This study is the first mycological evaluation of bat guano and the air around it in Harmanecká and Driny Caves in Slovakia. These caves are the most important underground localities of bats in Slovakia. Samples were collected in July 2014 and cultivated for fungi. Harmanecká Cave had seven species of filamentous fungi and one yeast-like fungus isolated from bat guano, compared to six species of filamentous fungi in guano from Driny Cave. Air samples from Harmanecká Cave had twelve species of fungi, compared to nine species from Driny Cave. Fungal density was higher in guano from Driny Cave (4720.1 CFU/g guano) than from Harmanecká Cave (3498.3 CFU/g). The pattern was reversed with fungi from the air. Fungal density in air from Harmanecká Cave (211.3 CFU/m 3 ) was higher than that from Driny Cave (175.7 CFU/m 3 ). Penicillium granulatum was the most frequently isolated fungal species, except in the guano of Driny Cave, where Mucor hiemalis was most common. Bat guano is a very good substrate for the development and survival of fungi in the caves, and it can be a reservoir of fungi harmful for bats. However, air samples from both caves contained more species of fungi than the bat guano, because the majority of fungi are transferred to underground ecosystems with air bioaerosols from the external environment.

Harmanecká Cave is located in the Harmanec Valley to the northwest of Banská Bystrica city, in the southern part of the Great Fatra Mountains, Slovakia. This cave is the most important underground locality of bat occurrence in Slovakia (population of 1000 to 1500 individuals). The study aimed at mycological evaluation of the air, the water, and the rock surface of Harmanecká Cave in Slovakia. The samples were taken on 24 July 2014. To examine the air, the Air Ideal 3P sampler was used. Microbiological evaluation of the rock surface was performed using swab sampling and the water by using the serial dilution technique. The authors observed a relationship between air temperature and the concentration of fungi. The concentration of airborne fungi increased with the increase in the air temperature and decreased with distance from the entrance to the cave. The density of airborne fungi isolated from the outdoor air samples was 810.5 colony-forming units (CFU) per 1 m3 of air and from 27.4 to 128.5 CFU for the indoor air samples. From the rock surface inside the cave, 45.0 to 106.6 CFU per 1 cm2 were isolated, whereas from the water, 29.9 CFU per 1 ml were isolated. Seven species of filamentous fungi were isolated from the external air samples and 12 species of filamentous fungi and 3 species of yeast-like fungi from the internal air samples. From the surface of the rocks inside the cave, 5 species of filamentous fungi and 1 species of yeast-like fungi were cultured, whereas from the water samples, 6 species of filamentous fungi were cultured. Cladosporium spp. were the fungi most frequently isolated from the external air; from the internal air, Penicillium urticae was most frequently isolated; from the rock surface, it was Gliocladium roseum; and from the water, it was P. chrysogenum. The species found in the cave can be pathogenic for humans and animals, especially for immunocompromised persons, and they can also cause biodegradation of the rocks. However, the concentration of airborne fungi inside the cave did not exceed official limits and norms stated as dangerous for the health of tourists.

This paper is the first aero-mycological report from Demänovská Ice Cave. Fungal spores were sampled from the internal and external air of the cave in June, 2014, using the impact method with a microbiological air sampler. Airborne fungi cultured on PDA medium were identified using a combination of classical phenotypic and molecular methods. Altogether, the presence of 18 different fungal spores, belonging to 3 phyla, 9 orders and 14 genera, was detected in the air of the cave. All of them were isolated from the indoor samples, and only 9 were obtained from the outdoor samples. Overall, airborne fungal spores belonging to the genus Cladosporium dominated in this study. However, the spores of Trametes hirsuta were most commonly found in the indoor air samples of the cave and the spores of C. herbarum in the outdoor air samples. On the other hand, the spores of Alternaria abundans, Arthrinium kogelbergense, Cryptococcus curvatus, Discosia sp., Fomes fomentarius, Microdochium seminicola and T. hirsuta were discovered for the first time in the air of natural and artificial underground sites. The external air of the cave contains more culturable airborne fungal spores (755 colony-forming units (CFU) per 1 m3 of air) than the internal air (from 47 to 273 CFU in 1 m3), and these levels of airborne spore concentration do not pose a threat to the health of tourists. Probably, the specific microclimate in the cave, including the constant presence of ice caps and low temperature, as well as the location and surrounding environment, contributes to the unique species composition of aeromycota and their spores in the cave. Thus, aero-mycological monitoring of underground sites seems to be very important for their ecosystems, and it may help reduce the risk of fungal infections in humans and other mammals that may arise in particular due to climate change.

Keratinolytic and keratinophilic fungi, such as dermatophytes, are frequently a cause of infections in humans and animals. Underground ecosystems are inhabited by various animals and are of interest for tourists. Therefore, the main goal of our research was the first evaluation of sediment and soil samples taken inside and outside the Harmanecká Cave in Slovakia for the occurrence of keratinolytic and keratinophilic fungi. Tests with Vanbreuseghema bait, as well as phenotyping and molecular methods, showed that all of the sampling sites contained ten isolates, all of the same species of keratinophilic fungi, belonging to the Microsporum cookei clade and with close affinities to Paraphyton cookei (Ajello) Y. Gräser, Dukik & de Hoog. Our research showed that, dependent on the medium, its mycelium varied in color and showed different growth rates. It also produced metabolites alkalizing DTM (dermatophyte test medium) medium. It dissolved keratin in in vitro hair perforation tests and was able to utilize most substrates in the API® 20C AUX, except for MDG (α-methyl-D-glucoside). In addition, the vegetative structures of mycelium were viable after storage at temperatures from −72 to −5 °C for 56 days, and actively grew after 28 days at a temperature range from 15 to 37 °C, with 25 °C being optimal. It showed weak, but active, growth at 5 and 10 °C after 56 days. We can assume that due to the low temperature in the caves, this fungus will not be able to actively grow rapidly on keratin substrates, but the contact with mammals, along with other favorable factors, might lead to an infection.

This paper is a speleomycological report from Driny Cave in the Lesser Carpathian Mountains, Slovakia. The samples were collected in July 2014 from one location outside and five locations inside the cave. To examine the air, the Air Ideal 3P sampler was used. Samples from the rock surfaces were collected using sterile swabs wetted in physiological saline (0.85% NaCl). The density of filamentous fungi isolated from the air inside and outside the cave ranged from 89.6 to 1284.7 colony-forming units per 1 m 3 of air and from 38.3 to 588.5 CFU per m 2 of the rock surface. Six species of filamentous fungi were isolated from the external air samples, and eleven species of filamentous fungi and three species of yeast-like fungi from the internal air samples. Fungi belonging to the Cladosporium genus were the most frequently isolated species from the internal and the external air. Six species of filamentous fungi and two species of yeast-like fungi were isolated from the surface of the rocks inside the cave and only two species from the samples collected outside the cave. Among the fungi isolated from the rock surfaces most frequently were Penicillium chrysogenum, P. granulatum, and Trichoderma harzianum. The concentration of airborne fungi inside the cave did not exceed official limits and norms stated as safe for health of tourists. However, the species found here can cause degradation of rock surfaces.