Worldwide, the number of infections caused by biofilm-forming fungal pathogens is very high. In human medicine, there is an increasing proportion of immunocompromised patients with prolonged hospitalization, and patients with long-term inserted drains, cannulas, catheters, tubes, or other artificial devices, that exhibit a predisposition for colonization by biofilm-forming yeasts. A high percentage of mortality is due to candidemia caused by medically important Candida species. Species of major clinical significance include C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, and C. auris. The association of these pathogenic species in the biofilm structure is a serious therapeutic problem. Candida cells growing in the form of a biofilm are able to resist persistent therapy thanks to a combination of their protective mechanisms and their ability to disseminate to other parts of the body, thus representing a threat from the perspective of a permanent source of infection. The elucidation of the key mechanisms of biofilm formation is essential to progress in the understanding and treatment of invasive Candida infections.

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.

Fungal skin diseases are well-recognized diseases with public health implications. The study provides a comprehensive overview and aims to determine the rate of positive fungal cultures to identify the most common fungal species in guinea pigs and rabbits and to determine the rate of asymptomatic carriers in healthy pet animals. This knowledge is essential for understanding disease transmission dynamics and epidemiological situation problems. A total of 167 animals (64 rabbits and 103 guinea pigs) were investigated in this study. The fungi of the genus Penicillium, Rhizopus, Mucor, Cladosporium, and Aspergillus were the most common in the examined animals, and they were isolated from 162 (97%) of the animals enrolled. No fungal growth was observed in 5 animals. In 15 cases (8.98%), we found pathogenic zoonotic dermatophytes (Trichophyton mentagrophytes), which caused several health problems in two humans in contact with affected animals. This study presents the prevalence of fungal flora in pet guinea pigs and rabbits in Slovakia.

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.

Malassezia (M.) pachydermatis, is often associated with secondary infection of the skin and external auditory canal in dogs and cats. The treatment of Malassezia infections is based on the local application of antifungals often combined with antiseptics. Due to increased resistance of yeast to commonly used antimycotics, especially in biofilm-forming cells, the use of natural substances, e.g. plant essential oils, appears as a new promised option. In this study, the efficacy of selected plant essential oils (EO) - oregano, rosemary, bergamot, clove, cinnamon, and thyme - in combination with chlorhexidine on both planktonic and biofilm-forming cells of M. pachydermatis, was investigated. The checkerboard test was used to determine the effect of chlorhexidine combined with individual EOs. According to the FICI (fractional inhibitory concentration index) in planktonic cells, most combinations showed additive effect, except for thyme and rosemary EO, where a synergistic effect was found (33.3 % and 16.7 % respectively). In the biofilm-forming cells, a synergistic effect was noted in chlorhexidine combined with bergamot EO, recorded in 6 isolates (33.3 %), and with thyme and oregano EO, detected in 3 isolates (16.7 %). A significant decrease (p ˂ 0.05) was found in FIC (fractional inhibitory concentration) compared to MIC (minimum inhibitory concentration), for both planktonic and biofilm-forming cells. Based on the obtained results, we can conclude that the combination of chlorhexidine with EOs achieved better efficiency than when using each agent alone and made it possible to reduce the concentration of both, and a sufficient antifungal and antibiofilm effect was achieved in M. pachydermatis strains.

Infections caused by Malassezia (M.) pachydermatis in dogs are mostly treated with azole antifungals. Excessive use of these drugs is usually associated with an increased incidence of resistant isolates, which can be prevented by combining commonly used antifungals with natural bioactive compounds. The present study aimed at testing the effectiveness of a combination of selected azole derivatives showing low antifungal activity against M. pachydermatis isolates, with plant essential oil components displaying the highest efficacy. Among the four azole antifungals tested (itraconazole, posaconazole, clotrimazole, and miconazole), clotrimazole (a mean MIC of 7.62 μg/mL at 72 h and 7.24 μg/mL at 96 h) and miconazole (a mean MIC of 1.71 μg/mL at 72 h and 2.33 μg/mL at 96 h) exhibited the lowest antifungal efficacy. Out of the four plant essential oil components tested (eugenol, terpinene-4-ol, geraniol, and limonene), eugenol (an average MIC of 378.57 μg/mL at 72 h and 1180 μg/mL at 92 h) showed the highest antifungal activity. The checkerboard method was used to assess the interaction of these agents. The fractional inhibitory concentration index (FICI) values for the combination of clotrimazole with eugenol reached 1.43 at 72 h and 0.70 at 96 h and for the combination of miconazole with eugenol, 1.30 at 72 h and 0.45 at 96 h. A higher effect of the combinations was recorded at 96 h, when the combination of clotrimazole with eugenol showed an additive effect in 66.67% of the isolates, and the combination of miconazole and eugenol brought a synergistic effect in 57.14% of the isolates. The obtained results indicate that eugenol is a suitable agent for enhancing the efficacy of poor azoles against M. pachydermatis.