Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of ...patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all
clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor
gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu
, Phe
, Phe
, and His
) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous
gene of a terbinafine-sensitive
(formerly
) strain. All of the generated
transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in
transformants expressing mutant forms of
SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.
The mechanisms of terbinafine resistance in a set of clinical isolates of
have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of ...TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of
to develop resistance through multidrug efflux transporters. The introduction of a
cDNA library into
allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the
genome, we performed a BLASTp analysis of
,
, and
on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in
Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both
and
were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of
in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in
, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.
Onychomycosis is mainly caused by two dermatophyte species,
and
. A study of nail invasion mechanisms revealed that the secreted subtilisin Sub6, which has never been detected under in vitro growth ...conditions, was the main protease secreted by
and
during infection. In contrast, most of the proteases secreted during the digestion of keratin in vitro were not detected in infected nails. The hypothesis that proteases isolated from dermatophytes grown in a keratin medium are virulence factors is no longer supported. Non-dermatophyte fungi can also be infectious agents in nails. It is necessary to identify the infectious fungus in onychomycosis to prescribe adequate treatment, as moulds such as
spp. and
spp. are insensitive to standard treatments with terbinafine or itraconazole, which are usually applied for dermatophytes. In these refractory cases, topical amphotericin B treatment has shown to be effective. Terbinafine treatment failure against dermatophytes is also possible, and is usually due to resistance caused by a missense mutation in the squalene epoxidase enzyme targeted by the drug.
resistance to terbinafine treatment is an emerging problem, and a switch to azole-based treatment may be necessary to cure such cases of onychomycosis.
Summary
Trichophyton rubrum is the most frequently isolated dermatophyte species in European countries. The lack or poor sporulation of T. rubrum has always been a major complication and a limiting ...factor when performing antifungal susceptibility testing. Therefore, we describe an in vitro method aiming to enhance sporulation of various T. rubrum isolates in order to perform antifungigrams. A combination of high CO2 tensions and incubation on PDA growth medium revealed to be optimal for sporulation of all tested T. rubrum isolates. This method was further used to examine in vitro the combined effects of amorolfine and azole derivatives against fungal growth using adapted checkerboard microdilution assays and an isobolographic approach of the data, adapted disc diffusion and Etest assays. Non‐antagonistic and synergistic effects were observed in these settings with amorolfine combined to each of the tested azole compounds. The optimised culture method appeared to be suitable for T. rubrum isolates for which antifungigrams were especially difficult to obtain because of the lack of sporulation.
Millions of people suffer from superficial infections caused by dermatophytes. Intriguingly, these filamentous fungi exclusively infect keratin-rich host structures such as hair, nails, and skin. ...Keratin is a hard, compact protein, and its utilization by dermatophytes for growth has long been discussed as a major virulence attribute. Here, we provide strong support for the hypothesis that keratin degradation is facilitated by the secretion of the reducing agent sulfite, which can cleave keratin-stabilizing cystine bonds. We discovered that sulfite is produced by dermatophytes from environmental cysteine, which at elevated concentrations is toxic for microbes and humans. We found that sulfite formation from cysteine relies on the key enzyme cysteine dioxygenase Cdo1. Sulfite secretion is supported by the sulfite efflux pump Ssu1. Targeted mutagenesis proved that dermatophyte mutants in either Cdo1 or Ssu1 were highly growth-sensitive to cysteine, and mutants in Ssu1 were specifically sensitive to sulfite. Most notably, dermatophyte mutants in Cdo1 and Ssu1 were specifically growth-defective on hair and nails. As keratin is rich in cysteine, our identified mechanism of cysteine conversion and sulfite efflux supports both cysteine and sulfite tolerance per se and progression of keratin degradation. These in vitro findings have implications for dermatophyte infection pathogenesis.
Background
An alarming increase in recalcitrant dermatophytosis has been witnessed in India over the past decade. Drug resistance may play a major role in this scenario.
Objectives
The aim of the ...present study was to determine the prevalence of in vitro resistance to terbinafine, itraconazole and voriconazole in dermatophytes, and to identify underlying mutations in the fungal squalene epoxidase (SQLE) gene.
Patients/Methods
We analysed skin samples from 402 patients originating from eight locations in India. Fungi were identified by microbiological and molecular methods, tested for antifungal susceptibility (terbinafine, itraconazole, voriconazole), and investigated for missense mutations in SQLE.
Results
Trichophyton (T.) mentagrophytes internal transcribed spacer (ITS) Type VIII was found in 314 (78%) samples. Eighteen (5%) samples harboured species identified up to the T interdigitale/mentagrophytes complex, and T rubrum was detected in 19 (5%) samples. 71% of isolates were resistant to terbinafine. The amino acid substitution Phe397Leu in the squalene epoxidase of resistant T mentagrophytes was highly prevalent (91%). Two novel substitutions in resistant Trichophyton strains, Ser395Pro and Ser443Pro, were discovered. The substitution Ala448Thr was found in terbinafine‐sensitive and terbinafine‐resistant isolates but was associated with increased MICs of itraconazole and voriconazole.
Conclusions
The high frequencies of terbinafine resistance in dermatophytes are worrisome and demand monitoring and further research. Squalene epoxidase substitutions between Leu393 and Ser443 could serve as markers of resistance in the future.
Background
Dermatophytes showing reduced sensitivity to antifungal agents have emerged in several countries. One terbinafine resistant strain of Trichophyton rubrum, TIMM20092, also showed reduced ...sensitivity to itraconazole (ITC) and voriconazole (VRC). The expression of two genes (TruMDR2 and TruMDR3) encoding multidrug transporters of the ABC family was found to be highly up‐regulated in this strain. Deletion of TruMDR3 in TIMM20092 abolished its resistance to VRC but only slightly reduced its resistance to ITC.
Objectives
We examined the potential of T rubrum to develop resistance to ITC by analysing the mechanism of ITC resistance in TIMM20092.
Methods
The deletion of TruMDR2 by gene replacement was performed in TIMM20092 and one TruMDR3‐lacking mutant (∆TruMDR3) previously generated from TIMM20092. TruMDR2 single and TruMDR2/TruMDR3 double mutants (∆TruMDR2 and ∆TruMDR2/3) were successfully obtained, respectively.
Results
The suppression of TruMDR2 was shown to abolish resistance to ITC in TIMM20092 and the TruMDR3‐lacking mutant, strongly suggesting that TruMDR2 is a major contributor to ITC resistance in TIMM20092.
Conclusions
Our study highlights the possible role of the ABC transporter TruMDR2 in ITC resistance of T. rubrum.
The co-cultivation of fungi has recently been described as a promising strategy to induce the production of novel metabolites through possible gene activation. A large screening of fungal co-cultures ...in solid media has identified an unusual long-distance growth inhibition between Trichophyton rubrum and Bionectria ochroleuca. To study metabolite induction in this particular fungal interaction, differential LC-MS-based metabolomics was performed on pure strain cultures and on their co-cultures. The comparison of the resulting fingerprints highlighted five de novo induced compounds, which were purified using software-oriented semipreparative HPLC-MS. One metabolite was successfully identified as 4″-hydroxysulfoxy-2,2″-dimethylthielavin P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated form, as well as three other related compounds, were found in the pure strain culture of B. ochroleuca.
Dermatophytes are the most common pathogenic agents of superficial mycoses in humans and animals. Knowledge of their epidemiology can facilitate the prevention of dermatophytosis and improve ...prophylactic measures. We sought to determine the incidence of the different dermatophyte species diagnosed in Lausanne (Switzerland) from 2001 to 2018. In total, 10,958 dermatophytes were isolated from patients and 459 from pets. Overall, 99% of tinea unguium and tinea pedis were caused by Trichophyton rubrum and Trichophyton interdigitale with a prevalence ratio of 3:1. Trichophyton violaceum and Trichophyton soudanense were mainly found in tinea capitis in patients of African and Mediterranean origin. Interestingly, while Epidermophyton floccosum and Trichophyton verrucosum were prevalent 50 years ago in an epidemiological analysis carried out in the same laboratory from 1967 to 1970, these two species were rarely detected from 2001 to 2018. Trichophyton mentagrophytes, Trichophyton benhamiae and Microsporum canis were the prevalent zoophilic pathogenic species in children and young adults. Our investigation of animal samples revealed the main reservoirs of these zoophilic species to be cats and dogs for T. mentagrophytes and M. canis, and Guinea pigs for T. benhamiae. This study provides an epidemiological overview of dermatophytoses in Switzerland to improve their surveillance.
Most inflammatory dermatophytoses in humans are caused by zoophilic and geophilic dermatophytes. Knowledge of the epidemiology of these fungi in animals facilitates the prevention of dermatophytosis ...of animal origin in humans. We studied the prevalence of dermatophyte species in domestic animals in Switzerland and examined the effectiveness of direct mycological examination (DME) for their detection compared to mycological cultures. In total, 3515 hair and skin samples, collected between 2008 and 2022 by practicing veterinarians, were subjected to direct fluorescence microscopy and fungal culture. Overall, 611 dermatophytes were isolated, of which 547 (89.5%) were from DME-positive samples. Cats and dogs were the main reservoirs of
and
, whereas
was predominantly found in guinea pigs. Cultures with
significantly (
< 0.001) outnumbered those with
in DME-negative samples (19.3% versus 6.8%), possibly because
can be asymptomatic in cats and dogs, unlike
, which is always infectious. Our data confirm DME as a reliable, quick, and easy method to identify the presence of dermatophytes in animals. A positive DME in an animal hair or skin sample should alert people in contact with the animal to the risk of contracting dermatophytosis.
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