Anaerobik Gut Fungus Piromyces sp.’den Elde Edilen İkincil Metabolitlerin Antimikrobiyal Potansiyelinin Araştırılması

Öz

Herbivorların sindirim sisteminde yaşayan anaerobik gut fungusları, lignoselülozik biyokütleyi parçalama yetenekleriyle bilinseler de, ikincil metabolit üretim kapasiteleri henüz tam olarak ortaya konulmamıştır. Bu çalışma, Tunceli yöresindeki sığır dışkılarından izole edilen Piromyces sp. suşlarının morfolojik ve moleküler karakterizasyonunu yaparak, patojen mikroorganizmalara karşı antibakteriyel etkilerini ortaya koymayı amaçlamaktadır. İzolasyonda Roll-Tube yöntemi kullanılmıştır. İzolatların tanımlanması morfolojik ve moleküler düzeyde doğrulanmıştır. En yüksek biyokütle artışını gösteren Piromyces sp. AGF‑3 suşundan 3, 5 ve 7 günlük kültürlerde etil asetat ekstraksiyonu ile sekonder metabolitler elde edilmiştir. Bu ekstraktların 15 patojen üzerindeki etkileri agar kuyusu difüzyon yöntemiyle değerlendirilmiştir. Moleküler analizler, izole edilen dört AGF örneğinin (AGF‑1–4) Piromyces cinsine ait olduğunu doğrulamıştır (%92,13 ile %97,44). Piromyces sp. AGF‑3 ekstraktları, test edilen 15 patojenin %73’ünde 3. ve 5. günlerde, %80’inde ise 7. günde antibakteriyel aktivite göstermiştir. En yüksek inhibisyon zonları; Salmonella kentucky (8,22±0,13 mm), Salmonella enteritidis (7,45±0,20 mm) ve Serratia marcescens (7,37±0,14 mm) üzerinde kaydedilmiştir. İnhibisyon etkisinin 3. günden 7. güne doğru artış göstermesi, metabolit sentezinin zamanla yoğunlaştığına işaret etmektedir. Bu çalışma, literatürde Piromyces türlerinin antibakteriyel özelliklerini değerlendiren ilk araştırma niteliğindedir. Bulgular, Piromyces sp. izolatlarının seçici ve zamana bağlı antibakteriyel aktivite sergilediğini göstermiştir. Ayrıca anaerobik gut fungusların yalnızca rumen ekolojisinde değil, yeni doğal antimikrobiyal bileşiklerin keşfinde de stratejik bir kaynak oluşturduğunu göstermektedir. Bu ikincil metabolitlerin kimyasal karakterizasyonu için HPLC ve GC-MS gibi ileri analitik yöntemleri kullanılarak, yeni doğal antimikrobiyal bileşiklerin ortaya çıkarılması büyük önem taşımaktadır.

Anahtar Kelimeler

• Anaerobik gut funguslar
• Piromyces
• Antimikrobiyal aktivite
• İkincil metabolitler
• Rumen mikrobiyotası.

Investigation of the Antimicrobial Potential of Secondary Metabolites Obtained from the Anaerobic Gut Fungus Piromyces sp.

Öz

Anaerobic gut fungi living in the digestive system of herbivores, although known for their ability to degrade lignocellulosic biomass, have not yet had their secondary metabolite production capacities fully explored. This study aims to reveal the antibacterial effects of Piromyces sp. strains isolated from cattle dung in the Tunceli region against pathogenic microorganisms through morphological and molecular characterization. The Roll-Tube method was used for isolation. Identification of the isolates was confirmed at both morphological and molecular levels. From Piromyces sp. AGF‑3, which showed the highest biomass production, secondary metabolites were obtained via ethyl acetate extraction from 3-, 5-, and 7-day cultures. The effects of these extracts on 15 pathogens were evaluated using the agar well diffusion method. Molecular analyses confirmed that the four AGF samples (AGF‑1–4) belonged to the genus Piromyces, with similarity rates ranging from 92.13% to 97.44%. Extracts of Piromyces sp. AGF‑3 exhibited antibacterial activity against 73% of the tested pathogens on days 3 and 5, and 80% on day 7. The highest inhibition zones were observed in Salmonella kentucky (8,22±0,13 mm), Salmonella enteritidis (7,45±0,20 mm), and Serratia marcescens (7,37±0,14 mm). The increase in inhibitory effect from day 3 to day 7 suggests that metabolite production intensifies over time. This study is the first in the literature to evaluate the antibacterial properties of Piromyces species. The findings indicate that Piromyces isolates exhibit selective and time-dependent antibacterial activity. Additionally, anaerobic gut fungi represent a strategic resource not only in rumen ecology but also in the discovery of novel natural antimicrobial compounds. The use of advanced analytical techniques such as HPLC and GC‑MS for the chemical characterization of these secondary metabolites is crucial for identifying new natural antimicrobial agents.

Anahtar Kelimeler

• Anaerobic gut fungi
• Piromyces
• Antimicrobial activity
• Secondary metabolites
• Rumen microbiota.

Kaynakça

1. Alves, M.J., Ferreira, I.C., Martins, A.N.A.B.E.L.A. ve Pintado, M. (2012). Antimicrobial activity of wild mushroom extracts against clinical isolates resistant to different antibiotics. Journal of Applied Microbiology. 113, 466-475.
2. Balouiri, M., Sadiki, M. ve Ibnsouda, S.K. (2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis. 6, 71-79.
3. Bano, S. A., Naz, S., Uzair, B., Hussain, M., Khan, M. M., Bibi, H., Habiba, U., Nisa, S., ve Israr, M. (2021). Detection of microorganisms with antibacterial activities from different industrial wastes and GC-MS analysis of crude microbial extract. Brazilian Journal of Biology, 83, e245585.
4. Barr, D.J.S., Kudo, H., Jakober, K.D. ve Cheng, K.J. (1989). Morphology and development of rumen fungi: Neocallimastix sp., Piromyces communis, and Orpinomyces bovis gen. nov., sp. nov. Canadian Journal of Botany. 67, 2815-2824.
5. Bhagat, N.R., Kumar, S., Kumari, R. ve Bharti, V.K. (2023). A review on rumen anaerobic fungi: current understanding on carbohydrate fermentation and roughages digestion in ruminants. Applied Biochemistry and Microbiology. 59, 231-249.
6. Cairns, T.C., Zheng, X., Zheng, P., Sun, J. ve Meyer, V. (2021). Turning inside out: Filamentous fungal secretion and its applications in biotechnology, agriculture, and the clinic. Journal of Fungi. 7, 535.
7. Calkins, S., Elledge, N.C., Hanafy, R.A., Elshahed, M.S. ve Youssef, N. (2016). A fast and reliable procedure for spore collection from anaerobic fungi: application for RNA uptake and long-term storage of isolates. J Microbiol Methods. 127, 206–213.
8. Cammack, K.M., Austin, K.J., Lamberson, W.R., Conant, G.C. ve Cunningham, H.C. (2018). Ruminant nutrition symposium: Tiny but mighty: The role of the rumen microbes in livestock production. Journal of Animal Science. 96, 752-770.

9. Conrado, R., Gomes, T.C., Roque, G.S.C. ve De Souza, A.O. (2022). Overview of bioactive fungal secondary metabolites: cytotoxic and antimicrobial compounds. Antibiotics. 11, 1604.
10. Cooper, D.G., Macdonald, C.R., Duff, S.J.B. ve Kosaric, N. (1981). Enhanced production of surfactin from Bacillus subtilis by continuous product removal and metal cation additions. Applied and Environmental Microbiology. 42, 408-412.
11. Cushnie, T.T. ve Lamb, A.J. (2005). Antimicrobial activity of flavonoids. International Journal of Antimicrobial Agents. 26, 343-356.
12. Çömlekcioğlu, U., Yazdıç, F.C., Keser, S., Kelleci, B.M., Battaloğlu, G. ve Özköse, E. (2012). Effects of carbon sources on enzyme production of Neocallimastix sp. and Orpinomyces sp. KSÜ Doğa Bilimleri Dergisi. 15, 30-37.
13. Çömlekçioğlu, U., Akyol, İ., Kar, B., Özköse, E. ve Ekinci, M.S. (2008). Anaerobik rumen funguslarının izolasyonu, tanımlanması ve kültür koleksiyonunun oluşturulması. Hayvansal Üretim Dergisi. 49, 28-34.
14. Dos Santos, A., Schultz, J., Almeida Trapp, M., Modolon, F., Romanenko, A., Kumar Jaiswal, A., ... ve Rosado, A.S. (2024). Investigating polyextremophilic bacteria in Al Wahbah crater, Saudi Arabia: A terrestrial model for life on Saturn’s moon Enceladus. Astrobiology. 24, 824-838.
15. Edwards, J. E., Forster, R. J., Callaghan, T. M., Dollhofer, V., Dagar, S. S., Cheng, Y., Chang, J., Kittelmann, S., Fliegerova, K., Puniya, A. K., Henske, J.K., Gilmor , S.P., O'Malley, M.A., Griffith, G.W. ve Smidt, H. (2017). PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: insights, challenges and opportunities. Frontiers in Microbiology, 8, 1657.
16. Elinç, C. ve Kar, B. (2025). Dağ Keçilerinden İzole Edilen Anaerobik Gut Fungusların Moleküler Tanımlanması. International Journal of Pure and Applied Sciences. 11, 604-615.
17. Eltem, R. ve Ucar, F. (1998). The determination of antimicrobial activity spectrum of 23 Bacillus strains isolated from Denizli-Acigol (Bitter Lake) which is soda lake (Na2SO4). J. KUKEM. 21, 57-64.
18. Gold, J.J., Heath, I.B. ve Bauchop, T. (1988). Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov., sp. nov., assigned to the Neocallimasticaceae. Biosystems. 21, 403-415.
19. Gruninger, R.J., Puniya, A.K., Callaghan, T.M., Edwards, J.E., Youssef, N., Dagar, S.S., ... ve ark. (2014). Anaerobic fungi (phylum Neocallimastigomycota): advances in understanding their taxonomy, life cycle, ecology, role and biotechnological potential. FEMS Microbiology Ecology. 90, 1–17.
20. Haitjema, C.H., Solomon, K.V., Henske, J.K., Theodorou, M.K. ve O'Malley, M.A. (2014). Anaerobic gut fungi: advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production. Biotechnology and Bioengineering. 111, 1471-1482.
21. Hanafy, R.A., Wang, Y., Stajich, J.E., Pratt, C.J., Youssef, N.H. ve Elshahed, M.S. (2023). Phylogenomic analysis of the Neocallimastigomycota: proposal of Caecomycetaceae fam. nov., Piromycetaceae fam. nov., and emended description of the families Neocallimastigaceae and Anaeromycetaceae. International Journal of Systematic and Evolutionary Microbiology. 73, 005735.
22. Hossain, T.J. (2024). Methods for screening and evaluation of antimicrobial activity: A review of protocols, advantages, and limitations. European Journal of Microbiology and Immunology. 14, 97-115.
23. Hungate, R.E. (1969). Chapter IV A roll tube method for cultivation of strict anaerobes. Methods in Microbiology. 3, 117-132.
24. Iwase, T.,Uehara, Y., Shinji, H., Tajima, A., Seo, H., Takada, K., Agata, T., ve Mizunoe, Y. (2010). Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. Nature, 465, 346-349.
25. Joblin, K.N. (1981). Isolation, enumeration, and maintenance of rumen anaerobic fungi in roll tubes. Applied and Environmental Microbiology. 42, 1119-1122.
26. Kalyani, P., Botsa, S.M., Laxmi, K.D. ve Anil, S. (2023). Optimization of cultural conditions for biomass and antibacterial metabolite production by Aspergillus fumigatus strain MF1. Hybrid Advances. 2, 100016.
27. Kamra, D.N. (2005). Rumen microbial ecosystem. Current Science. 89, 124-135.
28. Kar, B. (2024). Tunceli Yöresindeki At Dışkısı Örneklerinden İzole Edilen Anaerobik Gut Funguslarının (Piromyces spp.) Saf Kültürdeki Fibrolitik Aktiviteleri ve Moleküler Tanımlanması. International Journal of Pure and Applied Sciences. 10, 590-603.
29. Katarzyna, K. (2025). Advances in fungal natural products: Insights into bioactivity and therapeutic potential. Pharmaceuticals. 18, 1313. Knapp, S. (1995). Synthesis of complex nucleoside antibiotics. Chemical Reviews. 95, 1859-1876.
30. Liangzhi, L.I., Zheng, H. ve Yingjin, Y.U.A.N. (2007). Effects of propionate on streptolydigin production and carbon flux distribution in Streptomyces lydicus AS 4.2501. Chinese Journal of Chemical Engineering. 15, 143-149.
31. Liebetanz, E. (1910). Die parasitischen protozoen des wiederkäuermagens. Archiv für Protistenkunde. 19, 19-80.
32. Macielag, M.J. (2012). Chemical properties of antimicrobials and their uniqueness. Antibiotic Discovery and Development. 793-820.
33. McAllister, T.A., Thomas, K.D., Gruninger, R.J., Elshahed, M., Li, Y. ve Cheng, Y. (2025). International Symposium on Ruminant Physiology: Rumen fungi, archaea, and their interactions. Journal of Dairy Science. 108, 7545-7566.
34. McDonnell, G. ve Russell, A.D. (2001). Antiseptics and disinfectants: activity, action, and resistance. Clinical Microbiology Reviews. 14, 227-246.
35. Meili, C. H., Jones, A. L., Arreola, A. X., Habel, J., Pratt, C. J., Hanafy, R. A., Wang, Y., Yassin, A. S., TagElDein, M. A., Moon, C. D., Janssen, P. H., Shrestha, M., Rajbhandari, P., Nagler, M., Vinzelj, J. M., Podmirseg, S. M., Stajich, J. E., Goetsch, A. L., Hayes, J., Young, D., Fliegerova, K., Grilli, D. J., Vodička, R., Moniello, G., Mattiello, S., Kashef, M. T., Nagy, Y. I., Edwards, J. A., Dagar, S. S., Foote, A. P., Youssef, N. H., ve Elshahed, M. S. (2023). Patterns and determinants of the global herbivorous mycobiome. Nature Communications, 14, 3798.
36. Nash, K., Vinzelj, J., Pratt, C.J., Elshahed, M.S. ve Youssef, N.H. (2025). Piromyces struthionis, sp. nov., a new anaerobic gut fungus from the feces of ostriches. Mycologia. 117, 1044-1056.
37. Orpin, C.G. (1977). The rumen flagellate Piromonas communis: its life-history and invasion of plant material in the rumen. Microbiology. 99, 107-117.
38. Park, H.J. (2025). Influence of culture conditions on bioactive compounds in Cordyceps militaris: a comprehensive review. Foods. 14, 3408.
39. Paul, S.S., Deb, S.M., Punia, B.S., Singh, D. ve Kumar, R. (2010). Fibrolytic potential of anaerobic fungi (Piromyces sp.) isolated from wild cattle and blue bulls in pure culture and effect of their addition on in vitro fermentation of wheat straw and methane emission by rumen fluid of buffaloes. Journal of the Science of Food and Agriculture. 90, 1218-1226.
40. Perry, E.K., Meirelles, L.A. ve Newman, D.K. (2022). From the soil to the clinic: the impact of microbial secondary metabolites on antibiotic tolerance and resistance. Nature Reviews Microbiology. 20, 129-142.
41. Picard, K.T. (2017). Next-Generation Approaches to Understanding the Diversity and Evolution of Marine Fungi. Doktora Tezi. Duke University.
42. Rütten, A., Kirchner, T. ve Musiol-Kroll, E.M. (2022). Overview on strategies and assays for antibiotic discovery. Pharmaceuticals. 15, 1302. Schoch, C.L., Seifert, K.A., Huhndorf, S. ve ark. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci USA. 109, 6241–6246.
43. Sesli, E., Asan, A. ve Selçuk, F. (edlr.) Abacı Günyar, Ö., Akata, I., Akgül, H., Aktaş, S., Alkan, S., Allı, H., Aydoğdu, H., Berikten, D., Demirel, K., Demirel, R., Doğan, H.H., Erdoğdu, M., Ergül, C.C., Eroğlu, G., Giray, G., Halikî Uztan, A., Kabaktepe, Ş., Kadaifçiler, D., Kalyoncu, F., Karaltı, İ., Kaşık, G., Kaya, A., Keleş, A., Kırbağ, S., Kıvanç, M., Ocak, İ., Ökten, S., Özkale, E., Öztürk, C., Sevindik, M., Şen, B., Şen, İ., Türkekul, İ., Ulukapı, M., Uzun, Ya., Uzun, Yu.,Yoltaş, A. (2020). Türkiye Mantarları Listesi. İstanbul: Ali Nihat Gökyiğit Vakfı Yayınları. Syf 1177.
44. Ślusarczyk, J., Adamska, E. ve Czerwik-Marcinkowska, J. (2021). Fungi and algae as sources of medicinal and other biologically active compounds: A review. Nutrients. 13, 3178.
45. Swift, C. L., Louie, K. B., Bowen, B. P., Hooker, C. A., Solomon, K. V., Singan, V., Daum, C., Pennacchio, C. P., Barry, K., Shutthanandan, V., Evans, J. E., Grigoriev, I. V., Northen, T. R., ve O’Malley, M. A. (2021b). Cocultivation of anaerobic fungi with rumen bacteria establishes an antagonistic relationship. MBio, 12, e01441-21.
46. Swift, C. L., Louie, K. B., Bowen, B. P., Olson, H. M., Purvine, S. O., Salamov, A., Mondo, S. J., Solomon, K. V., Wright, A. T., Northen, T. R., Grigoriev, I. V., Keller, N. P., ve O’Malley, M. A. (2021a). Anaerobic gut fungi are an untapped reservoir of natural products. Proceedings of the National Academy of Sciences of the United States of America (PNAS), 118(18), e2019855118.
47. Torğut, G., Yazdıç, F.C. ve Gürler, N. (2022). Synthesis, characterization, pH‐sensitive swelling and antimicrobial activities of chitosan–graft‐poly (hydroxyethyl methacrylate) hydrogel composites for biomedical applications. Polymer Engineering & Science. 62, 2552-2559.
48. Wubah, D.A. (2004). Anaerobic zoosporic fungi associated with animals. Biodiversity of Fungi. 22, 501–510.
49. Yazdıç, F.C., Karaman, A. ve Yazdıç, F. (2018). Antimicrobial effect of secondary metabolites produced by some Bacillus strains developed in different energy sources. Journal of KUKEM. 21, 1-10.
50. Yazdıç, F.C., Yazdıç, F., Kar, B., Özköse, E. ve Ekinci, M.S. (2021). Anaerobik funguslarda hidrojenozomlar: Hidrojen üreten organeller. Mantar Dergisi. 12, 190-208.
51. Yazdic, F.C., Akyol, I. ve Ozkose, E. (2025). Production and Partial Characterisation of Bifunctional Cellulase Genes of Anaerobic Gut Fungus Orpinomyces sp. GMLF19. Applied Biochemistry and Microbiology. 61, 934-945.
52. Zhu, D., Adebisi, W.A., Ahmad, F., Sethupathy, S., Danso, B. ve Sun, J. (2020). Recent development of extremophilic bacteria and their application in biorefinery. Frontiers in Bioengineering and Biotechnology. 8, 483.