Biofilms provide resistance to antimicrobial agents and the body’s immune response in microorganisms that colonise the digestive tract of animals, in particular poultry. The study of biofilm formation of indicatory bacteria isolated from chickens under different keeping conditions allows assessing the impact of environmental factors on their phenotypic adaptation and potential risk to animal and human health. The purpose of the study was to determine the ability of Escherichia coli and Enterococcus faecalis bacteria isolated from chickens kept in a vivarium and on free range to form biofilms. Bacteriological, morphological, biochemical, and microscopic research methods were applied. The intensity of biofilm formation in indicatory microorganisms was assessed by the adsorption/resorption index of a 0.1% solution of crystal violet using polystyrene Petri dishes. The optical density was measured spectrophotometrically at a wavelength of 570 nm. It was found that E. coli, E. faecalis, Klebsiella spp. and Pseudomonas aeruginosa were isolated in samples from chickens kept in a vivarium, while in free-range chickens, representatives of the genus Klebsiella and Pseudomonas aeruginosa were not detected, indicating a lower presence of potential pathogens in natural conditions. All the cultures under study formed low- or medium-density biofilms. For E. coli isolates obtained from free-range chickens, the average value λ = 0.264 ± 0.09, while for vivarium isolates – λ = 0.187 ± 0.07. Cultures of E. faecalis biofilms were formed with an intensity of λ = 0.217 ± 0.04 in free-range chickens and λ = 0.137 ± 0.03 in vivarium chickens. Consequently, isolates obtained from natural conditions were characterised by a higher intensity of biofilm formation – by 41.2% (E. coli) and 58.4% (E. faecalis) in comparison with the conditions of a controlled microclimate. This may indicate a stimulating effect of environmental factors on the expression of adhesion and biofilm formation genes. However, all cultures under study were isolated from clinically healthy chickens, which indicates a commensal nature of the microbiome. The results obtained are important for assessing the risks of horizontal transfer of resistance genes and the formation of stable microbial biofilms in poultry farming
indicatory bacteria; Escherichia coli; Enterococcus faecalis; poultry keeping conditions; phenotypic adaptation; sanitary and microbiological monitoring
[1] ARRIVE. (n.d.). ARRIVE guidelines. Retrieved from https://arriveguidelines.org.
[2] Bezpalko, O., Ushkalov, A., Davydovska, L., Ushkalov, V., Machuskyy, O., Melnyk, V., Shevchenko, O., & Musiiets, I. (2024). Composition of indicator bacteria in industrial and garden keeping of chickens. One Health & Risk Management, 5(3), 42-51. doi: 10.38045/ohrm.2024.3.05.
[3] Chiang-Ni, C., Huang, J.Y., Hsu, C.Y., Lo, Y.C., Chen, Y.M., Lai, C.H., & Chiu, C.H. (2024). Genetic diversity, biofilm formation, and vancomycin resistance of clinical Clostridium innocuum isolates. BMC Microbiology, 24, article number 353. doi: 10.1186/s12866-024-03503-1.
[4] Cordero, M., Mitarai, N., & Jauffred, L. (2023). Motility mediates satellite formation in confined biofilms. The ISME Journal, 17(11), 1819-1827. doi: 10.1038/s41396-023-01494-x.
[5] de la Fuente-Núñez, C., Reffuveille, F., Fernández, L., & Hancock, R.E.W. (2013). Bacterial biofilm development as a multicellular adaptation: Antibiotic resistance and new therapeutic strategies. Current Opinion in Microbiology, 16(5), 580-589. doi: 10.1016/j.mib.2013.06.013.
[6] del Mar Cendra, M., & Torrents, E. (2021). Pseudomonas aeruginosa biofilms and their partners in crime. Biotechnology Advances, 49, article number 107734. doi: 10.1016/j. biotechadv.2021.107734.
[7] Directive 2010/63/EU of the European Parliament and of the Council “On the Protection of Animals Used for Scientific Purposes”. (2010, September). Retrieved from https://eur-lex. europa.eu/eli/dir/2010/63/oj/eng.
[8] DSTU 8534:2015. (2015). Food products. Method for detecting and determining the number of enterococci. Retrieved from https://surl.li/irxwgb.
[9] DSTU 8703-1:2017. (2017). Veterinary medicine. Diagnosis of infectious diseases. Part 1: Methods of sampling, packaging and transportation. Retrieved from https://online.budstandart.com/ua/ catalog/doc-page?id_doc=90401.
[10] DSTU 8703-2:2017. (2017). Veterinary medicine. Diagnosis of infectious diseases. Part 2: Safety requirements. Retrieved from https://online.budstandart.com/ua/catalog/doc-page.html?id_ doc=90410.
[11] EFSA, & ECDC. (2024). The European Union One Health 2023 zoonoses report. EFSA Journal, 22(1), article number e09106. doi: 10.2903/j.efsa.2024.9106.
[12] Geraldes, C., Tavares, L., Gil, S., & Oliveira, M. (2022). Enterococcus virulence and resistance traits associated with its permanence in the hospital environment. Antibiotics, 11(7), article number 857. doi: 10.3390/antibiotics11070857.
[13] Gupta, P., Sarkar, S., Das, B., Bhattacharjee, S., & Tribedi, P. (2016). Biofilm, pathogenesis and prevention – a journey to break the wall: A review. Archives of Microbiology, 198(1), 1-15. doi: 10.1007/s00203-015-1148-6.
[14] ISO 10273:2017. (2017). Microbiology of the food chain – horizontal method for the detection of pathogenic Yersinia enterocolitica. Retrieved from https://www.iso.org/standard/63508.html.
[15] ISO 11290-1:2017. (2017). Microbiology of the food chain – horizontal method for the detection and enumeration of Listeria monocytogenes and Listeria spp. – Part 1: Detection method. Retrieved from https://www.iso.org/standard/60313.html.
[16] ISO 21528-1:2017. (2017). Microbiology of the food chain – horizontal method for the detection and enumeration of Enterobacteriaceae – Part 1: Detection method. Retrieved from https:// online.budstandart.com/ua/catalog/doc-page.html?id_doc=108059.
[17] ISO 6579-1:2017. (2017). Microbiology of the food chain – horizontal method for the detection, enumeration and serotyping of SalmonellaPart 1: Detection of Salmonella spp. Retrieved from https://www.iso.org/ru/standard/56712.html.
[18] ISO 6887-1:2017. (2017). Microbiology of the food chain – preparation of test samples, initial suspension and decimal dilutions for microbiological examination – Part 1: General rules for the preparation of the initial suspension and decimal dilutions. Retrieved from https://www.iso.org/ ru/standard/63335.html.
[19] Kalantar-Neyestanaki, D., Mansouri, S., & Tadjrobehkar, O. (2023). Biofilm-associated genes in coagulase-negative staphylococci. BMC Microbiology, 23, article number 222. doi: 10.1186/ s12866-023-02959-x.
[20] Krawczyk, B., Wityk, P., Gałęcka, M., & Michalik, M. (2021). The many faces of Enterococcus spp. Microorganisms, 9(9), article number 1900. doi: 10.3390/microorganisms9091900.
[21] Kukhtyn, M., & Krushelnytska, N. (2014). Forming of biofilms of microorganisms obtained from milking equipment. Animal Biology, 16, 95-103.
[22] Lebreton, F., Willems, R.J.L., & Gilmore, M.S. (2014). Enterococcus diversity, origins in nature, and gut colonization. In M.S. Gilmore, D.B. Clewell, Y. Ike & N. Shankar (Eds.), Enterococci: From commensals to leading causes of drug resistant infection. Boston: Massachusetts Eye and Ear Infirmary.
[23] Li, J., et al. (2024) The intestinal microflora di-versity of aboriginal chickens in Jiangxi province, China. Poultry Science, 103(2), article number 103198. doi: 10.1016/j.psj.2023.103198.
[24] Lindstedt, B.A., Finton, M.D., Porcellato, D., & Brandal, L.T. (2018). High frequency of hybrid Escherichia coli strains with combined Intestinal Pathogenic Escherichia coli (IPEC) and Extraintestinal Pathogenic Escherichia coli (ExPEC) virulence factors isolated from human faecal samples. BMC Infectious Diseases, 18(1), article number 544. doi: 10.1186/s12879-0183449-2.
[25] Mahale, R.P., Princy, A., Maheshwarappa, Y.D., & Sumana, M.N. (2025). Comparative evaluation of biofilm forming capacity in uropathogenic and commensal E. coli. Frontiers in Cellular and Infection Microbiology, 15, article number 1570422. doi: 10.3389/fcimb.2025.1570422.
[26] Manders, T.T.M., de Louwere, J.J., Meijerhof, R., Nangsuay, A., van de Beek, M., van der GraafBloois, L., Zomer, A.L., Vargas, F., & de Wit, J.J. (2025). Severe effects of Escherichia coli and Enterococcus faecalis on hatchability and first-week performance following inoculation of 18-day-incubated embryonated broiler eggs. Poultry Science, 104(10), article number 105561. doi: 10.1016/j.psj.2025.105561.
[27] Order of the Cabinet of Ministers of Ukraine No. 1416-r “On Approval of the Strategy for Ensuring Biological Safety and Biological Protection According to the ‘One Health’ Principle for the Period until 2025 and Approval of the Action Plan for its Implementation”. (2019, November). Retrieved from https://zakon.rada.gov.ua/laws/show/1416-2019-%D1%80.
[28] Otokunefor, K., Melex, D., & Abu, G. (2020). Biofilm forming potential of Escherichia coli from various sources. Journal of Life and Bio Sciences Research, 1(2), 26-29. doi: 10.38094/ jlbsr1210.
[29] Pinto, S.C., Aleixo, J., Camela, K., Chilundo, A.G., & Bila, C.G. (2022). Seroprevalence of infectious bronchitis virus and avian reovirus in backyard chickens. Open Journal of Veterinary Research, 89(1), article number a2042. doi: 10.4102/ojvr.v89i1.2042.
[30] Ramos, S., Silva, V., Dapkevicius, M.L.E., Caniça, M., Tejedor-Junco, M.T., Igrejas, G., & Poeta, P. (2020). Escherichia coli as commensal and pathogenic bacteria among food-producing animals: Health implications of extended spectrum β-lactamase (ESBL) production. Animals, 10(12), article number 2239. doi: 10.3390/ani10122239.
[31] Sequeira, R.P., McDonald, J.A.K., Marchesi, J.R., & Clarke, T.B. (2020). Commensal bacteroidetes protect against Klebsiella pneumoniae colonization and transmission through IL-36 signalling. Nature Microbiology, 5, 304-313. doi: 10.1038/s41564-019-0640-1.
[32] Sharma, S., Mohler, J., Mahajan, S.D., Schwartz, S.A., Bruggemann, L., & Aalinkeel, R. (2023). Microbial biofilm: A review on formation, infection, antibiotic resistance, control measures, and innovative treatment. Microorganisms, 11(6), article number 1614. doi: 10.3390/ microorganisms11061614.
[33] Sonderholm, M., Bjarnsholt, T., Alhede, M., Kolpen, M., Jensen, P.O., Kühl, M., & Kragh, K.N. (2017). The consequences of being in an infectious biofilm: Microenvironmental conditions governing antibiotic tolerance. International Journal of Molecular Sciences, 18(12), article number 2688. doi: 10.3390/ijms18122688.
[34] Stepanovic, S., Vukovic, D., Dakic, I., Savic, B., & Svabic-Vlahovic, M. (2000). Modified microtiter-plate test for quantifying biofilm formation. Journal of Microbiological Methods, 40(2), 175-179. doi: 10.1016/S0167-7012(00)00122-6.
[35] Tran, N.B.V., Truong, Q.M., Nguyen, L.Q.A., Nguyen, N.M.H., Tran, Q.H., Dinh, T.T.P., Hua, V.S., Nguyen V.D., Lambert, P.A., & Nguyen, T.T.H. (2023). Prevalence and virulence of commensal Pseudomonas aeruginosa isolates from healthy individuals in Southern Vietnam (2018-2020). Biomedicines, 11(1), article number 54. doi: 10.3390/biomedicines11010054.
[36] Vygovska, L., Davydovska, L., Ushkalov, A., Melnyk, V., Ushkalov, V., & Shevchenko, O. (2025). Indicator microflora of ducks and chickens in home farm conditions. Veterinary Sciences and Practices, 20(1), 24-32. doi: 10.17094/vetsci.1577819.
[37] Xiaoxia, K., Xiaoxiao, Y., Yue, H., Conglin, G., Yuechen, L., Haiwei, J., Yuling, Q., & Li, W. (2023). Strategies and materials for the prevention and treatment of biofilms. Materials Today Bio, 23, article number 100827. doi: 10.1016/j.mtbio.2023.100827.