BACKGROUND: North American bat populations have suffered severe declines over the last decade due to the Pseudogymnoascus destructans fungus infection. The skin disease associated with this causative agent, known as white-nose syndrome (WNS), is specific to bats hibernating in temperate regions. As cultured fungal isolates are required for epidemiological and phylogeographical studies, the purpose of the present work was to compare the efficacy and reliability of different culture approaches based on either skin swabs or wing membrane tissue biopsies for obtaining viable fungal isolates of P. destructans. RESULTS: In total, we collected and analysed 69 fungal and 65 bacterial skin swabs and 51 wing membrane tissue biopsies from three bat species in the Czech Republic, Poland and the Republic of Armenia. From these, we obtained 12 viable P. destructans culture isolates. CONCLUSIONS: Our results indicated that the efficacy of cultures based on wing membrane biopsies were significantly higher. Cultivable samples tended to be based on collections from bats with lower body surface temperature and higher counts of UV-visualised lesions. While cultures based on both skin swabs and wing membrane tissue biopsies can be utilised for monitoring and surveillance of P. destructans in bat populations, wing membrane biopsies guided by UV light for skin lesions proved higher efficacy. Interactions between bacteria on the host's skin also appear to play an important role.

• Klíčová slova
• Bats, Fungal culture, Fungal infection, Pseudogymnoascus destructans skin microbiota, UV lesions,
• MeSH
• Chiroptera * mikrobiologie   MeSH
• hibernace * MeSH
• kožní nemoci * veterinární   MeSH
• kultivační média MeSH
• kůže patologie   MeSH
• reprodukovatelnost výsledků MeSH
• syndrom MeSH
• ultrafialové záření MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kultivační média MeSH

Co-existence of bats with a wide range of infectious agents relates to their co-evolutionary history and specific physiology. Here, we examined blood samples collected during hibernation and the post-hibernation period to assess the influence of trypanosomes and babesias on the health status of 50 Noctule bats (Nyctalus noctula) using nested PCR. The impact of blood parasites on health was assessed by analysis of haematology and blood chemistry parameters in 21 bats. Prevalence of trypanosomes (Trypanosoma dionisii and T. vespertilionis) and babesia (Babesia vesperuginis) was 44% and 8%, respectively. Analysis of blood parameters indicated impact of babesia on acid-base balance. Blood chemistry parameters showed a significant decrease in total dissolved carbon dioxide and bicarbonate, increased anion gap, and no change in blood pH, suggesting compensated metabolic acidosis. Adverse effects of babesia were only apparent in hibernating bats. Our results suggest differences in the pathogenicity of trypanosomes and babesia in bats. While trypanosomes in general had no significant impact on the health status, we observed alterations in the blood acid-base balance in Babesia-infected bats during hibernation. Despite being infected, Babesia-positive bats survived hibernation without showing any clinical signs.

• Klíčová slova
• Babesia vesperuginis, Chiroptera, Schizotrypanum, Trypanosoma dionisii, Trypanosoma vespertilionis, acid–base balance, blood chemistry, haematology,
• Publikační typ
• časopisecké články MeSH

European Bark Beetle Ips typographus is a secondary pest that affects dead and weakened spruce trees (Picea genus). Under certain environmental conditions, it has massive outbreaks, resulting in the attacks of healthy trees, becoming a forest pest. It has been proposed that the bark beetle's microbiome plays a key role in the insect's ecology, providing nutrients, inhibiting pathogens, and degrading tree defense compounds, among other probable traits yet to be discovered. During a study of bacterial associates from I. typographus, we isolated three strains identified as Pseudomonas from different beetle life stages. A polyphasic taxonomical approach showed that they belong to a new species for which the name Pseudomonas typographi sp nov. is proposed. Genome sequences show their potential to hydrolyze wood compounds and synthesize several vitamins; screening for enzymes production was verified using PNP substrates. Assays in Petri dishes confirmed cellulose and xylan hydrolysis. Moreover, the genomes harbor genes encoding chitinases and gene clusters involved in the synthesis of secondary metabolites with antimicrobial potential. In vitro tests confirmed the capability of the three P. typographi strains to inhibit several Ips beetles' pathogenic fungi. Altogether, these results suggest that P. typographi aids I. typographi nutrition and resistance to fungal pathogens.

• Klíčová slova
• Ips typographus, Pseudomonas, bacteriome, bark beetle, beneficial,
• Publikační typ
• časopisecké články MeSH

Antimicrobial resistance is a worldwide problem that threatens the effectiveness of treatments for microbial infection. Consequently, it is essential to study unexplored niches that can serve for the isolation of new microbial strains able to produce antimicrobial compounds to develop new drugs. Bark beetles live in phloem of host trees and establish symbioses with microorganisms that provide them with nutrients. In addition, some of their associated bacteria play a role in the beetle protection by producing substances that inhibit antagonists. In this study the capacity of several bacterial strains, isolated from the bark beetles Ips acuminatus, Pityophthorus pityographus Cryphalus piceae, and Pityogenes bidentatus, to produce antimicrobial compounds was analyzed. Several isolates exhibited the capacity to inhibit Gram-positive and Gram-negative bacteria, as well as fungi. The genome sequence analysis of three Pseudomonas isolates predicted the presence of several gene clusters implicated in the production of already described antimicrobials and moreover, the low similarity of some of these clusters with those previously described, suggests that they encode new undescribed substances, which may be useful for developing new antimicrobial agents. Moreover, these bacteria appear to have genetic machinery for producing antitumoral and antiviral substances. Finally, the strain IA19T showed to represent a new species of the genus Pseudomonas. The 16S rRNA gene sequence analysis showed that its most closely related species include Pseudomonas lutea, Pseudomonas graminis, Pseudomonas abietaniphila and Pseudomonas alkylphenolica, with 98.6, 98.5 98.4, and 98.4% identity, respectively. MLSA of the housekeeping genes gyrB, rpoB, and rpoD confirmed that strain IA19T clearly separates from its closest related species. Average nucleotide identity between strains IA19T and P. abietaniphila ATCC 700689T, P. graminis DSM 11363T, P. alkylphenolica KL28T and P. lutea DSM 17257T were 85.3, 80.2, 79.0, and 72.1%, respectively. Growth occurs at 4-37°C and pH 6.5-8. Optimal growth occurs at 28°C, pH 7-8 and up to 2.5% NaCl. Respiratory ubiquinones are Q9 (97%) and Q8 (3%). C16:0 and in summed feature 3 are the main fatty acids. Based on genotypic, phenotypic and chemotaxonomic characteristics, the description of Pseudomonas bohemica sp. nov. has been proposed. The type strain is IA19T (=CECT 9403T = LMG 30182T).

• Klíčová slova
• NRPS-PKS, antibiotic resistance, anticarcinogenic, antimicrobials, antiviral, bark beetles, genome mining, secondary metabolites,
• Publikační typ
• časopisecké články MeSH

Pathogenic and non-pathogenic related microorganisms differ in secondary metabolite production. Here we show that riboflavin overproduction by a fungal pathogen and its hyperaccumulation in affected host tissue exacerbates a skin infection to necrosis. In white-nose syndrome (WNS) skin lesions caused by Pseudogymnoascus destructans, maximum riboflavin concentrations reached up to 815 μg ml(-1), indicating bioaccumulation and lack of excretion. We found that high riboflavin concentrations are cytotoxic under conditions specific for hibernation, affect bats' primary fibroblasts and induce cell detachment, loss of mitochondrial membrane potential, polymerization of cortical actin, and cell necrosis. Our results explain molecular pathology of WNS, where a skin infection becomes fatal. Hyperaccumulation of vitamin B2 coupled with reduced metabolism and low tissue oxygen saturation during hibernation prevents removal of excess riboflavin in infected bats. Upon reperfusion, oxygen reacts with riboflavin resulting in dramatic pathology after arousal. While multiple molecules enable invasive infection, riboflavin-associated extensive necrosis likely contributes to pathophysiology and altered arousal pattern in infected bats. Bioaccumulation of a vitamin under natural infection represents a novel condition in a complex host-pathogen interplay.

• MeSH
• Ascomycota klasifikace   genetika   patogenita   MeSH
• buněčná adheze MeSH
• Chiroptera mikrobiologie   MeSH
• dermatomykózy mikrobiologie   MeSH
• elektronová mikroskopie MeSH
• faktory virulence metabolismus   MeSH
• fibroblasty cytologie   metabolismus   mikrobiologie   MeSH
• fylogeneze MeSH
• interakce hostitele a patogenu MeSH
• křídla zvířecí cytologie   mikrobiologie   ultrastruktura   MeSH
• kultivované buňky MeSH
• membránový potenciál mitochondrií MeSH
• riboflavin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory virulence MeSH
• riboflavin MeSH

Understanding the activity of bacteria in coniferous forests is highly important, due to the role of these environments as a global carbon sink. In a study of the microbial biodiversity of montane coniferous forest soil in the Bohemian Forest National Park (Czech Republic), we succeeded in isolating bacterial strain S55(T), which belongs to one of the most abundant operational taxonomic units (OTUs) in active bacterial populations, according to the analysis of RNA-derived 16S rRNA amplicons. The 16S rRNA gene sequence analysis showed that the species most closely related to strain S55(T) include Bryocella elongata SN10(T) (95.4% identity), Acidicapsa ligni WH120(T) (95.2% identity), and Telmatobacter bradus TPB6017(T) (95.0% identity), revealing that strain S55(T) should be classified within the phylum Acidobacteria, subdivision 1. Strain S55(T) is a rod-like bacterium that grows at acidic pH (3 to 6). Its phylogenetic, genotypic, phenotypic, and chemotaxonomic characteristics indicate that strain S55(T) corresponds to a new genus within the phylum Acidobacteria; thus, we propose the name Terracidiphilus gabretensis gen. nov., sp. nov. (strain S55(T) = NBRC 111238(T) = CECT 8791(T)). This strain produces extracellular enzymes implicated in the degradation of plant-derived biopolymers. Moreover, analysis of the genome sequence of strain S55(T) also reveals the presence of enzymatic machinery required for organic matter decomposition. Soil metatranscriptomic analyses found 132 genes from strain S55(T) being expressed in the forest soil, especially during winter. Our results suggest an important contribution of T. gabretensis S55(T) in the carbon cycle in the Picea abies coniferous forest.

• MeSH
• Acidobacteria genetika   izolace a purifikace   metabolismus   MeSH
• biodegradace MeSH
• biotransformace MeSH
• fylogeneze MeSH
• lesy MeSH
• molekulární sekvence - údaje MeSH
• půdní mikrobiologie * MeSH
• rostliny metabolismus   mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH