Extracellular phospholipase production by Malassezia pachydermatis strains and its inhibition by selected antimycotics and plant essential oil components.

Extracellular phospholipase (EPL) plays an important role in the pathogenesis of the yeast Malassezia pachydermatis. Currently, the attention of researchers is focused on studying the virulence factors involved in this process and searching solutions to reduce their activity. One of the options is the use of natural remedies as anti-virulence agents. This study is aimed at investigating the production of extracellular phospholipase in M. pachydermatis strains (18 samples) and followed by the time-dependent inhibitory effect of selected azole antifungals (itraconazole, posaconazole and voriconazole) and plant essential oil components (terpinen-4-ol, thymol, carvacrol, eugenol and geraniol), evaluated by Egg Yolk Agar plate method. Almost all strains (17 isolates, (94.4%) were found to be intense EPL producers. A significant, time-dependent inhibition of EPL was noted after 1-, 3- and 6-h exposure of Malassezia cells to itraconazole (26.4%, 47.2% and 50.9%, respectively) compared to exposure to posaconazole (26.4%, 28.3% and 28.3%, respectively) and voriconazole (18.8%, 20.8% and 35.8%, respectively). After one-hour exposure to plant essential oil components, the best inhibitory effect was recorded for eugenol (62.3%), followed by terpinen-4-ol and thymol (56.6%), geraniol (41.5%) and carvacrol (26.4%). A 3-h exposure revealed that thymol retained the best inhibitory effect (88.7%) on EPL production, followed by carvacrol (73.6%), eugenol (56.6%), terpinen-4-ol (52.8%) and geraniol (49.1%). After 6-h exposure, no growth of M. pachydermatis strains exposed to carvacrol was observed, and the inhibitory efficiency for the other tested essential oil (EO) components achieved 88.7%. The obtained results indicate the promising efficacy of plant essential oils components in the inhibition of virulence factors such as EPL production.

Prevalence and genetic diversity of azole-resistant Malassezia pachydermatis isolates from canine otitis and dermatitis: A 2-year study.

Despite previous reports on the emergence of Malassezia pachydermatis strains with decreased susceptibility to azoles, there is limited information on the actual prevalence and genetic diversity of azole-resistant isolates of this yeast species. We assessed the prevalence of azole resistance in M. pachydermatis isolates from cases of dog otitis or skin disease attended in a veterinary teaching hospital during a 2-year period and analyzed the ERG11 (encoding a lanosterol 14-α demethylase, the primary target of azoles) and whole genome sequence diversity of a group of isolates that displayed reduced azole susceptibility. Susceptibility testing of 89 M. pachydermatis isolates from 54 clinical episodes (1-6 isolates/episode) revealed low minimum inhibitory concentrations (MICs) to most azoles and other antifungals, but 11 isolates from six different episodes (i.e., 12.4% of isolates and 11.1% of episodes) had decreased susceptibility to multiple azoles (fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, and/or voriconazole). ERG11 sequencing of these 11 azole-resistant isolates identified eight DNA sequence profiles, most of which contained amino acid substitutions also found in some azole-susceptible isolates. Analysis of whole genome sequencing (WGS) results revealed that the azole-resistant isolates from the same episode of otitis, or even different episodes affecting the same animal, were more genetically related to each other than to isolates from other dogs. In conclusion, our results confirmed the remarkable ERG11 sequence variability in M. pachydermatis isolates of animal origin observed in previous studies and demonstrated the value of WGS for disentangling the epidemiology of this yeast species.

We analyzed the prevalence and diversity of azole-resistant Malassezia pachydermatis isolates in a veterinary hospital. A low prevalence of multi-azole resistance (c.10% of isolates and cases) was found. Whole genome and ERG11 sequencing of resistant isolates revealed remarkable genetic diversity.

Antifungal activity of azoles, allylamines, and 8-hidroxiquinolines, alone and in combination, against Malassezia pachydermatis in vitro and in vivo.

Malassezia pachydermatis is often reported as the causative agent of dermatitis in dogs. This study aims to evaluate the in vitro and in vivo antifungal activity of azoles and terbinafine (TRB), alone and in combination with the 8-hydroxyquinoline derivatives (8-HQs) clioquinol (CQL), 8-hydroxyquinoline-5-(n-4-chlorophenyl)sulfonamide (PH151), and 8-hydroxyquinoline-5-(n-4-methoxyphenyl)sulfonamide (PH153), against 16 M. pachydermatis isolates. Susceptibility to the drugs was evaluated by in vitro broth microdilution and time-kill assays. The Toll-deficient Drosophila melanogaster fly model was used to assess the efficacy of drugs in vivo. In vitro tests showed that ketoconazole (KTZ) was the most active drug, followed by TRB and CQL. The combinations itraconazole (ITZ)+CQL and ITZ+PH151 resulted in the highest percentages of synergism and none of the combinations resulted in antagonism. TRB showed the highest survival rates after seven days of treatment of the flies, followed by CQL and ITZ, whereas the evaluation of fungal burden of dead flies showed a greater fungicidal effect of azoles when compared to the other drugs. Here we showed for the first time that CQL is effective against M. pachydermatis and potentially interesting for the treatment of malasseziosis.

In Vitro Antimycotic Activity and Structural Damage against Canine Malassezia pachydermatis Strains Caused by Mexican Stingless Bee Propolis.

This work describes the antimycotic activity of propolis from the stingless bees Scaptotrigona mexicana and Tetragonisca angustula, collected from two Mexican regions (Veracruz and Chiapas, respectively), against three clinical isolates and the reference strain ATCC 14522 of Malassezia pachydermatis, the causative agent of canine otitis. The chemical components of the ethanolic extracts of propolis were determined by gas chromatography coupled with mass spectrometry (GC-MS), and sesquiterpenes were the predominant compounds. The antimycotic activity was evaluated by plate microdilution. The induced changes in the yeasts were evaluated by fluorescence microscopy and staining with calcofluor white and propidium iodide. The minimum inhibitory concentration (MIC) was 7.11 mg/mL, and the minimum fungicidal concentration was 21.33 mg/mL for both extracts. The EPPs of Scaptotrigona mexicana and Tetragonisca angustula caused substantial damage to yeast morphology, where the propidium iodide staining of the yeasts treated with both EEPs revealed the penetration of this marker, which indicates the destruction of the cell wall and plasma membrane of the fungi. This result suggests that these types of propolis could be used as alternative treatments for canine external otitis. To the best of our knowledge, this seems to be the first scientific report that has demonstrated structural damage in Malassezia pachydermatis by Mexican stingless bee propolis.

A rabbit model of ear otitis established using the Malassezia pachydermatis strain C23 from dogs.

Background and Aim: Fungal infections are a growing problem for both humans and animals due to the emergence of pathogenic strains resistant to modern antifungal treatments. To evaluate the efficacy of new antifungal drugs, it is essential to develop animal models that demonstrate typical responses to both the infection (pathogenesis and clinical course) and to the treatment, including adverse effects. In this study, we established a rabbit otitis model by infection of an aggressive multidrug-resistant strain from dogs, Malassezia pachydermatis C23, with no need for concomitant immunosuppression. Materials and Methods: Twenty healthy adult male gray giant rabbits (1 year old, 5.5 kg) were inoculated once with M. pachydermatis C23 at 108 colony-forming units/mL. We observed the clinical signs of the disease and collected ear smears and blood samples every 5 days. Results: The infection progressed rapidly and exhibited characteristic clinical signs without spontaneous recovery for at least 1 month. In fact, substantial deterioration was observed as evidenced by blood parameters. Conclusion: This rabbit otitis model established using an aggressive drug-resistant fungus strain without immunosuppression could prove valuable for testing novel antifungal agents.

Fungal diversity and drug susceptibility of the oral mycobiome of domestic dogs.

The purpose of this study was to characterize the variety and diversity of the oral mycobiome of domestic dogs and to identify the commensal and potentially pathogenic fungi present. Two hundred fifty-one buccal swabs from domestic dogs were obtained and struck onto a chromogenic fungal growth medium that distinguishes between fungal species based on colony color and morphology. After isolating and harvesting single colonies, genomic DNA was extracted from pure cultures. PCR was used to amplify a fungal-specific variable rDNA region of the genome, which was then sent for sequencing. Sequencing results were input into the NCBI BLAST database to identify individual components of the oral mycobiome of tested dogs. Of the 251 dogs swabbed, 73 had cultivable fungi present and 10 dogs had multiple fungal species isolated. Although the dogs did not show signs of oral infections at the time, we did find fungal species that cause pathogenicity in animals and humans. Among fungal isolates, Malassezia pachydermatis and species from the genus Candida were predominant. Following fungal isolate identification, antifungal drug susceptibility tests were performed on each isolate toward the medically important antifungal drugs including fluconazole, ketoconazole, and terbinafine. Drug susceptibility test results indicated that a large number of isolates had high MIC values for all three drugs. Exploring the oral mycobiome of dogs, as well as the corresponding drug susceptibility profiles, can have important implications for canine dental hygiene, health, and medical treatment. Identifying the microorganisms within the canine mouth can illustrate a common pathway for fungal pathogens of One Health concern to spread from our canine companions to humans.

In vitro effects of omeprazole in combination with antifungal compounds against Malassezia pachydermatis.

BACKGROUND: Fungal and yeast infections, including those caused by Malassezia spp., are becoming increasingly difficult to treat, likely due to the occurrence of drug resistance. OBJECTIVES: This study aimed to evaluate the antifungal effects of omeprazole (OME), a proton pump inhibitor, against antifungal-resistant Malassezia pachydermatis and to investigate the potential synergistic effects between OME and other antifungal compounds. METHODS: In total, 15 samples of M. pachydermatis isolated from the skin of dogs were tested. The susceptibility of M. pachydermatis to itraconazole, ketoconazole, miconazole, terbinafine and OME was assessed using a modified broth microdilution (BM) method. The in vitro efficacy of OME alone and in combination with other antifungal compounds was evaluated for all isolates using the BM chequerboard method. The data obtained were analysed using the fractional inhibitory concentration index (FICI). RESULTS: The minimum inhibitory concentration (MIC) values of antifungal compounds and OME against quality control strain (M. pachydermatis CBS1879) were lower than the MIC90 values of same drugs against clinically collected strains. There was no significant difference in MIC values between drugs alone and combination. According to the analysis by the FICI method, no interaction was observed with OME and antifungal compounds. CONCLUSIONS: Most M. pachydermatis strains were resistant to azole antifungal compounds. OME exerted antifungal effects against Malassezia spp. and even showed good effects on antifungal-resistant strains. No synergistic effects were observed between the antifungal compounds and OME.

Efficacy of manuka honey with conventional antifungals on Malassezia pachydermatis.

Yeast infections such as otitis externa and seborrheic dermatitis in dogs and cats are frequently associated with Malassezia pachydermatis secondary infection. It is part of the normal cutaneous microflora of most warm-blooded vertebrates, however, under certain conditions, it can become a causative agent of infection that needs to be treated pharmacologically. Azole derivatives are the drugs of the first choice. An interesting trend in developing resistance is the use of natural substances, which include manuka honey with confirmed antimicrobial properties. The main intention of this research was to evaluate the mutual effect of manuka honey in combination with four conventional azole antifungals - clotrimazole, fluconazole, itraconazole, and miconazole - on 14 Malassezia pachydermatis isolates obtained from dogs and 1 reference strain. A slightly modified M27-A3 method (CLSI 2008) and the checkerboard test (Nikolic et al. 2017) were used for this purpose. Our results show an additive effect of all 4 antifungals with manuka honey concurrent use. Based on the determined values of fractional inhibitory concentration index (FICI - 0.74±0.03 when manuka honey combined with clotrimazole, 0.96±0.08 with fluconazole, 1.0±0 with miconazole and 1.16±0.26 with itraconazole), it was found in all cases that the effect of substances used is more pronounced in mutual combination than when used separately.

Malassezia pachydermatis from brown bear: A comprehensive analysis reveals novel genotypes and distribution of all detected variants in domestic and wild animals.

Malassezia pachydermatis (phylum Basidiomycota, class Malasseziomycetes) is a zoophilic opportunistic pathogen with recognized potential for invasive infections in humans. Although this pathogenic yeast is widespread in nature, it has been primarily studied in domestic animals, so available data on its genotypes in the wild are limited. In this study, 80 yeast isolates recovered from 42 brown bears (Ursus arctos) were identified as M. pachydermatis by a culture-based approach. MALDI-TOF mass spectrometry (MS) was used to endorse conventional identification. The majority of samples exhibited a high score fluctuation, with 42.5% of isolates generating the best scores in the range confident only for genus identification. However, the use of young biomass significantly improved the identification of M. pachydermatis at the species confidence level (98.8%). Importantly, the same MALDI-TOF MS efficiency would be achieved regardless of colony age if the cut-off value was lowered to ≥1.7. Genotyping of LSU, ITS1, CHS2, and ß-tubulin markers identified four distinct genotypes in M. pachydermatis isolates. The most prevalent among them was the genotype previously found in dogs, indicating its transmission potential and adaptation to distantly related hosts. The other three genotypes are described for the first time in this study. However, only one of the genotypes consisted of all four loci with bear-specific sequences, indicating the formation of a strain specifically adapted to brown bears. Finally, we evaluated the specificity of the spectral profiles of the detected genotypes. MALDI-TOF MS exhibited great potential to detect subtle differences between all M. pachydermatis isolates and revealed distinct spectral profiles of bear-specific genotypes.

Standardization of Galleria mellonella as an Infection Model for Malassezia furfur and Malassezia pachydermatis.

Galleria mellonella larva has been widely exploited as an infection model for bacteria and fungi. Our laboratory uses this insect as a model for fungal infection caused by the genus Malassezia, in particular, systemic infections caused by Malassezia furfur and Malassezia pachydermatis, which are poorly understood. Here, we describe the G. mellonella larva inoculation process with M. furfur and M. pachydermatis and the posterior assessment of the establishment and dissemination of the infection in the larvae. This assessment was done through the evaluation of larval survival, melanization, fungal burden, hemocytes populations, and histological changes. This methodology allows for the identification of virulence patterns between Malassezia species and the impact of inoculum concentration and temperature.

In Vitro Activity of Farnesol against Malassezia pachydermatis Isolates from Otitis Externa Cases in Dogs.

Chronic otitis externa of dogs is a significant problem due to the prevalence and complexity of the treatment of such animals. There is evidence that in 60-80% of cases of infectious diseases microorganisms located in the biofilm phenotype play the main role. Microorganisms in the biofilm phenotype have a number of advantages, the most significant of which is considered to be increased resistance to various external factors. Among them, a special place is occupied by resistance to antibiotics. In recent decades, research has been conducted at an increasing scale on the role of biofilm infections in various pathologies in veterinary medicine. The etiology and therapy of dog otitis externa caused by Malassezia pachydermatis biofilm has not been fully studied. This is why we consider relevant the scientific and practical aspects of research on the etiology and therapy of dog otitis externa from the position of biofilm infection. In this work, it has been statistically proven that there is a relationship between the optical density of Malassezia pachydermatis biofilms and their sensitivity to drugs, and this relationship is statistically significant. In addition, we have demonstrated that Farnesol has a good antibiofilm effect at a concentration of more 1.6 µM/mL (24% OD decrease of biofilm), and its highest antibiofilm effect (71-55%-more than a half) was observed at a concentration of 200-12.5 µM/mL.

Standardization of in vitro dual-species biofilms of Staphylococcus pseudintermedius and Malassezia pachydermatis: a strategy to establish an ex vivo biofilm model.

Ex vivo experiments have been performed aiming at mimicking in vivo environments. The main aim of this research was to standardize in vitro dual-species biofilm formation by Staphylococcus pseudintermedius and Malassezia pachydermatis as a strategy to establish an ex vivo biofilm model. Initially, the in vitro formation of biofilms in co-culture was established, using YPD medium, inoculum turbidity of 0.5 on the McFarland scale and maturation periods of 96 h for M. pachydermatis and 48 h for S. pseudintermedius. Subsequently, biofilms were formed on porcine skin using the same conditions, under which a greater number of cells/ml was observed in in vitro dual-species than in in vitro mono-species biofilms. Furthermore, ex vivo biofilm images demonstrated the formation of a highly structured biofilm with the presence of cocci and yeasts surrounded by the matrix. Thus, these conditions optimized the growth of both microorganisms within biofilms in vitro and ex vivo.

Azole and terbinafine susceptibility testing of Malassezia pachydermatis in Japan.

Canine Malassezia dermatitis and otitis externa are generally treated by antifungal drugs. However, multi-drug-resistant strains of Malassezia pachydermatis have been reported worldwide. Given the presence of these multi-drug-resistant strains, it is unclear which antifungal agent is the most effective for canine Malassezia dermatitis and canine otitis. In this study, we attempted to determine the most effective drug against azole-resistant M. pachydermatis. Susceptibility to azoles and terbinafine (TBF) was assessed using a modified broth microdilution method. In all tested isolates, the minimum inhibitory concentration at 90% of organisms (MIC90) were 16 to >32 µg/mL for the azoles, and 2 µg/mL for TBF. All of the strains that showed low susceptibility to both itraconazole and miconazole were also relatively susceptible to TBF.

Is Vitamin D3 a Worthy Supplement Protecting against Secondary Infections in Dogs with Atopic Dermatitis?

Canine atopic dermatitis (CAD) is a common, chronic, inflammatory skin disease in dogs worldwide. This disease often predisposes for secondary organisms overgrowth and skin infections with pathogens, such as Staphylococcus pseudintermedius and Malassezia pachydermatis. Unfortunately, the causes of this disease in both humans and animals are not fully understood; therefore, the only possible option is a lifelong, symptomatic treatment. The management of CAD is mainly based on limiting contact with allergens and antipruritic therapy, most often with glucocorticoids and antihistamines. A serious problem in this situation is the fact, that long-term administration of glucocorticoids leads to side effects like polyuria, alopecia, increased susceptibility to infection, muscle atrophy, and many others. For this reason, great emphasis is placed on the development of replacement and supportive therapies. It is a well-documented fact that reduced concentrations of serum vitamin D3 contribute to the severity of atopic dermatitis symptoms in humans. Moreover, unlike the most commonly used therapeutic methods, of which the main goal is to ameliorate inflammation and pruritus, namely the symptoms of AD, vitamin D3 supplementation affects some underlying factors of this disease. Therefore, in this review, we summarize the current state of knowledge regarding the role of vitamin D3 in CAD, its protective effect against secondary bacterial and fungal infections, and the potential of its supplementation in dogs.

ERG11 Gene Variability and Azole Susceptibility in Malassezia pachydermatis.

Malassezia pachydermatis is part of the normal skin microbiota of various animal species but under certain circumstances becomes an opportunistic pathogen producing otitis and dermatitis. Commonly these Malassezia diseases are effectively treated using azoles. However, some cases of treatment failure have been reported. Alterations in the ERG11 gene have been associated with in vitro azole resistance in M. pachydermatis. In the present study, in vitro antifungal susceptibility of 89 different strains of M. pachydermatis isolated from different animal species and health status was studied. The susceptibility to fluconazole (FLZ), itraconazole (ITZ), ketoconazole and amphotericin B was tested by a disk diffusion method and 17 strains were also subjected to an ITZ E-test. Mueller-Hinton supplemented with 2% glucose and methylene blue was used as culture medium in both susceptibility assays. Multilocus sequence typing was performed in 30 selected strains using D1D2, ITS, CHS2 and ß-tubulin genes. Also, ERG11 gene was sequenced. The four antifungals tested were highly effective against most of the strains. Only two strains showed no inhibition zone to antifungals and a strain showed an increased MIC to ITZ. The study of the ERG11 sequences revealed a high diversity of DNA sequences and a total of 23 amino acid substitutions, from which only two have been previously described. Also, three deleterious substitutions (A302T, G459D and G461D) previously associated with azole resistance in this yeast were recovered. A correlation between certain genotypes and ERG11 mutations was observed. Some of the ERG11 mutations recovered were correlated with a reduced susceptibility to azoles.

In Vitro Biofilm Formation by Malassezia pachydermatis Isolates and Its Susceptibility to Azole Antifungals.

The yeast Malassezia pachydermatis, an opportunistic pathogen that inhabits the skin of various domestic and wild animals, is capable of producing a biofilm that plays an important role in antifungal resistance. The aim of this research study was to find the intensity of biofilm production by M. pachydermatis strains isolated from the ear canal of healthy dogs, and to determine the susceptibility of planktonic, adhered and biofilm-forming cells to three azole antifungals-itraco-nazole, voriconazole and posaconazole-that are most commonly used to treat Malassezia infections. Out of 52 isolates, 43 M. pachydermatis strains (82.7%) were biofilm producers with varying levels of intensity. For planktonic cells, the minimum inhibitory concentration (MIC) range was 0.125-2 µg/mL for itraconazole, 0.03-1 µg/mL for voriconazole and 0.03-0.25 µg/mL for posaconazole. Only two isolates (4.7%) were resistant to itraconazole, one strain (2.3%) to voriconazole and none to posaconazole. For adhered cells and the mature biofilm, the following MIC ranges were found: 0.25-16 µg/mL and 4-16 µg/mL for itraconazole, 0.125-8 µg/mL and 0.25-26 µg/mL for voriconazole, and 0.03-4 µg/mL and 0.25-16 µg/mL for posaconazole, respectively. The least resistance for adhered cells was observed for posaconazole (55.8%), followed by voriconazole (62.8%) and itraconazole (88.4%). The mature biofilm of M. pachydermatis showed 100% resistance to itraconazole, 95.3% to posaconazole and 83.7% to voriconazole. The results of this study show that higher concentrations of commonly used antifungal agents are needed to control infections caused by biofilm-forming strains of M. pachydermatis.

Medium-chain fatty acid esters are effective even in azole-resistant Malassezia pachydermatis.

BACKGROUND: Malassezia (M.) pachydermatis as a frequent reason for dermatological consultation in dogs and cats was recently shown to be lipid-dependent, too. Lipolytic activity is a prerequisite for activating antimicrobial effectivity of fatty acid esters. OBJECTIVES: It was therefore of interest whether it is possible to induce this mechanism in M. pachydermatis and to identify possible differences between minimal and strong lipid-dependent strains. METHODS: In an agar dilution test, the minimal inhibitory concentrations of six fatty acid esters were determined for seventeen M. pachydermatis strains. GC analysis of parent compounds and liberated fatty acids was used to quantify ester cleavage. RESULTS: Hydrolysis was observed in all test strains in a homogenous manner but was dependent on the chemical structure. Lowest MICs (500 ppm after 14 days of incubation) were obtained applying glyceryl monocaprylate and 3-hydroxylpropyl caprylate, while the corresponding esters of undecylenic acid showed nearly twice the value. As shown by GC analysis with the reference strains CBS 1879 and CBS 1892 and 3-hydroxypropyl caprylate, hydrolysis and caprylic acid formation starts immediately and was dependent on yeast density. Furthermore, nine azole-resistant strains isolated from dogs with treatment failures showed MIC values comparable to the other strains and no resistance to monohydric fatty acid esters. CONCLUSIONS: Medium-chain fatty acid esters may represent a new therapeutic option for veterinary use even in azole-resistant strains. The in vivo verification in M. pachydermatis-associated dermatitis in dogs and cats will be the next step for the successful development of new therapeutics.

Rapid Molecular Detection of Antifungal-Resistant Strains of Malassezia pachydermatis.

Azole resistance in Malassezia pachydermatis has been reported in isolates from canine skin worldwide. Decreased susceptibility of M. pachydermatis to azoles has been hypothesized to potentially result from mutations in the ERG11 gene, which encodes lanosterol 14α-demethylase. To sequence the mutation hotspots of ERG11 in the isolates, we prepared primers (MPERG11hot2S and MPERG11hot2R) based on the conserved sequences of M. pachydermatis ERG11. DNA samples from azole-resistant and -susceptible strains were amplified by PCR using the primer pair. PCR amplicons were sequenced and analyzed for single-nucleotide polymorphisms (SNPs) in the target gene. Seven of the tested azole-resistant strains (16 strains) harbored ERG11 SNPs at nucleotide 904 (G→A) or 905 (C→T), resulting in the replacement of Ala 302 with Thr or Val (Ala302Thr or Val). None of the tested azole-susceptible strains had a mutation at either of those residues. Our PCR method detected SNPs at the nucleotide-905 (C→T) hotspot mutation site in M. pachydermatis ERG11. Moreover, we discovered an additional hot spot site at nucleotide 904 (G→A).

Homeopathy treatment of Malassezia Pachydermatis in vitro

A common clinical occurrence in dogs is otitis externa caused by excessive growth of yeasts Malassezia pachydermatis, which can become chronic after wrong treatments, in which microbial resistance can occur. Homeopathic remedies can be considered a successful alternative, selecting the medicine through the similitude principle. Herein, 50 µL of a 1:1000 dilution of Malassezia pachydermatis suspension at 0.5 McFarland scale was used to seed the yeast into Sabouraud dextrose agar plates using a Drigalski spreader to proceed with colony unit counting. Before being seeded, the yeast suspensions were treated with 1% of different homeopathic treatments previously selected from a pilot study, which means Sulphur 6cH, Dolichos pruriens 6cH, and Kali carbonicum 6cH, being water, and succussed water used as controls. For comparison, a set of Sabouraud dextrose agar plates containing 1% Tween 80 was seeded in parallel. The treatments were made blind and evaluated in triplicate. Contaminated cultures were withdrawn. The number of colonies per plate was assessed, and smears were made from the cultures to classify yeast growth according to cytomorphology on ImageJ®software. The preliminary results show no significant effect of all tested medicines compared to the controls. High data variability was also observed, mainly in those cultures whose medium was prepared with Twin 80. In conclusion, at this point of the study, no evidence of the effects of the studied medicines on Malassezia pachydermatis growth in vitro could be identified. The analysis of cytomorphology is still in course.

Antifungal susceptibility of Malassezia pachydermatis isolated from the external auditive conduct from dogs, in central Chile.

Background: External otitis is common in dogs, and one of the main agents involved is Malassezia pachydermatis, a yeast belonging to the otic microbiota. Empirical treatment can fail; therefore, it is essential to know the antifungal susceptibility profile to prescribe appropriate treatment, a fact scarcely reported in Chile. Aim: This study aimed to determine the antifungal sensitivity of M. pachydermatis isolated from the external auditory canal of dogs in central Chile. Methods: Ear swabs from 30 dogs (15 healthy and 15 with external otitis) were used. Samples were subjected to cytology and fungal culture. The antifungal susceptibility was determined according to clinical and laboratory standards institute (CLSI) document M44A-2 using the disk diffusion test from amphotericin B, Caspofungin, fluconazole, nystatin, clotrimazole, and voriconazole were used. Results: The prevalence of M. pachydermatis was 66.7% from 8 healthy dogs and 12 with otitis. While fungal culture was not associated with the patient's clinical condition (p = 0.24), the yeast count by cytology was significantly higher in dogs with otitis (p = 0.003). The strains were sensitive to all antifungals except for Caspofungin, where 55% of the strains were resistant. Conclusion: Malassezia pachydermatis is isolated more frequently in dogs with otitis, and the ear cytological examination is useful to differentiate colonized dogs versus dogs with otitis. In addition, most antifungals in vitro are active against this yeast, except Caspofungin, an antifungal used in human medicine. This situation should be further monitored in epidemiological programs to evaluate the possible impact on Chilean public health.