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Simple Summary Fungi in underground environments usually occur as spores suspended in the air, because these ecosystems are characterized by harsh living conditions, e.g., constant low temperature, and a limited or complete lack of organic matter and light. Fungi may be potentially hazardous to mammals. Therefore, the goal of our research was the first report of aeromycota in the Brestovská Cave. The mycological quality of the air in the cave does not pose a risk to healthy tourists, but some fungal species may be potentially dangerous to people with a weakened immune system. A total of 18 cold-adapted fungal species were isolated during the study, and all of them were present inside the cave, but only seven were found outdoors. The cosmopolitan species dominated in the external air samples. Our research has allowed for the first detection of fungal species in the air inside the underground sites as well as species that have never been detected in any component of the underground ecosystems. There are many possible reasons explaining the detection of those species, but global warming is the most likely. Abstract Most underground ecosystems are heterotrophic, fungi in these objects are dispersed in the air in the form of spores, and they may be potentially hazardous to mammals. Research in underground sites has focused on mesophilic airborne fungi and only a few concerned cold-adapted species. Therefore, the goal of our research was the first report of psychrophilic and psychrotolerant aeromycota in the Brestovská Cave using culture-based techniques with genetic and phenotypic identification. Plates with PDA medium containing sampled biological material were incubated at 8 ± 0.5 °C. The density of mycobiota inside the cave ranged from 37.4 to 71 CFU 1 m−3 of air and 63.3 CFU 1 m−3 of air outside the cave. Thus, the level of fungal spores did not exceed the standards for the mycological quality of the air. A total of 18 species were isolated during the study, and some species may be potentially dangerous to people with weakened immune system. All fungal species were present inside the cave and only seven of them were outside. Cladosporium cladosporioides dominated in the external air samples and Mortierella parvispora was cultured most frequently from internal air samples. To our knowledge, this is the first discovery of the fungal species such as Coniothyrium pyrinum, Cystobasidium laryngis, Filobasidium wieringae, Leucosporidium drummii, M. parvispora, Mrakia blollopis, Nakazawaea holstii, and Vishniacozyma victoriae in the air inside the underground sites. Moreover, C. pyrinum, C. laryngis, L. drummii, M. blollopis, and N. holstii have never been detected in any component of the underground ecosystems. There are possible reasons explaining the detection of those species, but global warming is the most likely.

New fluconazole-loaded, 6-anhydro-α-L-galacto-β-D-galactan hydrogels incorporated with nanohydroxyapatite were prepared and their physicochemical features (XRD, X-ray Diffraction, SEM-EDS, Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy, ATR-FTIR, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy), fluconazole release profiles and enzymatic degradation were determined. Antifungal activity of pure fluconazole was tested using Candida species (C. albicans, C. tropicalis, C. glabarata), Cryptococcus species (C. neoformans, C. gatti) and Rhodotorula species (R. mucilaginosa, R. rubra) reference strains and clinical isolates. Standard microdilution method was applied, and fluconazole concentrations of 2–250 µg/mL were tested. Moreover, biofilm production ability of tested isolates was tested on the polystyrene surface at 28 and 37 ± 0.5 °C and measured after crystal violet staining. Strains with the highest biofilm production ability were chosen for further analysis. Confocal microscopy photographs were taken after live/dead staining of fungal suspensions incubated with tested hydrogels (with and without fluconazole). Performed analyses confirmed that polymeric hydrogels are excellent drug carriers and, when fluconazole-loaded, they may be applied as the prevention of chronic wounds fungal infection.

Epicoccum nigrum Link is a cosmopolitan species, and it has been described as both an in vitro and in vivo antagonist of many fungal pathogens of plants. However, there are no clear reports about the interactions between E. nigrum and various plant species, and about the effects of culture filtrates produced by this fungus on plants. Therefore, we assessed the interactions between E. nigrum and different plant species, such as sugar beet (Beta vulgaris L. ssp. vulgaris), spring wheat (Triticum aestivum L.), red clover (Trifolium pratense L.), and winter oilseed rape (Brassica napus L.). Additionally, we evaluated the effect of E. nigrum culture filtrates on garden cress (Lepidium sativum L.). Our study showed that the E. nigrum strains varied in terms of the color of excreted culture filtrates and showed different interactions with garden cress. Overall, fungal strains only affected adversely the sprout length in a significant way and, partially, the growth of the tested plant. In addition, we confirmed the suitability of the garden cress as a test plant in in vitro toxicological tests. Most strains of E. nigrum (61.1%) secreted enzymes expected to participate mainly in the later stages of the infection (amylases and proteases) and not those expected to operate in the early phases of host penetration (cellulases and pectinases) that were secreted by 33.3% of fungal strains. The group of pectinolytic enzymes represented the catalysts with the highest activity. Host specialization tests showed that E. nigrum was mainly re-isolated from the plant surface and the number of infected seedlings as well as the disease index depended on a studied plant species, with sugar beet and red clover being most sensitive to infection. In turn, the lowest value of the disease index caused by E. nigrum strains was recorded for spring wheat and winter oilseed rape. Overall, statistically significant differences in the growth of plant seedlings during the host specialization test were noted only for sugar beet and red clover seedlings. The seedlings of plants in the control group (without fungal inoculum) exhibited an increased length compared to those treated with E. nigrum inoculum. Our studies also showed that E. nigrum is probably a facultative saprotroph of plants and it may winter on red clover, which is presumably its main reservoirs, among the species considered.

Epicoccum nigrum Link is well known for producing biologically-active substances with activities against prokaryotic and eukaryotic cells. The major goal of this study was to assess E. nigrum as a potential in vitro agent against selected species of dermatophytes. The effects of the types of media used in this study on the interactions between the microscopic fungi were also examined. Epicoccum nigrum’s bioactive metabolites exhibited a strong growth inhibitory effect against the dermatophytes, suggesting its potential as a biocontrol agent. Notably, the strength of these interactions was dependent on the type of the medium. These secondary metabolites are not toxic against the higher eukaryotic organisms, which was further demonstrated by using the Galleria mellonella model.

Quaternary ammonium salts (QAS), as the surface active compounds, are widely used in medicine and industry. Their common application is responsible for the development of microbial resistance to QAS. To overcome, this issue novel surfactants, including gemini-type ones, were developed. These unique compounds are built of two hydrophilic and two hydrophobic parts. The double-head double-tail type of structure enhances their physicochemical properties (like surface activity) and biological activity and makes them a potential candidate for new drugs and disinfectants. Antimicrobial activity is mainly attributed to the biocidal action towards bacteria and fungi in their planktonic and biofilm forms, but the mode of action of gemini QAS is not yet fully understood. Moreover, gemini surfactants are of particular interest towards their application as gene carriers. Cationic charge of gemini QAS and their ability to form liposomes facilitate DNA compaction and transfection of the target cells. Multifunctional nature of gemini QAS is the reason of the long-standing research on mainly their structure-activity relationship.

The studies were aimed to contribute to the elucidation of the relationships between structure of the double-headed cationic surfactants – N,N-bis[3,3′-(dimethylamine)propyl]alkylamide dihydrochlorides and N,N-bis[3,3′-(trimethylammonio)propyl]alkylamide dibromides (alkyl: n-C9H19, n-C11H23, n-C13H27, n-C15H31), which are of particular interest, as they contain a labile amide group in the molecule and their antifungal activity. Therefore, the minimal inhibitory and fungicidal concentrations (MIC and MFC) of dicephalic surfactants against various fungi were tested using standardized methods. Most of the tested fungi were resistant to the Cn(TAPABr)2 compounds. The strongest growth inhibition was caused by Cn(DAPACl)2 series, which MICs ranged from 6.5 to 16 μM. The influence of dicephalic surfactants on Candida albicans biofilm and adhesion to the various surfaces was investigated with crystal violet staining or colony counting. The reduction of fungal adhesion was also observed, especially to the glass surface. One of the compounds (C14(DAPACl)2) caused DNA leakage from C. albicans cells. Further studies showed the impact of dicephalic surfactants on ROS production, accumulation of lipid droplets and filament formation. This study points to the possibility of application of dicephalic surfactants as the surface-coating agents to prevent biofilm formation or as disinfectants. The results give an insight into the possible mechanism of action of newly synthesized dicephalic surfactants in yeast cells.

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.

Bats can contribute to an increase of aeromycota in underground ecosystems and might be a vector/reservoir of microorganisms, however, there is no information about the number and species composition of fungi around hibernating bats. One of the most common species in Europe with direct human contact is the greater mouse-eared bat (Myotis myotis). The goal of our research was the first report of the airborne fungi present in the close vicinity of hibernating M. myotis in the Nietoperek bat reserve (Western Poland) by the use of culture-based techniques and genetic and phenotypic identifications. Aerobiological investigations of mycobiota under hibernating bats were performed on two culture media (PDA and YPG) and at two incubation temperatures (7 and 24 ± 0.5 °C). Overall, we detected 32 fungal species from three phyla (Ascomycota, Basidiomycota, and Zygomycota) and 12 genera. The application of YPG medium and the higher incubation temperature showed higher numbers of isolated fungal species and CFU. Penicillium spp. were dominant in the study, with spores found outside the underground hibernation site from 51.9% to 86.3% and from 56.7% to 100% inside the bat reserve. Penicillium chrysogenum was the most frequently isolated species, then Absidia glauca, Aspergillus fumigatus, A. tubingensis, Mortierella polycephala, Naganishia diffluens, and Rhodotorula mucilaginosa. Temperature, relative humidity, and the abundance of bats correlated positively with the concentration of airborne fungal propagules, between fungal species diversity, and the concentration of aeromycota, but the number of fungal species did not positively correlate with the number of bats. The air in the underground site was more contaminated by fungi than the air outside, however, the concentration of aeromycota does not pose a threat for human health. Nevertheless, hibernating bats contribute to an increase in the aeromycota and as a vector/reservoir of microscopic fungi, including those that may cause allergies and infections in mammals, and should be monitored.

We investigated the influence of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)- N,N,N-trimethyl ammonium chloride) on yeast cells of the parental strain and the IM-resistant mutant (EO25 IMR) growth. The phenotype of this mutant was pleiotropic. The IMR mutant exhibited resistance to ethanol, osmotic shock and oxidative stress, as well as increased sensitivity to UV. Moreover, it was noted that mutant EO25 appears to have an increased resistance to clotrimazole, ketoconazole, fluconazole, nystatin and cycloheximide. It also tolerated growth in the presence of crystal violet, DTT and metals (selenium, tin, arsenic). It was shown that the presence of IM decreased ergosterol level in mutant plasma membrane and increased its unsaturation. These results indicate changes in the cell lipid composition. Western blot analysis showed the induction of Pma1 level by IM. RT-PCR revealed an increased PMA1 expression after IM treatment.

Aims The studies were aimed to contribute to the elucidation of the relationships between structure of the double-headed cationic surfactants - N,N-bis[3,3’-(dimethylamine)- propyl]alkylamide dihydrochlorides and N,N-bis[3,3’-(trimethylammonio)propyl]alkylamide dibromides (alkyl: n-C9H19, n-C11H23, n-C13H27, n-C15H31) and their antibacterial and biofilm preventing activity. Methods and Results The minimal inhibitory and bactericidal concentrations (MIC and MBC) of dicephalic surfactants against Staphylococcus epidermidis and Pseudomonas aeruginosa were tested using standard methods. P. aeruginosa was resistant to studied compounds but MBC values against S. epidermidis reached 0.48 – 0.01 mmol l−1. The influence of dicephalic surfactants on bacterial biofilm and adhesion to the various surfaces was investigated with crystal violet staining or colony counting. The reduction of bacterial adhesion was observed, especially in the case of glass and stainless steel. The condensation of the DNA was shown in the ethidium bromide intercalation assay. Conclusions Dicephalic surfactants exhibited antibacterial activity against S. epidermidis. The activity of studied compounds depended on the hydrocarbon chain length and the counterion. Surfactants deposited on different materials reduced S. epidermidis adhesion, dependently on the surfactant structure and the substratum. Dicephalic surfactants showed the ability of DNA compaction. Significance and Impact of the Study This study points the possibility of application of dicephalic surfactants as the surface-coating agents to prevent biofilm formation. These compounds efficiently condensed DNA and are potential candidates for further studies towards the transfection. This article is protected by copyright. All rights reserved.

The antibacterial activity of alanine-derived gemini quaternary ammonium salts (chlorides and bromides) with various spacer and alkyl chain lengths was investigated. The studied compounds exhibited a strong bactericidal effect, especially bromides with 10 and 12 carbon alkyl chains and 3 carbon spacer groups (TMPAL-10 Br and TMPAL-12 Br), with a short contact time. Both salts dislodged biofilms of Pseudomonas aeruginosa and Staphylococcus epidermidis, and were lethal to adherent cells of S. epidermidis. Bromide with 2 carbon spacer groups and 12 carbon alkyl chains (TMEAL-12 Br) effectively reduced microbial adhesion by coating polystyrene and silicone surfaces. The results obtained suggest that, after further studies, gemini QAS might be considered as antimicrobial agents in medicine or industry.