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.

Biofilm Formation in Medically Important Candida Species.

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.

Resistance of Primary Photosynthesis to Photoinhibition in Antarctic Lichen Xanthoria elegans: Photoprotective Mechanisms Activated during a Short Period of High Light Stress.

The Antarctic lichen, Xanthoria elegans, in its hydrated state has several physiological mechanisms to cope with high light effects on the photosynthetic processes of its photobionts. We aim to investigate the changes in primary photochemical processes of photosystem II in response to a short-term photoinhibitory treatment. Several chlorophyll a fluorescence techniques: (1) slow Kautsky kinetics supplemented with quenching mechanism analysis; (2) light response curves of photosynthetic electron transport (ETR); and (3) response curves of non-photochemical quenching (NPQ) were used in order to evaluate the phenomenon of photoinhibition of photosynthesis and its consequent recovery. Our findings suggest that X. elegans copes well with short-term high light (HL) stress due to effective photoprotective mechanisms that are activated during the photoinhibitory treatment. The investigations of quenching mechanisms revealed that photoinhibitory quenching (qIt) was a major non-photochemical quenching in HL-treated X. elegans; qIt relaxed rapidly and returned to pre-photoinhibition levels after a 120 min recovery. We conclude that the Antarctic lichen species X. elegans exhibits a high degree of photoinhibition resistance and effective non-photochemical quenching mechanisms. This photoprotective mechanism may help it survive even repeated periods of high light during the early austral summer season, when lichens are moist and physiologically active.

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.

Pedobacter fastidiosus sp. nov., isolated from glacial habitats of maritime Antarctica.

Strains P8930T and 478 were isolated from Antarctic glaciers located on James Ross Island and King George Island, respectively. They comprised Gram-stain-negative short rod-shaped cells forming pink pigmented colonies and exhibited identical 16S rRNA gene sequences and highly similar MALDI TOF mass spectra, and hence were assigned as representatives of the same species. Phylogenetic analysis based on 16S rRNA gene sequences assigned both isolates to the genus Pedobacter and showed Pedobacter frigidisoli and Pedobacter terrae to be their closest phylogenetic neighbours, with 97.4 and 97.2 % 16S rRNA gene sequence similarities, respectively. These low similarity values were below the threshold similarity value of 98.7%, confirming the delineation of a new bacterial species. Further genomic characterization included whole-genome sequencing accompanied by average nucleotide identity (ANI) and digital DNA-DNA hybridization calculations, and characterization of the genome features. The ANI values between P8930T and P. frigidisoli RP-3-11T and P. terrae DSM 17933T were 79.7 and 77.6 %, respectively, and the value between P. frigidisoli RP-3-11T and P. terrae DSM 17933T was 77.7 %, clearly demonstrating the phylogenetic distance and the novelty of strain P8930T. Further characterization included analysis of cellular fatty acids, quinones and polar lipids, and comprehensive biotyping. All the obtained results proved the separation of strains P8930T and 478 from the other validly named Pedobacter species, and confirmed that they represent a new species for which the name Pedobacter fastidiosus sp. nov. is proposed. The type strain is P8930T (=CCM 8938T=LMG 32098T).

Comparison of antifungal activity of selected essential oils against Fusarium graminearum in vitro.

INTRODUCTION: Fusaria are microscopic filamentous fungi which are spread in soil, in various organic substrates, and include more than 80 phytopathogenic species which are predominantly hosted by cereals, fruits and vegetables. Many of these species, under certain conditions, are capable of synthesizing secondary metabolites, mycotoxins. At present, various substances are used for their elimination and one of the solutions appears to be essential oils. In the presented study, the antifungal activity of essential oils was researched in vitro. MATERIAL AND METHODS: In this study, two standard fungal isolates Fusarium graminearum CCM F-683 and Fusarium graminearum CCM 8244 (Brno, Czech Republic) were used. The antifungal effect of 6 tested essential oils (Syzygium aromaticum, Origanum vulgare, Thymus vulgaris, Hyssopus officinalis , Ocimum basilicum, Myristica fragrans) was determined using the broth microdilution method, which allows reading of the MIC (minimum inhibitory concentration). According to the results obtained, the growth inhibition of Fusarium graminearum was determined by assay for the inhibition of radial growth of the mycelium. RESULTS: The inhibitory effects of thymus, oregano, basil, myristica, hyssop and syzygium essential oil (EO) on mycelial growth of Fusarium graminearum CCM F-683 and CCM 8244 were investigated. The best antifungal activity against the both strains of Fusarium graminearum (37.4%; 40.7%) was demonstrated by Origanum vulgare EO at the concentration 100 µg/mL. Among the four tested oils, three (Syzygium aromaticum, Thymus vulgaris, Origanum vulgare) achieved the best inhibitory effect (100%) at concentrations 500 µg/mL and 1000 µg/mL. CONCLUSIONS: In the protection of plants against pathogenic fungi, essential oils appear to be a suitable substitute for synthetic chemicals.

Antibiofilm activity of selected plant essential oils from the Lamiaceae family against Candida albicans clinical isolates.

INTRODUCTION: The virulence of Candida albicans is conditioned by several virulence factors, one of which is the formation of biofilm which reduces the sensitivity of the yeast to conventional antimycotics. This study determines the antifungal and antibiofilm activity of five essential oils (EOs) of the Lamiaceae family: Salvia officinalis, Thymus vulgaris, Rosmarinus officinalis, Origanum vulgare, and Hyssopus officinalis. MATERIAL AND METHODS: In the preliminary research, the antifungal effect of eachof the EOs was tested in the concentration range of 200-0.4 mg/mL on planktonic Candida albicans (C. albicans) cells. A total of 13 C. albicans clinical isolates and one reference strain were evaluated on biofilm formation. RESULTS: Nine isolates (69.2%) showed weak biofilm production and four strains (30.8%) were detected as moderate biofilm producers. The EOs of Thymus vulgaris and Origanum vulgare were seen as effective antifungal agents on planktonic cells with the MIC 0.4 mg/mL. The highest average MIC values were recorded in Salvia officinalis EO (24.0 and 14.8 mg/mL). All isolates were used to determine EOs efficacy on the inhibition of adherence phase and biofilm formation. The biofilm production of C. albicans after exposition by EOs was quantitatively examined by crystal violet dye. CONCLUSIONS: The most effective for adherence phase and biofilm formation were EOs of Origanum vulgare (0.1 mg/mL and 0.3 mg/mL) and Thymus vulgaris (0.1 mg/mL and 0.4 mg/mL). The obtained results show that EOs of Thymus vulgaris and Origanum vulgare are potential agents for antifungal treatment or prophylaxis by reducing the resistance of pathogen.

Photosynthetic performance of Antarctic lichen Dermatocarpon polyphyllizum when affected by desiccation and low temperatures.

Lichens are symbiotic organisms that are well adapted to desiccation/rehydration cycles. Over the last decades, the physiological background of their photosynthetic response-specifically activation of the protective mechanism during desiccation-has been studied at the level of photosystem II of the lichen photobiont by means of several biophysical methods. In our study, the effects of desiccation and low temperatures on chlorophyll fluorescence and spectral reflectance parameters were investigated in Antarctic chlorolichen Dermatocarpon polyphyllizum. Lichen thalli were collected from James Ross Island, Antarctica, and following transfer to a laboratory, samples were fully hydrated and exposed to desiccation at temperatures of 18, 10, and 4 °C. During the desiccation process, the relative water content (RWC) was measured gravimetrically and photosynthetic parameters related to the fast transient of chlorophyll fluorescence (OJIP) were measured repeatedly. Similarly, the change in spectral reflectance parameters (e.g., NDVI, PRI, G, NPCI) was monitored during thallus dehydration. The dehydration-response curves showed a decrease in a majority of the OJIP-derived parameters (e.g., maximum quantum yield of photosystem II photochemistry: FV/FM, and performance index: PI in D. polyphyllizum, which were more apparent at RWCs below 20%. The activation of protective mechanisms in severely dehydrated thalli was documented by increased thermal dissipation (DI0/RC) and its quantum yield (Phi_D0). Low temperature accelerated these processes. An analysis of the OJIP shape reveals the presence of K-bands (300 µs), and L-bands (80 µs), which can be attributed to dehydration-induced stress. Spectral reflectance indices decreased in a majority of cases with an RWC decrease and were positively related to the OJIP-derived parameters: FV/FM (capacity of photosynthetic processes in PSII), Phi_E0 (effectiveness of electron transport), and PI_tot (total performance index), which was more apparent in NDVI. A negative relation was found for NPCI. These indices could be used in follow-up ecophysiological photosynthetic studies of lichens that are undergoing rehydration/dehydration cycles.

Diclofenac as an environmental threat: Impact on the photosynthetic processes of Lemna minor chloroplasts.

Mechanisms of pharmaceuticals action on biochemical and physiological processes in plants that determine plant growth and development are still mostly unknown. This study deals with the effects of non-steroidal anti-inflammatory drug diclofenac (DCF) on photosynthesis as an essential anabolic process. Changes in primary and secondary photosynthetic processes were assessed in chloroplasts isolated from Lemna minor exposed to 1, 10, 100, and 1000 µM DCF. Decreases in the potential and effective quantum yields of photosystem II (FV/FM by 21%, ΦII by 44% compared to control), changes in non-photochemical fluorescence quenching (NPQ), and a substantial drop in Hill reaction activity (by 73%), especially under 1000 µM DCF, were found. Limitation of electron transport through photosystem II was confirmed by increased fluorescence signals in steps J and I (by 50% and 23%, respectively, under 1000 µM DCF) in OJIP fluorescence transient. Photosystem I exhibited changes only in the redox state of P700 reaction centres (decrease in Pm by 10%, increase in reduced P700 by 5% under 1000 µM DCF). Similarly, RuBisCO activity was only lowered by 30% under 1000 µM DCF. In contrast, a significant increase in reactive oxygen and nitrogen species (by 116% and 157%, respectively) was observed under 10 µM DCF, and lipid peroxidation increased even at 1 µM DCF (by nearly seven times compared to the control). Results demonstrate the ability of environmentally relevant DCF concentrations to induce oxidative stress in isolated duckweed chloroplasts; however, photosynthetic processes were affected considerably only by the highest DCF treatments.

Reprint of Efficient fungal UV-screening provides a remarkably high UV-B tolerance of photosystem II in lichen photobionts.

Lichen photobionts in situ have an extremely UV-B tolerant photosystem II efficiency (Fv/Fm). We have quantified the UV-B-screening offered by the mycobiont and the photobiont separately. The foliose lichens Nephroma arcticum and Umbilicaria spodochroa with 1: intact or 2: removed cortices were exposed to 0.7 Wm-2 UV-BBE for 4 h. Intact thalli experienced no reduction in Fv/Fm, whereas cortex removal lowered Fv/Fm in exposed photobiont layers by 22% for U. spodochroa and by 14% for N. arcticum. We also gave this UV-B dose to algal cultures of Coccomyxa and Trebouxia, the photobiont genera of N. arcticum and U. spodochroa, respectively. UV-B caused a 56% reduction in Fv/Fm for Coccomyxa, and as much as 98% in Trebouxia. The fluorescence excitation ratio (FER) technique comparing the fluorescence from UV-B or UV-A-excitation light with blue green excitation light using a Xe-PAM fluorometer showed that these photobiont genera did not screen any UV-B or UV-A The FER technique with a Multiplex fluorometer estimated the UV-A screening of isolated algae to be 13-16%, whereas intact lichens screened 92-95% of the UV-A. In conclusion, the cortex of N. arcticum and U. spodochroa transmitted no UV-B and little UV-A to the photobiont layer beneath. Thereby, the upper lichen cortex forms an efficient fungal solar radiation screen providing a high UV-B tolerance for studied photobionts in situ. By contrast, isolated photobionts have no UV-B screening and thus depend on their fungal partners in nature.

Efficient fungal UV-screening provides a remarkably high UV-B tolerance of photosystem II in lichen photobionts.

Lichen photobionts in situ have an extremely UV-B tolerant photosystem II efficiency (Fv/Fm). We have quantified the UV-B-screening offered by the mycobiont and the photobiont separately. The foliose lichens Nephroma arcticum and Umbilicaria spodochroa with 1: intact or 2: removed cortices were exposed to 0.7 Wm-2 UV-BBE for 4 h. Intact thalli experienced no reduction in Fv/Fm, whereas cortex removal lowered Fv/Fm in exposed photobiont layers by 22% for U. spodochroa and by 14% for N. arcticum. We also gave this UV-B dose to algal cultures of Coccomyxa and Trebouxia, the photobiont genera of N. arcticum and U. spodochroa, respectively. UV-B caused a 56% reduction in Fv/Fm for Coccomyxa, and as much as 98% in Trebouxia. The fluorescence excitation ratio (FER) technique comparing the fluorescence from UV-B or UV-A-excitation light with blue green excitation light using a Xe-PAM fluorometer showed that these photobiont genera did not screen any UV-B or UV-A The FER technique with a Multiplex fluorometer estimated the UV-A screening of isolated algae to be 13-16%, whereas intact lichens screened 92-95% of the UV-A. In conclusion, the cortex of N. arcticum and U. spodochroa transmitted no UV-B and little UV-A to the photobiont layer beneath. Thereby, the upper lichen cortex forms an efficient fungal solar radiation screen providing a high UV-B tolerance for studied photobionts in situ. By contrast, isolated photobionts have no UV-B screening and thus depend on their fungal partners in nature.

Flavobacterium chryseum sp. nov. and Flavobacterium psychroterrae sp. nov., novel environmental bacteria isolated from Antarctica.

A group of rod-shaped, aerobic, Gram-stain-negative, gliding bacteria producing flexirubin-type pigment was isolated from environmental samples collected in Antarctica in 2009-2014. Phylogenetic analysis of the almost complete 16S rRNA gene sequences revealed two separated branches belonging to the genus Flavobacterium. Group I (n=8), represented by strain CCM 8826T, shared the highest sequence similarity to Flavobacterium collinsii 983-08T (98.8 %) and Flavobacterium saccharophilum DSM 1811T (98.4 %), and group II (n=4) represented by strain CCM 8827T shared the highest similarity to Flavobacterium aquidurense WB 1.1-56T (99.6 %). High genetic homogeneity of both groups, separation from each other and from phylogenetically close Flavobacterium species was verified by the rep-PCR fingerprinting method. DNA-DNA hybridization confirmed low genomic relatedness between strain CCM 8826T and F. collinsii 983-08T and F. saccharophilum DSM 1811T (18 and 28 %, respectively) and between strain CCM 8827T and F. aquidurense WB 1.1-56T (27 %). Chemotaxonomic analyses of strains CCM 8826T and CCM 8827T revealed the respiratory quinone to be MK-6, the major identified polar lipid was phosphatidylethanolamine and the predominant polyamine was sym-homospermidine. The common major fatty acids were C15 : 0 iso, C17 : 0 iso 3OH, C15 : 1 iso G, Summed Feature 3 (C16 : 1ω7c/C16 : 1ω6c), C15 : 0 iso 3OH and additionally, C15 : 0 anteiso among group II members. All analyses confirmed that strains of group I and II represent two novel species of the genus Flavobacterium, for which the names Flavobacterium chryseum sp. nov. (type strain CCM 8826T=P3160T=LMG 30615T) and Flavobacterium psychroterrae sp. nov. (type strain CCM 8827T=P3922T=LMG 30616T) are proposed.

Species composition of Malassezia yeasts in dogs in Slovakia.

Malassezia (M.) pachydermatis is the lipophilic yeast, which is normally present on the skin and in the ear canal of dogs but under certain conditions it may cause dermatitis and otitis. There is less known about the occurrence of lipid-dependent Malassezia species in dogs. The aim of this study was to detect whether lipid-dependent yeasts are part of the normal microflora in dogs. Two groups of animals were selected for comparison. The group of healthy dogs contained samples of 118 individuals and the group of dogs with cutaneous lesions or otitis externa comprised 328 dogs. The isolates of Malassezia were identified by using genotypic methods that allow the precise identification. M. pachydermatis was the most frequently isolated species in this study (121 isolates). Only four isolates were identified as M. furfur and one isolate was identified as M. nana.

A dip in the chlorophyll fluorescence induction at 0.2-2 s in Trebouxia-possessing lichens reflects a fast reoxidation of photosystem I. A comparison with higher plants.

An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2-2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100-1800 micromol photons m(-2) s(-1)). Simultaneous measurements of FI and 820-nm transmission kinetics (I(820)) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2-0.4 s) is accompanied by a decrease of I(820), i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4-2 s) of the dip is not paralleled by the change in I(820). These results were compared with that measured with pea leaves-representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I-P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 microM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I(820) kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin-NADP(+)-oxidoreductase or Mehler-peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed.