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.

CzechGlobe Global Change Research Institute of the Czech Academy of Sciences Bělidla 986 4a 603 00 Brno Czech Republic

Department of Botany and Zoology Masaryk University Kotlářská 267 2 611 37 Brno Czech Republic

Department of Ecology and Diseases of Zoo Animals Game Fish and Bees University of Veterinary Sciences Brno Palackého tř 1946 1 612 42 Brno Czech Republic

Department of Vertebrate Ecology and Palaeontology Institute of Environmental Biology Wrocław University of Environmental and Life Sciences Kożuchowska 5B 51 631 Wrocław Poland

Department of Zoology Yerevan State University 1 Alex Manoogian 0025 Yerevan Armenia

Institute of Vertebrate Biology Czech Academy of Sciences Květná 8 603 65 Brno Czech Republic

Laboratory of Fungal Genetics and Metabolism Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 142 20 Prague 4 Czech Republic

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Barton HA, Northup DE. Geomicrobiology in cave environments: past, current and future perspectives. J Cave Karst Stud. 2007;69:163–178.

Gabriel CR, Northup DE. Microbial ecology: caves as an extreme habitat. Springerbriefs in Microbiology. 2013;1:85–108. doi: 10.1007/978-1-4614-5206-5_5. DOI

Hershey OS, Barton HA. The Microbial Diversity of Caves. In: Moldovan O, Kováč Ľ, Halse S, editors. Cave Ecology. Ecological Studies (Analysis and Synthesis) Cham: Springer; 2018. p. 235.

Barton HA, Jurado V. What’s up down there? Microbial diversity in caves Microbe. 2007;2:132–138.

Hawksworth DL. Lichenization: the origins of a fungal life-style. In: Upreti DK, Divakar PK, Shukla V, Bajpai R, editors. Recent Advances in Lichenology. Modern methods and approaches in lichen systematics and culture techniques, 2. New Delhi: Springer; 2015. pp. 1–10.

Wrzosek M, Ruszkiewicz-Michalska M, Sikora K, Damszel M, Sierota Z. The plasticity of fungal interactions. Mycol Prog. 2017;16:101–108.

Verant ML, Boyles JG, Waldrep W, Jr, Wibbelt G, Blehert DS. Temperature-dependent growth of Geomyces destructans, the fungus that causes bat white-nose syndrome. PLoS One. 2012;7(9):e46280. PubMed PMC

Raudabaugh DB, Miller AN. Fungal Planet description sheets. PLoS One. 2013;54:868–950.

Smyth C, Schlesinger S, Overton B, Butchkoski C. The Alternative Host Hypothesis and Potential Virulence Genes in Geomyces destructans. Bat Research News. 2013;54:17–24.

Blehert DS, Hicks AC, Behr M, Meteyer CU, Berlowski-Zier BM, Buckles EL, Coleman JTH, Darling SR, Gargas A, Niver R, Okoniewski JC, Rudd RJ, Stone WB. Bat white-nose syndrome: an emerging fungal pathogen? Science. 2009;323(5911):227–227. PubMed

Langwig KE, Frick WF, Bried JT, Hicks AC, Kunz TH, Marm Kilpatrick A. Sociality, density-dependence and microclimates determine the persistence of populations suffering from a novel fungal disease, white-nose syndrome. Ecol Lett. 2012;15(9):1050–1057. PubMed

Frick WF, Puechmaille SJ, Hoyt JR, Nickel BA, Langwig KE, Foster JT, Barlow KE, Bartonička T, Feller D, Haarsma AJ, Herzog C, Horáček I, Van der Kooij J, Mulkens B, Petrov B, Reynolds R, Rodrigues L, Stihler CW, Turner GG, Kilpatrick AM. Disease alters macroecological patterns of North American bats. Glob Ecol Biogeogr. 2015;24(7):741–749.

Hoyt JR, Kilpatrick AM, Langwig KE. Ecology and impacts of white-nose syndrome on bats. Nat Rev Microbiol. 2021;19(3):196–210. PubMed

Meteyer CU, Buckles EL, Blehert DS, Hicks AC, Green DE, Shearn-Bochsler V, Thomas NJ, Gargas A, Behr MJ. Histopathologic criteria to confirm white-nose syndrome in bats. J Vet Diagn Invest. 2009;21(4):411–414. PubMed

Turner GG, Meteyer CU, Barton H, Gumbs JF, Reeder DM, Overton B, Bandouchova H, Bartonička T, Martínková N, Pikula J, Zukal J, Blehert DS. Nonlethal screening of bat-wing skin with the use of ultraviolet fluorescence to detect lesions indicative of white-nose syndrome. J Wildl Dis. 2014;50(3):566–573. PubMed

Makimura K, Murayama SY, Yamaguchi H. Detection of a wide range of medically important fungi by polymerase chain reaction. J Med Microbiol. 1994;40(5):358–364. PubMed

Sandhu GS, Kline BC, Stockman L, Roberts GD. Molecular probes for diagnosis of fungal infections. J Clin Microbiol. 1995;33(11):2913–2919. PubMed PMC

Brandt ME, Park BJ. Think fungus - prevention and control of fungal infections. Emerg Infect Dis. 2013;19:1688–1689. doi: 10.3201/eid1910.131092. PubMed DOI PMC

Leopardi S, Blake D, Puechmaille SJ. White-Nose Syndrome fungus introduced from Europe to North America. Curr Biol. 2015;25:217–219. doi: 10.1016/j.cub.2015.01.047. PubMed DOI

Ghosh PN, Fisher MC, Bates KA. Diagnosing emerging fungal threats: a one health perspective. Front Genet. 2018;9:376. PubMed PMC

Eloff JN, Masoko P, Picard J. Resistance of animal fungal pathogens to solvents used in bioassays. S Afr J Bot. 2007;73(4):667–669.

O’Donoghue AJ, Knudsen GM, Beekman C, Perry JA, Johnson AD, DeRisi JL, Craik CS, Bennett RJ. Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome. Proc Natl Acad Sci. 2015;112(24):7478–7483. PubMed PMC

Macheleidt J, Mattern DJ, Fischer J, Netzker T, Weber J, Schroeckh V, Valiante V, Brakhage AA. Regulation and role of fungal secondary metabolites. Annu Rev Genet. 2016;50(1):371–392. PubMed

Flieger M, Bandouchova H, Cerny J, Chudickova M, Kolarik M, Kovacova V, Martinkova N, Novak P, Sebesta O, Stodulkova E, Pikula J. Vitamin B2 as a virulence factor in Pseudogymnoascus destructans skin infection. Sci Rep. 2016;6(1):1–12, 33200. PubMed PMC

Chaturvedi V, DeFiglio H, Chaturvedi S. Phenotype profiling of white-nose syndrome pathogen Pseudogymnoascus destructans and closely-related Pseudogymnoascus pannorum reveals metabolic differences underlying fungal lifestyles. F1000Res. 2018;7:665. PubMed PMC

Ren P, Rajkumar SS, Zhang T, Sui H, Masters PS, Martinkova N, Kubátová A, Pikula J, Chaturvedi S, Chaturvedi V. A common partitivirus infection in United States and Czech Republic isolates of bat white-nose syndrome fungal pathogen Pseudogymnoascus destructans. Sci Rep. 2020;10(1):1–11. PubMed PMC

Veselská T, Homutová K, Fraile PG, Kubátová A, Martínková N, Pikula J, Kolařík M. Comparative eco-physiology revealed extensive 1 enzymatic curtailment, lipases production and strong conidial resilience of the bat pathogenic fungus Pseudogymnoascus destructans. Sci Rep. 2020;10(1):16530, 113. PubMed PMC

Myers JM, Ramsey JP, Blackman AL, Nichols AE, Minbiole KP, Harris RN. Synergistic inhibition of the lethal fungal pathogen Batrachochytrium dendrobatidis: the combined effect of symbiotic bacterial metabolites and antimicrobial peptides of the frog Rana muscosa. J Chem Ecol. 2012;38(8):958–965. doi: 10.1007/s10886-012-0170-2. PubMed DOI

Hoyt JR, Cheng TL, Langwig KE, Hee MM, Frick WF, Kilpatrick AM. Bacteria isolated from bats inhibit the growth of Pseudogymnoascus destructans, the causative agent of white-nose syndrome. PLoS One. 2015;10:e0121329. PubMed PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Johannes S, Rueden C, Saalfeld S, Schmid S, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–682. PubMed PMC

Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Window 95/98/NT. Nucleic Acids Symp. 1999;Series 41:95–98.

Gams W. Gliederungsprinzipien in der gattung Mortierella. Nova Hedwigia. 1970;18:30-43.

Fassatiová O. Moulds and filamentous fungi in technical microbiology. Amsterdam, Netherlands: Elsevier; 1986.

Samson RA, Hoekstra ES, Lund F, Filtenborg O, Frisvad JC. Methods for the detection, isolation and characterisation of food-borne fungi. Introduction to food-and airborne fungi. 7. 2004. pp. 283–297.

Palmer JM, Kubatova A, Novakova A, Minnis AM, Kolarik M, Lindner DL. Molecular characterization of a heterothallic mating system in Pseudogymnoascus destructans, the fungus causing white-nose syndrome of bats. G3:Genes, Genomes, Genetics. 2014;4(9):1755–1763. PubMed PMC

Zukal J, Bandouchova H, Brichta J, Cmokova A, Jaron KS, Kolarik M, Kovacova V, Kubátová A, Nováková A, Orlov O, Pikula J, Presetnik P, Šuba J, Zahradníková A, Jr, Martínková N. White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America. Sci Rep. 2016;6:13. PubMed PMC

Frick WF, Pollock JF, Hicks AC, Langwig KE, Reynolds DS, Turner GG, Butchkoski CB, Kunz TH. An emerging disease causes regional population collapse of a common North American bat species. Science. 2010;329:679–682. PubMed

Cheng TL, Reichard JD, Coleman J, Weller TJ, Thogmartin WE, Reichert BE, Bennett AB, Broders HG, Campbell J, Etchison K, Feller DJ, Geboy R, Hemberger T, Herzog C, Hicks AC, Houghton S, Humber J, Kath JA, King RA, Loeb SC, … Frick WF. The scope and severity of white-nose syndrome on hibernating bats in North America. Conserv Biol. 2021;35(5):1586–1597. PubMed PMC

Pikula J, Amelon SK, Bandouchova H, Bartonička T, Berkova H, Brichta J, Hooper S, Kokurewicz T, Kolarik M, Köllner B, Kovacova V, Linhart P, Piacek V, Turner GG, Zukal J, Martínková N. White-nose syndrome pathology grading in Nearctic and Palearctic bats. PLoS One. 2017;12:21. doi: 10.1371/journal.pone.0180435. PubMed DOI PMC

Reeder DM, Frank CL, Turner GG, Meteyer CU, Kurta A, et al. Frequent Arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome. PLoS One. 2012;7(6):e38920. PubMed PMC

McGuire LP, Turner JM, Warnecke L, et al. White-nose syndrome disease severity and a comparison of diagnostic methods. EcoHealth. 2016;13:60–71. PubMed

Blehert DS, Lorch JM. Laboratory maintenance and culture of Pseudogymnoascus destructans, the fungus that causes Bat White-Nose Syndrome. Curr Protoc. 2021;1(1):e23. doi: 10.1002/cpz1.23. PubMed DOI

Gargas A, Trest MT, Christensen M, Volk TJ, Blehert DS. Geomyces destructans sp. nov. associated with bat white-nose syndrome. Mycotaxon. 2009;108:147–154. doi: 10.5248/108.147. DOI

Verant ML, Bohuski EA, Richgels KLD, Olival KJ, Epstein JH, Blehert DS. Determinants of Pseudogymnoascus destructans within bat hibernacula: Implications for surveillance and management of white-nose syndrome. J Appl Ecol. 2018;55:820–829. PubMed PMC

Martínková N, Škrabánek P, Pikula J. Modelling invasive pathogen load from non-destructive sampling data. J Theor Biol. 2019;464:98–103. PubMed

Hoyt JR, Langwig KE, Okoniewski J, Frick WF, Stone WB, Kilpatrick AM. Long-term persistence of Pseudogymnoascus destructans, the causative agent of white-nose syndrome, in the absence of bats. EcoHealth. 2015;12(2):330–333. PubMed

Hayman DTS, Pulliam JRC, Marshall JC, Cryan PM, Webb CT. Environment, host, and fungal traits predict continental-scale White-nose syndrome in bats. Sci Adv. 2016;2:1–13. PubMed PMC

Martínková N, Pikula J, Zukal J, Kovacova V, Bandouchova H, Bartonička T, Botvinkin AD, Brichta J, Dundarova H, Kokurewicz T, Irwin NR, Linhart P, Orlov OL, Piacek V, Škrabánek P, Tiunov MP, Zahradníková A. Hibernation temperature-dependent Pseudogymnoascus destructans infection intensity in Palearctic bats. Virulence. 2018;9(1):1734–1750. PubMed PMC

Zukal J, Bandouchova H, Bartonicka T, Berkova H, Brack V, Brichta J, Dolinay M, Jaron KS, Kovacova V, Kovarik K, Martínková N, Ondracek K, Rehak Z, Turner GG, Pikula J. White-nose syndrome fungus: a generalist pathogen of hibernating bats. Plos One. 2014;9(5):e97224. doi: 10.1371/journal.pone.0097224. PubMed DOI PMC

Muller LK, Lorch JM, Lindner DL, O’Connor M, Gargas A, Blehert DS. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans. Mycologia. 2013;105(2):253–259. PubMed

Kovacova V, Zukal J, Bandouchova H, Botvinkin AD, Harazim M, Martínková N, Orlov O, Piacek V, Shumkina AP, Tiunov MK, Pikula J. White-nose syndrome detected in bats over an extensive area of Russia. BMC Vet Res. 2018;14(1):1–9. PubMed PMC

Lorch JM, Muller LK, Russell RE, O'Connor M, Lindner DL, Blehert DS. Distribution and Environmental Persistence of the Causative Agent of White-Nose Syndrome, Geomyces destructans, in Bat Hibernacula of the Eastern United States. Appl Environ Microbiol. 2013;79(4):1293-1301. PubMed PMC

Grice EA, Segre JA. The skin microbiome. Nat Rev Microbiol. 2011;9(4):244–253. PubMed PMC

Ross AA, Rodrigues Hoffmann A, Neufeld JD. The skin microbiome of vertebrates. Microbiome. 2019;7(1):79. PubMed PMC

Kobayashi DY, Crouch JA. Bacterial/fungal interactions: from pathogens to mutualistic endosymbionts. Annu Rev Phytopathol. 2009;47:63–82. PubMed

Wargo MJ, Hogan DA. Fungal-bacterial interactions: a mixed bag of mingling microbes. Curr Opin Microbiol. 2006;9(4):359–364. PubMed

Gram L, Melchiorsen J, Spanggaard B, Huber I, Nielsen TF. Inhibition of Vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish. Appl Environ Microbiol. 1999;65(3):969–973. PubMed PMC

Vanderwolf KJ, Campbell LJ, Taylor DR, Goldberg TL, Blehert DS, Lorch JM. Mycobiome traits associated with disease tolerance predict many western North American bat species will be susceptible to white-nose syndrome. Microbiology Spectr. 2021;9:e00254–e321. PubMed PMC

Remold SK, Purdy-Gibson ME, France MT, Hundley TC. Pseudomonas putida and Pseudomonas fluorescens species group recovery from human homes varies seasonally and by environment. PLoS One. 2015;10(5):e0127704. PubMed PMC

Ingraham JL. Growth of psychrophilic bacteria. J Bacteriol. 1958;76(1):75. PubMed PMC

Negi YK, Prabha D, Garg SK, Kumar J. Genetic diversity among cold-tolerant fluorescent Pseudomonas isolates from Indian Himalayas and their characterization for biocontrol and plant growth-promoting activities. J Plant Growth Regul. 2011;30(2):128–143.

Lemieux-Labonté V, Dorville NAS, Willis CKR, Lapointe FJ. Antifungal potential of the skin microbiota of hibernating big brown bats (Eptesicus fuscus) infected with the causal agent of white-nose syndrome. Front Microbiol. 2020;11:1776. PubMed PMC

Garcia-Fraile P, Chudíčková M, Benada O, Pikula J, Kolařík M. Serratia myotis sp. nov. and Serratia vespertilionis sp. nov. isolated from bats hibernating in caves in the Czech Republic. Int J Syst Evol Microbiol. 2015;65(1):90–94. PubMed

Bach E, Sant’Anna V, Daroit DJ, Corrêa APF, Segalin J, Brandelli A. Production, one-step purification, and characterization of a keratinolytic protease from Serratia marcescens P3. Process Biochem. 2012;47(12):2455–2462.

Li Z, Li A, Dai W, Leng H, Liu S, Jin L, Sun K, Feng J. Skin microbiota variation among bat species in China and their potential defense against pathogens. Front Microbiol. 2022;13:808788. PubMed PMC

Collado J, Platas G, Paulus B, Bills GF. High-throughput culturing of fungi from plant litter by a dilution-to-extinction technique. FEMS Microbiol Ecol. 2007;60(3):521–533. PubMed

Donaldson ME, Davy CM, Vanderwolf KJ, Willis CK, Saville BJ, Kyle CJ. Growth medium and incubation temperature alter the Pseudogymnoascus destructans transcriptome: implications in identifying virulence factors. Mycologia. 2018;110(2):300–315. PubMed

Zhang T, Victor TR, Rajkumar SS, Li X, Okoniewski JC, Hicks AC, Davis AD, Broussard K, LaDeau SL, Chaturvedi S, Chaturvedi V. Mycobiome of the Bat White Nose Syndrome Affected Caves and Mines Reveals Diversity of Fungi and Local Adaptation by the Fungal Pathogen Pseudogymnoascus (Geomyces) destructans. PloS One. 2014;9(9):e108714. PubMed PMC

Insińska-Rak M, Golczak A, Sikorski M. Photochemistry of riboflavin derivatives in methanolic solutions. J Phys Chem A. 2012;116(4):1199–1207. PubMed

Rajamani S, Bauer WD, Robinson JB, Farrow JM, III, Pesci EC, Teplitski M, Gao M, Sayre RT, Phillips DA. The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor. Mol Plant Microbe Interact. 2008;21(9):1184–1192. PubMed PMC

Fanning S, Mitchell AP. Fungal biofilms. PLoS Pathog. 2012;8(4):e1002585. PubMed PMC

Cogen AL, Nizet V, Gallo RL. Skin microbiota: a source of disease or defence? Br J Dermatol. 2008;158(3):442–455. PubMed PMC

Lorch JM, Meteyer CU, Behr MJ, Boyles JG, Cryan PM, Hicks AC, Ballmann AE, Coleman JTH, Redell DN, Reeder DM, Blehert DS. Experimental infection of bats with Geomyces destructans causes white-nose syndrome. Nature. 2011;480(7377):376–378. PubMed