Mycopathologia https://doi.org/10.1007/s11046-022-00696-9 (0123456789().,-volV) ( 01234567 89().,-volV) ORIGINAL ARTICLE ERG11 Gene Variability and Azole Susceptibility in Malassezia pachydermatis Leyna Dı́az . Gemma Castellá . M. Rosa Bragulat . F. Javier Cabañes Received: 15 September 2022 / Accepted: 18 November 2022 Ó The Author(s) 2022 Abstract Malassezia pachydermatis is part of the strains were also subjected to an ITZ E-test. Mueller– normal skin microbiota of various animal species but Hinton supplemented with 2% glucose and methylene under certain circumstances becomes an opportunistic blue was used as culture medium in both susceptibility pathogen producing otitis and dermatitis. Commonly assays. Multilocus sequence typing was performed in these Malassezia diseases are effectively treated using 30 selected strains using D1D2, ITS, CHS2 and b- azoles. However, some cases of treatment failure have tubulin genes. Also, ERG11 gene was sequenced. The been reported. Alterations in the ERG11 gene have four antifungals tested were highly effective against been associated with in vitro azole resistance in M. most of the strains. Only two strains showed no pachydermatis. In the present study, in vitro antifungal inhibition zone to antifungals and a strain showed an susceptibility of 89 different strains of M. pachyder- increased MIC to ITZ. The study of the ERG11 matis isolated from different animal species and health sequences revealed a high diversity of DNA sequences status was studied. The susceptibility to fluconazole and a total of 23 amino acid substitutions, from which (FLZ), itraconazole (ITZ), ketoconazole and ampho- only two have been previously described. Also, three tericin B was tested by a disk diffusion method and 17 deleterious substitutions (A302T, G459D and G461D) previously associated with azole resistance in this yeast were recovered. A correlation between certain Handling Editor: Takashi Sugita. genotypes and ERG11 mutations was observed. Some of the ERG11 mutations recovered were correlated Supplementary Information The online version contains with a reduced susceptibility to azoles. supplementary material available at https://doi.org/10.1007/ s11046-022-00696-9. Keywords Antifungal susceptibility  Azole drugs  L. Dı́az  G. Castellá (&)  M. R. Bragulat  ERG11 mutations  Malassezia pachydermatis F. J. Cabañes Veterinary Mycology Group, Department of Animal Health and Anatomy, Universitat Autònoma de Barcelona, Bellaterra, Catalonia, Spain e-mail: Gemma.Castella@uab.cat Introduction G. Castellá Malassezia pachydermatis is a lipophilic yeast that Grup de Micologia Veterinària, Departament de Sanitat i d’Anatomia Animals, Facultat de Veterinària, Universitat belongs to the normal skin microbiota of various Autònoma de Barcelona, 08193 Bellaterra, Barcelona, animal species and colonizes the skin and mucosal Spain 123 Mycopathologia sites of healthy dogs. Although M. pachydermatis has KTZ, ITZ and miconazole but the efficacy of FLZ is been considered classically the only non-lipid-depen- variable [8, 11, 19]. Some sporadic reports of thera- dent species of the genus Malassezia, they also lack peutic failure with azoles in canine M. pachydermatis the genes coding for a fatty acid synthase and thus, are dermatitis associated with increased tolerance to unable to synthetize long-chained fatty acids (C14 or azoles in vitro might reflect the chronic and relapsing C16) de novo [1, 2]. Malassezia pachydermatis can course of Malassezia diseases that often need frequent utilize lipid fractions within the peptone component of and lengthy treatments [16, 20–23]. SGA, thus they also need a few lipids to be able to There are various mechanisms of azole antifungal grow [2–4]. resistance in Malassezia species. The drug efflux Despite being part of the microbiota, Malassezia pump is associated to azole resistance in M. pachy- may act as an opportunistic pathogen under certain dermatis [14, 25]. Mutations in the coding region of circumstances that allow the overgrowth of the yeast the ERG11 gene, encoding a lanosterol 14a-demethy- population. Malassezia pachydermatis is a common lase, which is the target enzyme of azole antifungals, responsible agent of otitis and dermatitis in dogs [5, 6]. are considered a mechanism of resistance [26–28]. In humans, it has also been reported to produce Kim et al. [29] demonstrated that tandem quadrupli- systemic infections particularly in neonates and cation of the genomic region of the ERG11 gene immunocompromised patients receiving parenteral contributes to azole resistance in M. pachydermatis. nutrition [7]. The treatment of Malassezia diseases is At the present, only two M. pachydermatis strains currently based on the use of topical and systemic have demonstrated to be resistant to azoles. The strains antifungal therapy. Topical agents include chlorhex- were isolated in Japan [23] and Italy [22]. Both idine, clotrimazole, enilconazole, ketoconazole, isolates showed high minimum inhibitory concentra- miconazole and nystatin that can be used in various tions (MICs) against azoles but only in one case this forms such as shampoos, sprays, or ointments [8]. resistance was linked to ERG11 mutations [23]. Azoles like ketoconazole (KTZ) or itraconazole (ITZ) Although some mutations have been detected in this are commonly used as systemic agents for the gene, its intrinsic diversity in M. pachydermatis treatment of Malassezia dermatitis and otitis. Flu- yeasts, that are mainly susceptible to antifungals, is conazole (FLZ) can be used for the treatment of yet unknown. Malassezia skin diseases like seborrheic dermatitis Even though there is not a reference method for [9, 10]. Amphotericin B (AMB) and liposomal susceptibility testing of M. pachydermatis available amphotericin B are indicated in the treatment of yet, on this study we evaluate the susceptibility of systemic cases of Malassezia in humans [8, 11]. different strains of this yeast against three different The Clinical and Laboratory Standards Institute azoles (FLZ, ITZ, and KTZ) and AMB. We included a (CLSI) developed in 2002 a reference broth microdi- wide variety of strains recovered from different animal lution method for evaluating the susceptibility of species with different health status and collected over Candida spp. and Cryptococcus neoformans [12]. This different years. Also, we study the genetic variability method is inapplicable to the genus Malassezia of ERG11 gene in these strains. Gene mutations and because of their lipid-dependency, their slower growth antifungal susceptibility testing results was rate and tendency to form clusters [13, 14]. Thus, it has investigated. been subsequently adapted to Malassezia by various researchers modifying the media, time of incubation and inoculum but it has not been yet standardized Materials and Methods [13, 15–17]. Also, disk diffusion method and the E-test gradient have been adapted to evaluate the suscepti- Strains bility of Malassezia against antifungal compounds [14, 18]. A total of 89 strains of M. pachydermatis (Table S1) Although antimicrobial resistance is a global seri- were selected from our culture collection for suscep- ous threat to human and animal health, recent studies tibility testing. Strains were isolated between 1994 and have showed that most wild-type Malassezia yeast 2021 and included different animal species: dog remain susceptible to the most used azoles such as (n = 73), cat (n = 12), pig (n = 1), cow (n = 1), goat 123 Mycopathologia Table 1 Interpretative Inhibitory zone diameter (mm) criterion of zones of inhibition RoscoÓ Bio-RadÓ S SDD R S SDD R Amphotericin B C 15 14–10 B 10 [ 10 – B 10 S, susceptible; SDD, Fluconazole C 19 18–15 B 14 C 19 18–15 B 14 susceptible dose dependent; Itraconazole C 23 22–14 B 13 – – – R, resistant Ketoconazole C 28 27–21 B 20 C 20 20–10 B 10 (–) No Data (n = 1), and horse (n = 1). The M. pachydermatis GM) was the media selected for the disk diffusion neotype strain CBS1879 was also included. The method [13, 30]. For each suspension tube, 5 plates of strains were stored at - 80 8C [24]. the media were streaked using a sterile cotton swab The 73 strains of dogs included four strains isolated dipped into the inoculum suspension. One of the plates from healthy dogs, eight strains from dogs with was a growth control and on the remaining four, one dermatitis, 59 from dogs with otitis externa and two disk of antifungal per plate was placed. Two com- from otitis media. According to the data provided by mercial antifungal disks were used comprising AMB, the clinicians when sending the samples, the strains FLZ, ITZ and KTZ (Neo-Sensitabs, Rosco) at con- from otitis externa were classified into chronic otitis centrations of 10, 25, 10 and 15 lg/disk respectively externa (lasting more than three months, n = 25), and FLZ, KTZ and AMB (Bio-Rad) at concentrations acute otitis externa (lasting less than six weeks, of 25, 50 and 100 lg/disk, respectively. Plates were n = 17), purulent otitis externa (n = 6) and recurrent then incubated at 358C, and inhibition zone sizes were otitis externa (n = 4). The strains isolated from cats measured at 48, 72 and 96 h. The inhibition zone included five strains recovered from cats with der- diameter was determined after incubating at 358C for matitis and six strains from animals with otitis, from 72 h. Candida parapsilopsis ATCC 90,028 and Pichia which two were otitis media and four otitis externa. kundriavzevii ATCC 6258 were included as quality The strains from otitis externa were classified into control strains in both disk diffusion tests. acute otitis externa (n = 2), chronic otitis externa The interpretative criterion was according to the (n = 1) and purulent otitis externa (n = 1). One strain manufacturer’s guidelines for yeasts, as shown in from a healthy cat was also included. The strains from Table 1. cow, horse, pig, and goat were all obtained from Susceptibility against ITZ was also studied using healthy animals. the E-test method on 17 out of the 89 strains. The 17 strains were selected based on their diverse response to Susceptibility Testing antifungals by disk diffusion method, but also differ- ent animal species were included. The strain selection Four different antifungals were selected for suscepti- included strains obtained from healthy animals bility testing: amphotericin B (AMB), fluconazole (n = 7), from animals with otitis externa (n = 8) and (FLZ), itraconazole (ITZ) and ketoconazole (KTZ). animals with dermatitis (n = 2). Also, the M. pachy- Susceptibility of the strains was tested by a disk dermatis neotype strain CBS1879 was included. E-test diffusion method. For the disk diffusion method, two was performed under the same conditions that the disk stock inoculum suspensions were prepared from 3-day diffusion method. One E-test gradient stripe for ITZ old cultures on SGA (Oxoid) at 358C from each strain. (Biomérieux) was placed on each plate. The plates The stock inoculum suspensions were performed in were then incubated at 358C and examined at 48, 72 3 mL of distilled water supplemented with 0.004% and 96 h. The MICs were determined after 72 h of Tween 80 to reduce clump formation and were incubation. Also, some strains were evaluated using adjusted to a density of 1 McFarland standards SGA supplemented with 1% Tween 80 [23] to (3 9 108 CFU/ml). Mueller–Hinton supplemented compare the results obtained. with 2% glucose and 0.5 mg/l methylene blue (MH- 123 Mycopathologia ERG11 Gene Amplification and Sequencing based on four target regions (D1/D2 region of the 26S rRNA gene, the ITS-5.8S rRNA gene, the b-tubulin A total of 31 strains were selected to sequence their gene and the CHS2 gene). ERG11 gene, including the neotype strain of M. Sequences of the four genes of 16 strains had been pachydermatis CBS1879 and the 17 strains evaluated characterized previously [34]. The four genes of 15 by E-test. The strains selected included different remaining strains were amplified and sequenced as animal species, dog (n = 23), cat (n = 4), pig (n = 1), described previously [35]. To illustrate the phyloge- horse (n = 1), goat (n = 1) and cow (n = 1). These netic relatedness between our strains a minimum strains were recovered from nine healthy animals, 17 spanning tree was constructed using the PHYLOViZ animals with otitis externa, one animal with otitis 2.0 software [36]. Each genotype cluster was identified media and four animals with dermatitis. The strains by using the goeBURST algorithm version. were selected based on their susceptibility results against the four antifungals tested. Strains without an Statistical Analysis inhibition zone, a reduced inhibition zone in the disk diffusion method and a high MIC to ITZ were Statistical analyses were conducted by Minitab 17 included. Also, strains with wider inhibition zone Statistical software (Minitab). The results of both diameters were included. dilutions of each strain were compared using the The DNA was extracted directly from 5-day-old Student’s t-test. To compare the means of inhibition cultures grown in SGA according to the FastDNA Spin zone diameter between strains obtained from healthy kit protocol (MP Bio-medicals, Biolink, Barcelona, animals and strains obtained from animals with Spain). The ERG11 gene was amplified using the dermatitis/otitis the ANOVA test was used. primers pairs MALAERG1S/R, MALAERG2S/R, MALAERG3S/R, MALAERG4S/R described by Kano et al. [23]. Reaction mixtures contained 5.0 ll Results of template DNA, 5.0 ll of 10 9 PCR buffer, 0.2 mM of each dNTP, 1.5 mM MgCl2, 0.2 lM each primer Susceptibility Testing and 1.25 U of Taq polymerase in a final volume of 50 ll. The amplification process consisted of a pre- All the strains tested showed growth on the media denaturation step at 95 8C for 10 min, followed by 35 selected (MH-GM). Susceptibility results were deter- cycles of denaturation at 95 8C /15 s, annealing at 55 mined after 72 h of incubation at 358C, since it was the 8C /30 s and extension at 72 8C /1 min, and a final optimal time of growth for all the strains included. At extension of 5 min at 72 8C. Both strands of purified 48 h not all the strains had growth and no differences gene fragments were sequenced with BigDye Termi- were observed between inhibition zone diameters at nator v3.1. cycle sequencing kit (Applied Biosystems) 72 and 96 h. The results of both disk diffusion on an Applied Biosystems 3730 DNA Analyzer methods at 72 h are summarized in Table 2. No (Applied Biosystems). Sequence alignments were significant differences were observed between both carried out using Clustal X v2.0.12. software [31]. dilutions of each strain (P [ 0.05). The PROVEAN software [32] was used to study the In general, according to the manufacturer’s break- effect on the protein of the amino acid substitutions. points for yeasts, the four antifungals tested were Maximum likelihood analysis of the ERG11 active against most of the strains regardless of their sequences was conducted using MEGA 11 software origin. However, a few exceptions were observed. [33] with 1,000 bootstrap replicates. Clades that were With Rosco antifungal disks, five strains were classi- supported by bootstrap values of C 70% were fied as susceptible dose dependent (SDD) and five as regarded as strongly supported. resistant (R) to FLZ. One strain was classified as R to KTZ, one as SDD to ITZ, and four as SDD and one as Multilocus Sequences Typing R to AMB. One strain (MA165) was classified as R to ITZ, KTZ and AMB, and one strain (MA1386) was The strains selected for ERG11 gene sequencing were classified as R to FLZ and SDD to AMB. With Bio- also chosen for a multilocus sequence typing system Rad antifungal disks, four strains were classified as 123 Mycopathologia Table 2 Mean (± SD) inhibition diameters at 72 h and range to fluconazole (FLZ), itraconazole (ITZ), ketoconazole (KTZ) and amphotericin B (AMB) by both disk diffusion tests performed in strains from healthy animals, animals with otitis and animals with dermatitis HEALTHY (n = 9) OTITIS (n = 67) DERMATITIS (n = 13) TOTAL (n = 89) p value n X ± SD range n X ± SD range n X ± SD range n X ± SD range FLZ R 25 lg 4 32.25 ± 2.90 29.00–36.00 67 30.50 ± 10.31 7.50–56.50 12 30.38 ± 11.76 0- 42.00 83 30.57 ± 10.22 0–56.50 0.945 B 25 lg 8 35.19 ± 3.94 28.00–39.00 66 33.68 ± 12.82 5–73.00 12 35.75 ± 17.06 0- 68.00 86 34.11 ± 12.49 0–73.00 0.775 ITZ R 10 lg 8 36.44 ± 3.97 29.50–42.00 67 39.43 ± 6.80 19.0–58.00 12 39.88 ± 5.41 28.00–47.50 87 39.22 ± 6.43 19.00– 0.433 58.00 KTZ R 15 lg 8 48.94 ± 2.80 44.00–53.00 67 53.78 ± 7.88 20.00–73.00 12 53.71 ± 3.86 45.50–59.00 87 53.32 ± 7.22 20.00–73.00 0.199 B 50 lg 8 56.94 ± 3.45 53.00–63.00 67 62.79 ± 6.45 38.50–78.00 13 66.85 ± 5.12 57.50–77.00 88 62.86 ± 6.46 38.50–78.00 0.002 AMB R 10 lg 4 16.63 ± 0.75 15.50–17.00 67 17.33 ± 2.99 0–22.00 12 17.88 ± 1.55 15.00–20.00 83 17.37 ± 2.76 0–22.00 0.706 B 100 lg 8 19.88 ± 1.22 18.00–22.00 67 19.88 ± 2.73 13.00–27.50 13 19.46 ± 2.20 16.00–23.00 88 19.82 ± 2.54 13.00–27.50 0.530 R, RoscoÓ; B, Bio-RadÓ 123 Mycopathologia Table 3 Strains of Malassezia pachydermatis used, including of the disk diffusion method and itraconazole minimum animal host, health status, corresponding type of ERG11 inhibitory concentration (MIC) using E-test method at 72 h sequence, amino acid substitutions, mean inhibition diameters Strain Host Health ERG11 Amino acid substitutions Mean inhibition diameters (mm) MIC status DNA values at 72 h (lg/ Sequence ml) type FLZ FLZ ITZ KTZ KTZ E-test (R) (B) (R) (R) (B) ITZ CBS1879 Dog OE Ia – 28 36.5 37.5 49 54.5 0.002 CBS6535 Dog H Ia – 26 36.5 34 50 55 0.003 MA13 Dog H Ia – 36 36 40 53 63 0.003 MA52 Dog H Ia – 34 37 38.5 51.5 58.5 0.002 MA56 Dog H Ia – 35 35.5 38 47.5 59.5 0.003 a MA195 Dog OE I – 56.5 47 57 64 63.5 0.002 MA1595 Cow H Ia – 36 39 35 49.5 57 – CBS1884 Dog OE IIa E181Q 35 42.5 42 51.5 69.5 0.002 MA94 Horse H IIa E181Q 31.5 39 34.5 49 53 0.002 MA312 Cat OE XIa E181Q 36 38.5 37.5 48.5 62 0.003 MA587 Cat OM IIa E181Q 37.5 54.5 36.5 46.5 38.5 – MA579 Cat D XVIIIa I25V, E181Q 31.5 41 40 54 67 0.002 MA1716 Dog D XXIa A302T, G459D – 6.5 – – 59 0.125 MA10 Dog OE (C) VIIIa I25V, E181Q, T354I 31 28 58 47 49 – MA140 Cat H IVa I25V, E181Q, T354I 32.5 28 42 47 56 0.002 MA1401 Dog OE (R) XVIa V33I, E181Q, R202H 23 30 34 45 60 – MA475 Pig H Vb R84K, D166E, E181Q, D405N 29 30.5 29.5 44 53 0.002 MA7 Dog OE (R) VIb A17T, R84K, R175H, Q178R, E181Q 21 17.5 30 50 61 – MA8 Dog OE (C) VIIb A17T, R84K, R175H, Q178R, E181Q 40.5 41.5 45.5 59 60 – MA856 Dog OE (A) XIVb A17T, R84K, R175H, Q178R, E181Q 20 24 33 39.5 63.5 – MA944 Dog OE (C) XIVb A17T, R84K, R175H, Q178R, E181Q 15.5 27.5 29 45 59 – MA968 Dog OE (P) XIVb A17T, R84K, R175H, Q178R, E181Q 25 25 25 44.5 68.5 – MA1382 Dog OE (C) XVb A17T, R84K, R175H, Q178R, E181Q 16 18.5 41 54 59.5 0.002 MA107 Goat H IIIc E181Q, N212S, E290D, Y352F, – – – – – – H399R MA165 Dog OE (C) IXb A17T, R84K, F143S, R175H, Q178R, 21 33.5 19 20 66.5 0.002 E181Q MA356 Dog O XIIb A17T, R84K, Q178R, E181Q, Y352F, 23.5 27.5 39 51 53.5 0.002 H399R MA280 Dog OE (C) Xc I25S, W52L, R84K, L86F, E181Q, 24.5 9.5 46 62.5 64 0.002 N212S, E290D, Y352F, H399R MA361 Dog OE (C) XIIIc I25S, W52L, R84K, L86F, E181Q, 18 20 34 51 63 – N212S, E290D, Y352F, H399R MA1289 Dog D XIXc I25S, W52L, R84K, L86F, E181Q, 19 19 40 52.5 65 – N212S, E290D, Y352F, H399R MA1478 Dog OE (A) XVIIc I25S, W52L, R84K, L86F, E181Q, 8 5.5 32 59 58.5 – N212S, S226L, E290D, Y352F, H399R 123 Mycopathologia Table 3 continued Strain Host Health ERG11 Amino acid substitutions Mean inhibition diameters (mm) MIC status DNA values at 72 h (lg/ Sequence ml) type FLZ FLZ ITZ KTZ KTZ E-test (R) (B) (R) (R) (B) ITZ MA1429 Dog D XXc I25S, W52L, R84K, L86F, E181Q, 0 0 32.5 47 64.5 – N212S, E290D, A306S, Y352F, H399R, G461D In bold, deleterious mutations OE, otitis externa; OM, otitis media; A, acute; C, chronic; P, purulent; R, recurrent; H, healthy; D, dermatitis; FLZ, fluconazole; ITZ, itraconazole; KTZ, ketoconazole; R, RoscoÓ; B, Bio-RadÓ Subclades from the phylogenetic analysis of ERG11 gene: asubclade I; bsubclade III; csubclade II – Not tested SDD and seven as R to FLZ. These strains were for each of the 31 strains (Table 3). The ERG11 recovered from animals with chronic otitis externa or sequence included 1623 bp. The sequences of the dermatitis. All strains recovered from healthy animals strains shared 96.0–100% sequence similarity to M. were classified as susceptible according to the man- pachydermatis neotype strain CBS1879 and were ufacturer’s breakpoints for yeasts. Strain MA1429, clustered into 21 different genetic types. Representa- isolated from a dog with dermatitis, showed the tive nucleotide sequences of the different genetic types complete absence of inhibition zone to FLZ and strain determined in this study have been deposited at the MA165, isolated from a dog with chronic otitis GenBank database under accession numbers externa, showed no inhibition zone to AMB. ON814677- ON814697. No differences were observed in the inhibition zone Of the 31 strains of M. pachydermatis, seven of diameters between strains recovered from healthy them belonged to the genetic type I, including the animals and strains from animals with otitis or neotype strain CBS1879. All strains of genetic type I dermatitis except for the KTZ Bio-Rad antifungal were isolated from healthy animals except two disks (Table 2). Strains recovered from chronic otitis (MA195 and CBS1879). Genetic type II included externa and otitis media showed smaller inhibition three strains recovered from different animal species diameters to KTZ than strains from purulent and acute and genetic type XIV included three strains recovered otitis externa (P = 0.010). from dogs with otitis. The rest of genetic types were Most of the strains selected for the E-test method unique for each strain. using MH-GM as culture medium, showed MICs The predicted ERG11 amino acid sequences con- between 0.002 and 0.003 lg/ml to ITZ (Table 3). The sisted of 540 amino acids. Some silent polymorphisms only exception was strain MA1716 that showed a MIC were identified, and comparison of the deduced amino of 0.125 lg/ml (Fig. 1). This strain was also tested acid sequences revealed 14 unique amino acid using SGA supplemented with 1% Tween 80 and a sequence types. Strains belonging to genetic type I MIC of 8-12 lg/ml was obtained. The neotype strain had no amino acid substitution. Strains from genetic CBS 1879 showed a MIC of 0.002 lg/ml and 0.38 lg/ type II and XI showed the same amino acid sequence ml in MH-GM and SGA ? 1% Tween 80, with one amino acid substitution (E181Q). Strains respectively. from genetic type IV and VIII showed the same amino acid sequence with three substitutions (I25V, E181Q, ERG11 Sequence and T354I). Strains from genetic type X, XIII and XIX showed the same amino acid sequence with nine Amplification of the complete ERG11 gene and substitutions (I25S, W52L, R84K, L86F, E181Q, screening for amino acid substitutions was performed N212S, S226L, E290D, Y352F, H399R). Strains from 123 Mycopathologia Fig. 1 E-test assay of two isolates of M. pachydermatis against itraconazole. A) Reference strain CBS1879 in MH-GM (left) and SGA ? Tween 80 (right) culture media. B) Strain MA1716 in MH-GM (left) and SGA ? Tween 80 (right) culture media genetic type VI, VII, XIV and XV showed the same (94% bootstrap) grouped strains from healthy animals amino acid sequence with five substitutions (A17T, and animals with otitis or dermatitis. Subclade II (96% R84K, R175H, Q178R, and E181Q). The number of bootstrap) grouped mainly strains isolated from dogs amino acid substitutions of the rest of genetic types with otitis and dermatitis and one strain from a healthy varied between the strains and ranged from 2 to 11 goat. A third subclade, subclade III (81% bootstrap) substitutions per strain. A total of 23 amino acid grouped strains isolated from dogs with otitis and one substitutions were identified. Fourteen amino acid strain from a healthy pig. substitutions were considered as neutral: A17T, I25V, I25S, V33I, R84K, L86F, D166E, R175H, Q178R, Multilocus Sequence Typing E181Q, N212S, E290D, T354I, H399R, D405N. All these mutations were recovered from strains isolated Sequence types obtained for each gene are listed in from different animal species and health status. Nine Supplementary Table S2. Six different sequence types amino acid substitutions were considered as deleteri- of D1/D2 region were recovered, five from them were ous substitutions (W52L, F143S, R202H, S226L, previously described and one is new from this study A302T, A306S, Y352F, G459D, G461D) and were (VI). Eleven out of the 30 strains (36.66%) had recovered from strains isolated mainly from dogs with sequence type I which was the most abundant. Strains otitis or dermatitis. Mutations A302T, G459D were from healthy animals only showed sequence types I or detected in strain MA1716 with a MIC of 0.125 lg/ml II. and mutations W52L, A306S, Y352F, and G461D The ITS 5.8S rRNA was successfully amplified and were detected in strain MA1429 with no inhibition sequenced. Fifteen different sequence types were zone to FLZ. recovered from which four were new (XIV, XV, Maximum likelihood analysis of the ERG11 gene is XVI and XVII). Ten strains had one of the new ITS shown in Fig. 2. All M. pachydermatis sequences sequence types. The sequence type I was the majority grouped in a supported clade. The strains were (n = 5; 16.6%) and was recovered from strains from grouped in three different subclades. Subclade I dogs. 123 Mycopathologia Fig. 2 Molecular phylogenetic tree inferred from maximum likelihood analysis of ERG11 sequences of M. pachydermatis strains. Bootstrap values [ 70% in 1,000 replications are shown at the nodes. Sequences of Cryptococcus neoformans, Candida albicans, Candida tropicalis and Saccharomyces cerevisiae were selected as outgroup for the tree construction Ten different sequence types of b-tubulin were MIC to ITZ. A second cluster (II) included seven recovered from which two were new ones (X and XI). strains of seven different genotypes with five to 11 Sequence type VIII was the most abundant and was amino acid mutations. One strain within this cluster recovered from eight strains (26%) isolated from dogs (MA1429) showed no inhibition zone to FLZ. Also, a with otitis or dermatitis. third cluster (III) of seven strains of three different Regarding CHS2 gene, nine different sequence genotypes was obtained with five to six amino acid types were recovered in total, and all of them have substitutions. One strain was considered as R to KTZ been described previously. Sequence type I was and SDD to ITZ, and two strains were considered SDD majority and was obtained from a total of nine strains to FLZ. (30%) of dogs (n = 6), horse (n = 1), cat (n = 1) and cow (n = 1). The sequences obtained in this study have been Discussion deposited at the GenBank database under accession numbers ON787824 (D1D2), ON791562-ON791565 Antifungal susceptibility testing for M. pachydermatis (ITS), and ON814675- ON814676 (b-tubulin). must be interpreted with caution neither breakpoints When the four loci (D1D2, ITS, CHS2 and b- nor a reference method have been yet stablished for tubulin) were combined, a total of 25 genotypes were this yeast species. This means that any classification of identified. Among all genotypes, 22 were only found the strains into susceptible, intermediate, and resistant once. Two genotypes were shared by two strains, and remains speculative. However, according to the man- one genotype was shared by five strains. When the ufacturer’s breakpoints available for yeasts the four presence of amino acid substitutions was considered, antifungals tested in our study were active against the graphing algorithm analysis revealed small clus- most of the strains selected. Only one strain in our ters of ERG11-mutated isolates. As shown in Fig. 3, a study showed an increased MIC to ITZ. As reported by cluster of 12 strains of 10 different genotypes (I) with a some authors [8, 16, 37, 38], M. pachydermatis is few amino acid substitutions (0–3) was observed. All highly susceptible to KTZ and ITZ which is consistent the strains within this group showed wide inhibition with the results observed in our study. Since now, only diameters to azoles except for one strain (MA1716) two M. pachydermatis isolates have demonstrated to with a small inhibition diameter to FLZ and a higher be resistant to both ITZ and KTZ [22, 23]. In our study, 123 Mycopathologia Fig. 3 Minimum spanning trees using the goeBURST algorithm of 31 Malassezia pachydermatis strains based on analysis of the four loci used for genotyping. Each circle represents a unique genotype. The diameter of each circle corresponds to the number of isolates sharing the same genotype and the numbers within circles represent strain number. The color of the circle is related to the presence of amino acid substitutions in ERG11: black for strains with 6 to 11 amino acid substitutions, dark gray for strains with 4 to 5 amino acid substitutions, light gray for strains with 1 to 3 amino acid substitutions and white for strains without any amino acid substitution only one strain showed no inhibition zone to FLZ. higher for isolates from animals with otitis/dermatitis Fluconazole consistently returns significantly higher [21, 38, 44]. Also, in our study, differences were MICs when compared to other antifungals tested observed in the inhibition diameters between strains among studies with M. pachydermatis [17, 39, 40]. It from chronic otitis externa and otitis media, and has been demonstrated that M. pachydermatis isolates strains from purulent and acute otitis externa. The can become resistant during treatment with FLZ by mean inhibition diameters to KTZ were higher in the inducing resistance in vitro to this antifungal [41]. strains from acute and purulent otitis externa. A study Thus, the clinical utility of this azole in dogs and cats is by Chiavassa et al. [20] compared the MIC values of questionable [8, 42]. Bernardo et al. [43] found a M. two different antifungal agents between isolates of M. pachydermatis isolate resistant to AMB. However, pachydermatis from chronic otitis and acute otitis due to the potential toxicity of AMB its use in externa. The results showed that the isolates from veterinary medicine is limited to serious progressive chronic otitis externa had MIC values higher than the or disseminated systemic mycoses [8]. isolates from acute otitis externa. It was hypothesized In our study, all the strains with a reduced inhibition that those increased MICs were a result to the exposure zone or no inhibition zone were recovered from of isolates to antifungal agents. In our study, strains animals with otitis and/or dermatitis. These results MA1716, with an increased MIC to ITZ, and agree with the ones obtained in three different studies MA1429, with complete absence of inhibition zone where the MICs of various antifungals agents were to FLZ, were recovered from dogs with previous 123 Mycopathologia antifungal treatments with ITZ and miconazole, pachydermatis associated with azole resistance respectively. [51, 52]. One of the mutations present in our study, In the E-test method, only one strain (MA1716) G461D, recovered from a strain with no inhibition showed higher MICs in MH-GM and SGA ? 1% zone to FLZ, was also observed in a clinical isolate Tween 80 and thus, could be considered to have a with in vitro resistance to ravuconazole [51]. reduced susceptibility against ITZ. An increase of The other mutation, G459D, was reported in 64-fold of the MIC was observed when the media used miconazole tolerant clones of the CBS1879, selected was SGA with lipid supplementation. This increase in by serial passage on miconazole supplemented media MIC values when the culture medium has a lipid [52]. This mutation was also present in a supplementation was also observed in M. pachyder- strain (MA1716) with a higher MIC to ITZ and with matis CBS1879 (Fig. 1). This medium was also used a reduced inhibition zone to FLZ in our study. This to assess susceptibility to ITZ and KTZ by the E-test strain also showed a mutation at point A302T. This technique [23]. Different testing variables are known point mutation but with a different amino acid change to have an impact on in vitro determinations as lipid (A302V) has been described by Kano et al. [23] in an supplementation enhancing the yeast growth [8, 45]. isolate with proven in vitro resistance to ITZ and KTZ. Thus, the isolates could appear to be susceptible or The different amino acid change could explain the resistant only by modifying test conditions. Due to results obtained in susceptibility testing of our strain. this, it is essential to establish a set of standardized Another point mutation described by Kano et al. [23] criteria for in vitro susceptibility testing of Malassezia and associated with azole resistance, M138V, was not spp. [14, 46]. detected in our study. Mutations of the ERG11 gene could reduce the Point mutations of the ERG11 gene leading to susceptibility of fungi to azoles [26–28, 47–49]. In our amino acid substitutions that induce antifungal resis- study, a high variability of this gene was observed. tance have also been observed in other Malassezia Sequence differences in the ERG11 gene among species [47, 53]. In M. furfur a point mutation Y67F strains ranged from 0.1 to 4.0%, which is greater than (130 in M. pachydermatis) is associated with flucona- those described with CHS2 (1.9 to 3.4%) or b-tubulin zole resistance [53]. In M. globosa three-point muta- (0.3 to 3.4%) [34]. Indeed, only 6 of the strains tions Y127F, A169S and K176N (synonymous with displayed the sequence of the M. pachydermatis 130, 172 and 179 in M. pachydermatis) are associated neotype strain CBS1879. This strain has been previ- with azole resistance [47]. However, none of these ously used as comparison to detect possible amino point mutations were observed in the strains of this acid mutations [23]. The analysis of the ERG11 gene study. grouped M. pachydermatis strains in three different Some of the amino acid substitutions observed in clades. This aggrupation correlates quite well with the our study had been described in other fungal species groups defined by multilocus sequencing. associated to a reduced antifungal susceptibility. In Even though many polymorphisms were observed, Candida albicans, point mutations at positions 54, not all lead into amino acid substitutions. Our data 145, 226 and 307 (synonymous with 52, 143, 202, and clearly show that in M. pachydermatis point mutations 226 in M. pachydermatis, respectively) were associ- leading to amino acid changes are a frequent event in ated with a reduced azole susceptibility [54–57]. ERG11 gene. A total of 23 different amino acid These point mutations are deleterious in M. pachy- substitutions were recovered from which nine were dermatis and were observed in our study in some deleterious substitutions. The analysis of multilocus strains. Also, the presence of two amino acid substi- groups using ERG11 amino acid substitutions as tutions in combination G307S ? G450E in C. albi- discriminant parameter revealed some closely related cans was reported to increase by 16-fold the MICs to genotypes carrying more amino acid substitutions. FLZ [56]. This combination is synonymous with the This suggested a correlation between certain geno- combination A306S ? G461D in M. pachydermatis types of M. pachydermatis and in vitro susceptibility observed in one strain of our study (MA1429) with a results, in agreement with other authors [38, 50]. disk diffusion with no inhibition zone to FLZ. Of the amino acid substitutions identified, only two The relationship between ERG11 amino acid (G459D, G461D) have been reported previously in M. mutations and drug resistance has been reported in 123 Mycopathologia many pathogenic fungi such as Aspergillus fumigatus, showed a reduced susceptibility to some antifungals, Candida spp. and Cryptococcus spp. [58]. This is not further studies would be necessary to completely the case of M. pachydermatis. Since now, few strains understand the role of these mutations in the suscep- of M. pachydermatis have demonstrated to be resistant tibility against antifungal agents. to azoles and only four amino acid mutations in ERG11 have been described [23, 51, 52]. None of Acknowledgements The authors thanks Laura Ordeix for a sample from a dog with recurrent dermatitis kindly provided for these substitutions have been confirmed to cause azole our work. The authors thank Carolina Gómez from the resistance using in vitro experiments and it’s not Veterinary Mycology Group of Universitat Autònoma de known which part of the protein is involved probably Barcelona (UAB) for valuable technical assistance. because there is no experimentally determined three- dimensional structure of the ERG11 protein available. Author Contributions GC, MRB, and FJC contributed to the study conception and design. Material preparation, data Based on the three-dimensional structure of C. albi- collection and analysis were performed by LD, GC, MRB, and cans ERG11, the mutations G448D and G450D in C. FJ C. The first draft of the manuscript was written by LD, and all albicans (synonymous with G459D and G461D in M. authors commented and contributed to the preparation of the pachydermatis) are located near the heme-binding site final manuscript. All authors read and approved the final manuscript. and near the end of helix I of the protein, respectively [59, 60]. Also, mutation F145L (synonymous with Funding Open Access Funding provided by Universitat F143S in M. pachydermatis) is located near the Autonoma de Barcelona. Financial support came from Servei substrate channel [60]. Based on the three-dimen- Veterinari de Bacteriologia i Micologia from the UAB. sional structure of C. neoformans CYP51, residues Declarations A317 and F158 in C. neoformans (synonymous with A306S and F143S in M. pachydermatis, respectively) Conflict of interest The authors declare that they have no are in the heme binding site and mutation A313 in C. conflict of interest. neoformans (A302T in M. pachydermatis) is part of Open Access This article is licensed under a Creative Com- one of the active site cavities [61]. However, the mons Attribution 4.0 International License, which permits use, possibility that other deleterious substitutions, which sharing, adaptation, distribution and reproduction in any med- are located outside of the active side, may also ium or format, as long as you give appropriate credit to the contribute to fungal resistance via other structural original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The changes cannot be ruled out. images or other third party material in this article are included in The presence or absence of amino acid substitutions the article’s Creative Commons licence, unless indicated may not be the solely cause of antifungal resistance otherwise in a credit line to the material. If material is not [25, 55]. Other mechanisms of resistance against included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds azoles have been suggested in M. pachydermatis as the permitted use, you will need to obtain permission directly efflux pumps [14, 25]. Other possibility for these from the copyright holder. To view a copy of this licence, visit reduced susceptibilities is a chromosomal rearrange- http://creativecommons.org/licenses/by/4.0/. ment that leads to an overexpression of the ERG11 and ERG4 genes. Kim et al. [29] found a tandemly quadruplicated region in chromosome 4 of two M. pachydermatis isolates with high in vitro MICs of References KTZ. Thus, an overexpression of this region was observed that could be responsible of the higher MIC. 1. Xu J, Saunders CW, Hu P, et al. 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In vitro evaluation of Malassezia pachy- self-archiving of the accepted manuscript version of this article dermatis susceptibility to azole compounds using E-test and is solely governed by the terms of such publishing agreement CLSI microdilution methods. Med Mycol. and applicable law. 2012;50:795–801. 123