity, permeability, and structure (27). The ergosterol biosynthetic pathway includes nine nonessential genes, including ERG3 and ERG6 (28). Deletions and mutations in some nonessential ergosterol biosynthesis genes cause fungi to become resistant to antifungal drugs such as azoles and polyanes, which disrupt normal sterol biosynthesis (10). Among the three T. verrucosum variant isolates, the MIC₅₀ of thiabendazole was the highest for T. album, which had the highest ERG3 and ERG6 detection ratios. The MIC₉₀ of enilconazole was the lowest for T. ochraceum, in which ERG3 and ERG6 were not detected. This result is similar to that of a previous study (29), which reported that strains with polymorphic ERG genes are resistant to drugs, whereas strains without this gene family are sensitive to all antifungals.

In this study, seven SUB genes in T. verrucosum variant isolates were detected. Only SUB6 was detected in all isolated strains. Other genes showed a 55.6–88.9% positive ratio. This value was slightly higher than that in a previous study, which showed a 50–60% detection rate of SUB3-7 in animal samples (30). However, this rate is lower than that in Lemsaddek et al.’s study (31), in which the seven SUB genes were 100% positive in all T. verrucosum variants. The SUB gene is one of the most important virulence genes for initial invasion (11). SUB6 is a key element in the induction of inflammation by Trichophyton rubrum (32). SUB5 has been significantly associated with T. rubrum infection in humans (31). However, the absence of SUB1-3 in Microsporum canis isolates is not indispensable for dermatophyte invasion (33). SUB4 plays a less significant role in infection (34). In our study, SUB6 was the most prevalent, followed by SUB2. Significantly different ratios of SUB5 were found among the three variants. Overall, pathogenicity may not be related to specific genes but to the accumulation and synergistic effects of their products

In conclusion, to the best of our knowledge, this is the first report on the antifungal susceptibility and the presence or absence of SUB and ERG in T. verrucosum variants isolated from animals clinically infected with dermatophytes and farm environments in the Republic of Korea. The detection rate of dermatophytes was 60.1%, and 27 T. verrucosum variants were isolated and classified. In the antifungal susceptibility test, one strain had a high MIC of enilconazole, suggesting that antifungal susceptibility test should be performed before applying antifungal drugs. We identified drug-resistance genes in T. verrucosum variants. In addition, we have provided information about key virulence genes. However, this study involved a limited number of isolates; further research is required to screen antifungal susceptibility and determine relationship with ERG. Overall, our results on T. verrucosum variants may help prevent and manage dermatophytosis in cattle.

Sample size determination and sample collection

The sample size was determined using the following formula (35), with an expected disease prevalence of 10%, an accepted absolute error of 5%, and a confidence level of 95% with a simple random sampling design.

$$\text{n}=\frac{{Z}^{2} P(1-P)}{{d}^{2}}$$

where n = Sample size, Z = level of confidence, P = Expected prevalence, and d = Precision.

The calculated minimum sample size for this study was 138. A total of 178 samples, including cattle (n = 139) and environmental (fences and water bowls, n = 39) samples, were collected from 35 farms in 11 Korean cities (Boseong, Cheonan, Damyang, Geochang, Gimje, Hadong, Hamyang, Muju, Pyeongchang, Sangju, Wanju). Samples of animals clinically infected with dermatophytes only were chosen by a veterinarian. Clinically symptomatic animal samples were collected from the skin at the junction between healthy and skin lesion regions using a brush, and fences and water bowls were randomly sampled using a brush in pens with symptomatic animals. To remove the contamination, both sources were cleaned with ethyl alcohol before sampling, and samples were collected in a sterile, labelled, closed envelope, and immediately transferred to the laboratory for mycological examination. Data related to the cattle, including age and sex as risk factors, were recorded (Table 1). All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the National Institute of Animal Science of the Republic of Korea (approval number: NIAS-2020127).

Direct examination

Samples were placed on a clean glass slide; a drop of 20% KOH was added with mild heat and left for 30 min, after which the samples were covered with a cover slide and examined by microscopy (BX-53-P, Olympus, Tokyo, Japan) at magnifications of ×10 and ×40 to determine the presence of fungal hyphae, macroconidia, and arthrospores.

Culture and isolation of fungi

Samples were incubated on Sabouraud’s dextrose agar (SDA) supplemented with chloramphenicol, cycloheximide, thiamine, and inositol at 37°C for four weeks and observed periodically for the appearance of fungal growth. Suspicious colonies were transferred to fresh agar and incubated under the same conditions, and the color, texture, surface topography, reverse side colony, and margin were examined to identify the T. verrucosum variant. Morphologically, T. verrucosum is classified into three variants: T. ochraceum, flat and verrucose but with an ochraceous pigment; T. album, heaped and folded with a waxy appearance; T. discoides, disc-shaped with a small central knob as described in Georg 1950 (4).

Phylogenetic analysis

Suspicious fungal colonies were subjected to DNA extraction by boiling, and PCR was performed using the ITS primers (ITS1 and ITS4). All PCR amplicons were sequenced using SolGent Co., Ltd (Daejeon, Korea). Sequences were analyzed using BLAST from the National Centre for Biotechnology Information (NCBI) database (USA). Phylogenetic analysis of the ITS sequences of the 27 T. verrucosum isolates was performed using the neighbor-joining (NJ) method with 1000 bootstrap replications using the Kimura two-parameter model. The NJ tree was constructed using MEGA7 software. T. verrucosum strains and other Trichophyton spp. obtained from the NCBI Database were used as reference strains for phylogenetic construction.

Antifungal susceptibility test of dermatophyte isolates

Preparation of fungal inocula

Fungal suspensions of the 27 T. verrucosum isolates were prepared from freshly grown 12-day-old cultures on SDA plates at 37°C. Briefly, fungal colonies were gently probed using a sterile wet cotton swab and resuspended in 2 ml of Sabouraud’s dextrose broth (SDB). The fungal suspension was filtered through a sterile 100-mesh filter. The number of conidia in the insula was counted using a hemocytometer, and the final inoculum was adjusted to approximately 1 × 10⁸ conidia/ml.

Preparation of antifungal agents and antifungal susceptibility test

The efficacies of five antifungal agents (thiabendazole, enilconazole, natamycin, resorcinol, and iodine) against 27 T. verrucosum isolates were evaluated using a spot culture growth inhibition assay (SPOTi). For each drug, serial two-fold dilutions were prepared from a stock solution in 1% DMSO. Each dilution was dispensed into SDA to prepare the two-fold dilution series, and the wells of a 96-well plate were filled with 200 µl of SDB using a multichannel pipette. The final drug concentration in agar ranged from 64 to 0.0625 µg/ml for the antifungal agents; 10 µl of the prepared fungal inoculum was spotted in each well of agar containing the antifungal agents using a multichannel pipette. The plates were incubated at 37°C for seven days, and T. verrucosum growth was visually assessed for seven days.

Molecular identification of T. verrucosumgenes

Primer design

With data from the NCBI, Primer3Plus analysis software was used to create seven subtilisin genes (SUB1-7) from conserved areas of homologous genes in other dermatophytes. Three ergosterol genes (ERG3, ERG6, and ERG11) were used in a previous study (36). Primer-BLAST, available at http://www.ncbi.nlm.nih.gov, was used to determine the efficacy of each primer (Supplementary Table 1, Additional File1).

Isolation of genomic DNA from samples.

Total genomic DNA extraction was performed using the method described by Tartor et al. (13). Briefly, a total of 178 clinical samples were used for DNA extraction. About 25 mg of each sample was collected into a 2-ml Eppendorf tube containing tungsten carbide beads (Qiagen, Hilden, Germany), 180 ml of ALT buffer, and 20 µl of protease (QIAamp DNA mini kit, Qiagen, Germany) and incubated overnight at 55°C. Tissues were disrupted using a tissuelyzer (Qiagen, Germany) for 2 min at 30 Hz. Total DNA extraction was continued according to the QIAamp DNA mini-kit protocol. DNA was eluted in 50 µl of elution buffer, and the concentration and quality of DNA were assessed using an Epoch spectrophotometer (BioTek, Vermont, USA).

Amplification reaction of T. verrucosum genes

To evaluate the potential pathogenicity of the T. verrucosum isolates, fungal genomic DNA was subjected to PCR assays to detect three drug-resistance genes and seven virulence genes. The PCR assays were performed using a PCR premix (Bioneer Inc., Daejeon, Korea) containing 1 µl of each primer (10 pmol) and 4 µl of DNA template with nuclease-free water (total reaction volume, 20 µl). The PCR was performed using an AllinOne thermal cycler (Bioneer, Korea) with the following cycling conditions: 95°C for 5 min (initial denaturing), followed by 40 cycles of 95°C for 45 s (denaturing), 52°C for 45 s (annealing), 72°C for 1 min (elongation), followed by 7 min at 72°C (final extension). The resulting PCR amplicons were electrophoresed on 1.5% (w/v) agarose gels using RedSafe™ (iNtRON Biotechnology, Seongnam, Korea) and visualized under UV light.

Statistical analysis

Risk factor analysis was performed considering sample type, age, and sex using the Student’s t-test.

The Geometric means of antifungal susceptibility result was analyzed by ANOVA using descriptive statistics. The Chi-square test was used to analyze the data to show statistical independence between ERG and SUB genes in three T. verrucosum isolates. Results with p-values < 0.05 were considered significant.

ERG: Ergosterol biosynthesis; MIC: Minimum inhibitory concentration; SUB: Subtilisin; T. verrucosum: Trichophyton verrucosum

Ethics approval and consent to participate: This research was approved by the Institutional Animal Care and Use Committee (IACUC) at the National Institute of Animal Science, the Republic of Korea (approved number: NIAS-2020127). In addition all methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all farmers who participated in this study, and all sampling was performed by an expert veterinarian working at the farm.
Consent for publication: Not applicable
Availability of data and materials: The datasets generated during the current study are available in GenBank accession number OR058550-OR058576
Competing interests: Authors have no competing interests to declare.
Funding: This study was supported by the 2023 RDA Fellowship Program of the National Institute of Animal Science, Rural Development Administration, the “Cooperative Research Program for Agriculture Science and Technology Development (Project title: The improvement of animal diseases control in National Institute of Animal Science, Project No. PJ01567708)”, and Rural Development Administration, Republic of Korea.
Authors' contributions: HGL, YHJ, AC, JKO, and TYH conceived and designed the experiment. HGL and AC performed the experiments and analyzed the data HGL, JKO, and TYH drafted and revised the manuscript. All authors read and approved the final manuscript.
Acknowledgements: The authors thank the veterinarian who worked at the farm for help during the sampling.

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No competing interests reported.

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Drug-resistance genes and antifungal susceptibility of Trichophyton verrucosum variants isolated from bovine skin lesions and farm environments

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Abstract

Figures

Background

Results

Dermatophyte identification

Antifungal activity

Virulence genes

Discussion

Conclusion

Methods

Sample size determination and sample collection

Direct examination

Culture and isolation of fungi

Phylogenetic analysis

Antifungal susceptibility test of dermatophyte isolates

Preparation of fungal inocula

Preparation of antifungal agents and antifungal susceptibility test

Primer design

Statistical analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1