This study aims to determine the bacterial flora on the skin surface of the juvenile forms of Pelophylax ridibundus inhabiting three different altitudes and examine potential correlations between bacterial diversity, ecological location, and factors. It was attempted to characterize thirty-two bacteria isolated from the Melet River, Sülük Lake, and Çambaşı Pond through combined biochemical and molecular methods. Canonical correspondence analysis showed that the most important ecological factors for microorganisms to settle on frog skin were determined as water conductivity and dissolved oxygen amount. The most frequently isolated bacteria belonged to the genera Erwinia and Pseudomonas. Altitude positively affected Exiguobacterium. This first report of skin cultivable bacteria from P. ridibundus juvenile forms natural population improves our knowledge of amphibian skin bacterial flora. This study contributes to a better understanding of their ecology and how this species has survived in an environment modulated by altitude.

Faculty of Education Science Education Department Ordu University Ordu Turkey

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Abarca Alvarado JG, Whitfield SM, Zúñiga Cháves I, Alvarado Barboza G, Kerby JL, Murillo Cruz C, Pinto Tomás AA (2021) Genotyping and differential bacterial inhibition of Batrachochytrium dendrobatidis in threatened amphibians in Costa Rica. Microbiol. https://doi.org/10.1099/mic.0.001017 DOI

Alexiev A, Chen MY, Korpita T, Weier AM, McKenzie VJ (2023) Together or alone: evaluating the pathogen inhibition potential of bacterial cocktails against an amphibian pathogen. Microbiol Spectr 11(2):e01518-e1522. https://doi.org/10.1128/spectrum.01518-22 PubMed DOI PMC

Bian DR, Xue H, Piao CG, Li Y (2020) Stenotrophomonas cyclobalanopsidis sp. nov., isolated from the leaf spot disease of Cyclobalanopsis patelliformis. Anton Leeuw Int J G 113(10):1447–1454. https://doi.org/10.1007/s10482-020-01453-y

Busse HJ (2016) Review of the taxonomy of the genus Arthrobacter, emendation of the genus Arthrobacter sensu lato, proposal to reclassify selected species of the genus Arthrobacter in the novel genera Glutamicibacter gen. nov., Paeniglutamicibacter gen. nov., Pseudoglutamicibacter gen. nov., Paenarthrobacter gen. nov. and Pseudarthrobacter gen. nov., and emended description of Arthrobacter roseus. Int J Syst Evol Microbiol 66:9–37. https://doi.org/10.1099/ijsem.0.000702 PubMed DOI

Campbell LJ, Garner TW, Hopkins K, Griffiths AG, Harrison XA (2019) Outbreaks of an emerging viral disease covary with differences in the composition of the skin microbiome of a wild United Kingdom amphibian. Front Microbiol 10:1245. https://doi.org/10.3389/fmicb.2019.01245 PubMed DOI PMC

Canik Orel D (2020) Erwinia persicina as the new causal agent of lettuce soft rot. Eur J Plant Pathol 158(1):223–235. https://doi.org/10.1007/s10658-020-02068-9 DOI

Carvalheira A, Silva J, Teixeira P (2021) Acinetobacter spp. in food and drinking water–a review. Food Microbiol 95:103675. https://doi.org/10.1016/j.fm.2020.103675

Cevallos MA, Basanta MD, Bello-López E, Escobedo-Muñoz AS, González-Serrano FM, Nemec A, Romero-Contreras J, Serrano M, Rebollar EA (2022) Genomic characterization of antifungal Acinetobacter bacteria isolated from the skin of the frogs Agalychnis callidryas and Craugastor fitzingeri. FEMS Microbiol Ecol 98(12):1–11. https://doi.org/10.1093/femsec/fiac126 DOI

Choi SY, Yoon KH, Lee JI, Mitchell RJ (2015) Violacein: properties and production of a versatile bacterial pigment. BioMed Res Int 2015. https://doi.org/10.1155/2015/465056

Corno G, Coci M, Giardina M, Plechuk S, Campanile F, Stefani S (2014) Antibiotics promote aggregation within aquatic bacterial communities. Front Microbiol 5:297. https://doi.org/10.3389/fmicb.2014.00297 PubMed DOI PMC

Cüce H, Kalıpcı E, TAŞ B, Yılmaz M (2020) Evaluation of the impacts on water quality from meteorological changes due to differences in altitude by GIS: a comparison for two morphologically different lakes. The Black Sea J Sci 10(1):1–26. https://doi.org/10.31466/kfbd.649297

Cicek K, Ayaz D, Afsar M, Bayrakcı Y, Pekşen ÇA, Cumhuriyet O, İsmail IB, Yenmiş M, Ustundag E, Tok CV, Bilgin CC, Akçakaya HR (2021) Unsustainable harvest of water frogs in southern Turkey for the European market. Oryx 55(3):364–372. https://doi.org/10.1017/S0030605319000176 DOI

Dökenel G, Özer S (2019) Bacterial agents isolated from cultured marsh frog (Pelophylax ridibundus, Pallas 1771). Ege JFAS 36(2):115–124. https://doi.org/10.12714/egejfas.2019.36.2.03

Duman M, Mulet M, Altun S, Saticioglu IB, Gomila M, Lalucat J, Garcia-Valdes E (2021) Pseudomonas piscium sp. nov., Pseudomonas pisciculturae sp. nov., Pseudomonas mucoides sp. nov. and Pseudomonas neuropathica sp. nov. isolated from rainbow trout. Int J Syst Evol Microbiol 71:004714. https://doi.org/10.1099/ijsem.0.005125

Eda ARA (2022) Metagenomics analysis of cutaneous microbiome of New Zealand’s endemic frogs (Genus Leiopelma) associated with Batrachochytrium dendrobatidis. Dissertation, University of Otago

Frühling A, Schumann P, Hippe H, Sträubler B, Stackebrandt E (2002) Exiguobacterium undae sp. nov. and Exiguobacterium antarcticum sp. nov. Int J Syst Evol Microbiol 52(4):1171–1176. https://doi.org/10.1099/00207713-52-4-1171

Gyobu Y, Miyadoh S (2001) Proposal to transfer Actinomadura carminata to a new subspecies of the genus Nonomuraea as Nonomuraea roseoviolacea subsp. Carminata comb. nov. Int J Syst Evol Microbiol 51:881–889. https://doi.org/10.1099/00207713-51-3-881 PubMed DOI

Hacioglu N, Gul C, Tosunoglu M (2015) Bacteriological screening and antibiotic–heavy metal resistance profile of the bacteria isolated from some amphibian and reptile species of the Biga stream in Turkey. Int J Environ Ecol Eng 9(4):422–426. https://doi.org/10.5281/zenodo.1100709 DOI

Holden WM, Hanlon SM, Woodhams DC, Chappell TM, Wells HL, Glisson SM, McKenzie VJ, Knight R, Parris MJ, Rollins-Smith LA (2015) Skin bacteria provide early protection for newly metamorphosed southern leopard frogs (Rana sphenocephala) against the frog-killing fungus, Batrachochytrium dendrobatidis. Biol Conser 187:91–102. https://doi.org/10.1016/j.biocon.2015.04.007 DOI

Huang XX, Shang J, Xu L, Yang R, Sun JQ (2021) Luteimonas deserti sp. nov., a novel strain isolated from desert soil. Int J Syst Evol Microbiol 71(10):005048. https://doi.org/10.1099/ijsem.0.005048

Ienes-Lima J, Prichula J, Abadie M, Borges-Martins M, Frazzon APG (2022) Skin cultivable bacterial of Melanophryniscus admirabilis (admirable red belly toad): the role of bacterial communities on species conservation. Research square (in press).  https://doi.org/10.21203/rs.3.rs-1483609/v1

Jaý M, Girault G, Perrot L, Taunay B, Vuilmet T, Rossignol F, Pitel P-H, Picard E, Ponsart C, Mick V (2018) Phenotypic and molecular characterization of Brucella microti-like bacteria from a domestic marsh frog (Pelophylax ridibundus). Front Vet Sci 5:283. https://doi.org/10.1099/00207713-52-4-1171 PubMed DOI PMC

Jukes TH, Cantor CR (1969) Evolution of protein molecules. Academic Press, London DOI

Kämpfer P, Wellner S, Lohse K, Martin K, Lodders N (2012) Duganella phyllosphaerae sp. nov., isolated from the leaf surface of Trifolium repens and proposal to reclassify Duganella violaceinigra into a novel genus as Pseudoduganella violceinigra gen. nov., comb. nov. Syst Appl Microbiol 35(1):19–23. https://doi.org/10.1016/j.syapm.2011.10.003

Khalifa AY, Bekhet G (2018) First isolation and characterization of the pathogenic Aeromonas veronii bv. veronii associated with ulcerative syndrome in the indigenous Pelophylax ridibundus of Al-Ahsaa. Saudi Arabia Microb Pathog 117:361–368. https://doi.org/10.1016/j.micpath.2017.10.019 PubMed DOI

Kumar S, Sketcher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. https://doi.org/10.1093/molbev/msw054

Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. John Wiley and Sons, New York, pp 115–175

Lauer A, Simon MA, Banning JL, Lam BA, Harris RN (2008) Diversity of cutaneous bacteria with antifungal activity isolated from female four-toed salamanders. ISME J 2(2):145–157. https://doi.org/10.1038/ismej.2007.110 PubMed DOI

Liu L, Feng Y, Wei L, Zong Z (2021) Genome-based taxonomy of Brevundimonas with reporting Brevundimonas huaxiensis sp. nov. Microbiol Spect 9(1):e00111–21. https://doi.org/10.1128/Spectrum.00111-21

Loch TP, Faisal M (2014) Chryseobacterium aahli sp. nov., isolated from lake trout (Salvelinus namaycush) and brown trout (Salmo trutta), and emended descriptions of Chryseobacterium ginsenosidimutans and Chryseobacterium gregarium. Int J Syst Evol Microbiol 64(Pt_5):1573–1579. https://doi.org/10.1099/ijs.0.052373-0

Madison JD, Berg EA, Abarca JG, Whitfield SM, Gorbatenko O, Pinto A, Kerby JL (2017) Characterization of Batrachochytrium dendrobatidis inhibiting bacteria from amphibian populations in Costa Rica. Front Microbiol 8:290. https://doi.org/10.3389/fmicb.2017.00290 PubMed DOI PMC

Mogadem A, Almamary MA, Mahat NA, Jemon K, Ahmad WA, Ali I (2021) Antioxidant activity evaluation of FlexirubinType pigment from Chryseobacterium artocarpi CECT 8497 and related docking study. Molecules 26(4):979. https://doi.org/10.3390/molecules26040979 PubMed DOI PMC

Mulet M, Duman M, Altun S, Saticioglu IB, Gomila M, Matthijs S, Lalucat J, García-Valdés E (2021) Pseudomonas arcuscaelestis sp. nov., isolated from rainbow trout and water. Int J Syst Evol Microbiol 71(7):004860. https://doi.org/10.1099/ijsem.0.004860

Nechwatal J, Theil S (2019) Erwinia persicina associated with a pink rot of parsley root in Germany. J Plant Dis Prot 126(2):161–167. https://doi.org/10.1007/s41348-018-0200-6 DOI

Ngo HT, Yin CS (2016) Luteimonas terrae sp. nov., isolated from rhizosphere soil of Radix ophiopogonis. Int J Syst Evol Microbiol 66(5):1920–1925. https://doi.org/10.1099/ijsem.0.000901

Oh WT, Jun JW, Giri SS, Yun S, Kim HJ, Kim SG, Han SJ, Kwon J, Park SC (2020) Isolation of Chryseobacterium siluri sp. nov., from liver of diseased catfish (Silurus asotus). Heliyon 6(2):e03454. https://doi.org/10.1016/j.heliyon.2020.e03454

Peeters K, Verleyen E, Hodgson DA, Convey P, Ertz D, Vyverman W, Willems A (2012) Heterotrophic bacterial diversity in aquatic microbial mat communities from Antarctica. Polar Biol 35(4):543–554. https://doi.org/10.1007/s00300-011-1100-4 DOI

Perrin Y, Bouchon D, Delafont V, Moulin L, Héchard Y (2019) Microbiome of drinking water: a full-scale spatio-temporal study to monitor water quality in the Paris distribution system. Water Res 149:375–385. https://doi.org/10.1016/j.watres.2018.11.013 PubMed DOI

Proença DN, Fasola E, Lopes I, Morais PV (2021) Characterization of the skin cultivable microbiota composition of the frog Pelophylax perezi inhabiting different environments. Int J Environ Res Public Health 18(5):2585. https://doi.org/10.3390/ijerph18052585 PubMed DOI PMC

Ramírez-Bahena MH, Salazar S, Cuesta MJ, Tejedor C, Igual JM, Fernández-Pascual M, Peix Á (2016) Erwinia endophytica sp. nov., isolated from potato (Solanum tuberosum L.) stems. Int J Syst Evol Microbiol 66(2):975–981. https://doi.org/10.1099/ijsem.0.000820

Reddy CA, Beveridge TJ, Breznak JA, Marzluf G (2007) Methods for general and molecular microbiology. ASM Press. https://doi.org/10.1128/9781555817497 DOI

Rollins-Smith LA (2023) The importance of antimicrobial peptides (AMPs) in amphibian skin defense. Dev Comp Immunol 142:104657. https://doi.org/10.1016/j.dci.2023.104657

Ruuskanen MO, Vats D, Potbhare R, RaviKumar A, Munukka E, Ashma R, Lahti L (2022) Towards standardized and reproducible research in skin microbiomes. Environ Microbiol 24(9):3840–3860. https://doi.org/10.1111/1462-2920.15945 PubMed DOI PMC

Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 PubMed

Sarker S, Abraham TJ, Dash G, Nagesh TS (2019) Pathogenicity and pathology of Chryseobacterium sp. PLI2 in experimentally challenged ornamental goldfish, Carasius auratus (L.). Vet Arh 89(5):697–707. https://doi.org/10.24099/vet.arhiv.0476

Saticioglu IB, Duman M, Altun S (2020a) Determination of phenotypic and genome characteristics of Chryseobacterium sp. C-204 strain isolated from rainbow trout. Erciyes Üniv Vet Fak Derg 17(3):303–311. https://doi.org/10.32707/ercivet.828829

Saticioglu IB, Altun S, Duman M (2020b) Phenotypic, phylogenetic characterization and antimicrobial susceptibility determination of Chryseobacterium piscicola isolates recovered from diseased rainbow trout. J Anatol Environ Animal Sci 5(4):624–629. https://doi.org/10.35229/jaes.808537

Susilawati L, Iwai N, Komatsu K, Arie T (2021) Antifungal activity of bacteria isolated from Japanese frog skin against plant pathogenic fungi. Biol Control 153:104498. https://doi.org/10.1016/j.biocontrol.2020.104498

Tong Q, Cui LY, Bie J, Han XY, Hu ZF, Wang HB, Zhang JT (2021) Changes in the gut microbiota diversity of brown frogs (Rana dybowskii) after an antibiotic bath. BMC Vet Res 17:1–13. https://doi.org/10.1186/s12917-021-03044-z DOI

Turkherptil (2021) AdaMerOs Herptil Türkiye http://www.turkherptil.org/Turkiyeden.asp . Accessed 03 May 2023

TUİK (2018) Turkish Statistical Institute. Central Distribution System. Fisheries Statistics. https://biruni.tuik.gov.tr/medas/?kn=97&locale=tr . Accessed 04 September 2021

Türkiş L (2020) A preliminary study on lenght-weight relationship on marsh frog (Pelophylax ridibundus Pallas, 1771) and long-legged wood frog (Rana macrocnemis Boulenger, 1885). Dissertation, Ordu University.

Türkiş S (2013) Investigating some vegetation types’ bio-diversity in Mesudıye (Ordu) and around. Dissertation, Ondokuz Mayıs University.

Vijayakumar S, Biswas I, Veeraraghavan B (2019) Accurate identification of clinically important Acinetobacter spp.: an update. Future Sci OA 5(7):FSO395. https://doi.org/10.2144/fsoa-2018-0127

Wang X, Hu M, Xia Y, Wen X, Ding K (2012) Pyrosequencing analysis of bacterial diversity in 14 wastewater treatment systems in China. Appl Environ Microbiol 78(19):7042–7047. https://doi.org/10.1128/AEM.01617-12 PubMed DOI PMC

Yang X, Hou X, Wei L, Li Y, Qin M, Song T, Li Y (2020) Characterization of skin symbiotic bacteria of sympatric amphibians in Southeastern China. Asian Herpetol Res 11(4):381–393B. https://doi.org/10.16373/j.cnki.ahr.200033

Yılmaz E (2021) Holdridge ecological zones of Turkey in Last Glacial Maximum (LGM) using high resolution CCSM4 model data and comparison of current climate conditions. Turk J Geogr Sci 19(2):331–367. https://doi.org/10.33688/aucbd.880675

Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: a taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol 67:1613–1617. https://doi.org/10.1099/ijsem.0.001755 PubMed DOI PMC