Isolation and characterization of Bordetella bronchiseptica bacteriophages using prophage induction and enrichment methods
Abstract
Bordetella bronchiseptica is an important bacterial pathogen of respiratory infections in companion animals and a promising target for bacteriophage application. However, the efficiency of isolating specific bacteriophages from clinical material remains low, and the factors associated with successful phage isolation have not been sufficiently studied. The aim of this study was to optimize methods for the isolation of B. bronchiseptica bacteriophages and to determine the factors associated with the efficiency of their isolation from clinical material. A total of 437 dogs and cats with and without sym p toms of respiratory diseases were examined. The presence of B. bronc hiseptica was determined using polymerase chain reaction and bacteriological methods. PCR was used as a confirmatory method for the presence of the pathogen, whereas bacteriological isolation of the culture was evaluated as an indicator of the presence of a viable bacterial host required for bacteriophage replication. Phage isolation was performed using the direct isolation method, as well as with the application of prophage induction, preliminary enrichment, and chloroform treatment. According to the PCR results, B. bronchiseptica DNA was detected in 124 of 437 animals (28.4 % ), whereas viable cultures of B. bronchiseptica were isolated only in 53 cases (12.1 % ). The highest frequency of bacteriological isolation of the pathogen was established in animals with an acute course of respiratory infection (31.7%), whereas significantly lower frequencies were observed in animals with a chronic course of respiratory disease and in animals without clinical signs of respiratory pathology ( 6.7 % and 1.4 % , respectively ) . When using the direct isolation method, bacteriophages were detected only in 3 of 124 PCR-positive samples (2.4 % ). In contrast, the use of a combined protocol including prophage induction, preliminary enrichment, and chloroform treatment made it possible to increase the frequency of bacteriophage isolation to 8.9 % (11 of 124 samples), which statistically exceeded the efficiency of the classical direct isolation method (OR = 3.9; 95 % CI: 1.0–15.0; P = 0.048). In bacteriologically positive samples, bacteriophages were isolated significantly more frequently than in samples positive only according to PCR results (OR = 7.1; 95 % CI: 1.5–34.0). Of the 11 isolated bacteriophages, 10 were obtained from animals with an acute course of respiratory infection. The obtained results indicate that the key factors for the effective isolation of B. bronchiseptica bacteriophages are the presence of a viable bacterial host and the acute phase of the infectious process. To increase the frequency of bacteriophage isolation, it is advisable to collect clinical material from animals with an acute course of bordetellosis and to use a combined protocol including prophage induction, preliminary enrichment, and chlor o form treatment.References
Badhai, J., & Das, S. K. (2023). Genomic evidence and virulence properties decipher the extra-host origin of Bordetella bronchiseptica. Journal of Applied Microbiology, 134(9), lxad200.
Belcher, T., Dubois, V., Rivera-Millot, A., Locht, C., & Jacob-Dubuisson, F. (2021). Pathogenicity and virulence of Bordetella pertussis and its adaptation to its strictly human host. Virulence, 12(1), 2608–2632.
Belhart, K., Sisti, F., Gestal, M. C., & Fernández, J. (2023). Bordetella bronchiseptica diguanylate cyclase BdcB inhibits the type three secretion system and impacts the immune response. Scientific Reports, 13(1), 7157.
Bichet, M. C., Chin, W. H., Richards, W., Lin, Y. W., Avellaneda-Franco, L., Hernandez, C. A., Oddo, A., Chernyavskiy, O., Hilsenstein, V., Neild, A., Li, J., Voelcker, N. H., Patwa, R., & Barr, J. J. (2021). Bacteriophage uptake by mammalian cell layers represents a potential sink that may impact phage therapy. iScience, 24(4), 102287.
Bogach, M. V., Paliy, A. P., Perotsʼka, L. V., Pyvovarova, І. V., Stoyanova, V. Y., & Palii, A. P. (2020). The influence of hydro-meteorological conditions on the spread of chicken cestodiasis. Regulatory Mechanisms in Biosystems, 11(3), 414–418.
Chakraborty, N., Halder, S., Keswani, C., Vaca, J., Ortiz, A., & Sansinenea, E. (2024). New aspects of the effects of climate change on interactions between plants and microbiomes: A review. Journal of Basic Microbiology, 64(10), e2400345.
Chen, X., Wei, Y., & Ji, X. (2021). Research progress of prophages. Yi Chuan, 43(3), 240–248.
Chen, Y., Yang, L., Sun, E., Song, J., & Wu, B. (2019). Characterisation of a newly detected bacteriophage infecting Bordetella bronchiseptica in swine. Archives of Virology, 164(1), 33–40.
Chen, Y., Yang, L., Yang, D., Song, J., Wang, C., Sun, E., Gu, C., Chen, H., Tong, Y., Tao, P., & Wu, B. (2020). Specific integration of temperate phage decreases the pathogenicity of host bacteria. Frontiers in Cellular and Infection Microbiology, 10, 14.
Cornuault, J. K. (2024). CRISPRpi: Inducing and curing prophage using the CRISPR interference. Methods in Molecular Biology, 2793, 257–271.
Cornuault, J. K., & Moineau, S. (2021). Induction and elimination of prophages using CRISPR interference. The CRISPR Journal, 4(4), 549–557.
Costa, P., Pereira, C., Romalde, J. L., & Almeida, A. (2024). A game of resistance: War between bacteria and phages and how phage cocktails can be the solution. Virology, 599, 110209.
Cui, L., Kiga, K., Kondabagil, K., & Węgrzyn, A. (2024). Current and future directions in bacteriophage research for developing therapeutic innovations. Scientific Reports, 14(1), 24404.
Dear, J. D. (2020). Bacterial pneumonia in dogs and cats: An update. The Veterinary Clinics of North America, Small Animal Practice, 50(2), 447–465.
Dion, M. B., Oechslin, F., & Moineau, S. (2020). Phage diversity, genomics and phylogeny. Nature Reviews Microbiology, 18(3), 125–138.
Dong, J., Tsui, W. N. T., Leng, X., Fu, J., Lohman, M., Anderson, J., Hamill, V., Lu, N., Porter, E. P., Gray, M., Sebhatu, T., Brown, S., Pogranichniy, R., Wang, H., Noll, L., & Bai, J. (2022). Development of a three-panel multiplex real-time PCR assay for simultaneous detection of nine canine respiratory pathogens. Journal of Microbiological Methods, 199, 106528.
Faruk, O., Jewel, Z. A., Bairagi, S., Rasheduzzaman, M., Bagchi, H., Tuha, A. S. M., Hossain, I., Bala, A., & Ali, S. (2025). Phage treatment of multidrug-resistant bacterial infections in humans, animals, and plants: The current status and future prospects. Infectious Medicine, 4(1), 100168.
Ferriol-González, C., & Domingo-Calap, P. (2021). Phage therapy in livestock and companion animals. Antibiotics, 10(5), 559.
Guerrero-Bustamante, C. A., & Hatfull, G. F. (2024). Bacteriophage tRNA-dependent lysogeny: Requirement of phage-encoded tRNA genes for establishment of lysogeny. mBio, 15(2), e03260-23.
Hadzevych, D. V., Paliy, A. P., Hadzevych, O. V., Pavlichenko, O. V., Kovalenko, L. V., Dunaev, Y. K., & Gerilovych, I. O. (2026). Relationship between the Bvg+ phenotype of clinical isolates of Bordetella bronchiseptica and their immunogenic potential. Regulatory Mechanisms in Biosystems, 17(2), e26045.
Hosseindoust, A., Choi, Y., Ha, S., Tajudeen, H., Mun, J., Kinara, E., Kim, Y., & Kim, J. (2023). Anti-Bordetella bronchiseptica effects of targeted bacteriophages via microbiome and metabolic mediated mechanisms. Scientific Reports, 13(1), 21755.
Huang, X., Hou, Y., Zhao, M., Chen, J., Zhu, Z., Liu, H., Wang, M., Hua, L., Chen, H., Wu, B., & Peng, Z. (2025). Identification of a broad-spectrum lytic Bordetella phage and assessments of its potential for combating Bordetella infections. Virology, 608, 110545.
Huang, Y., Wang, W., Zhang, Z., Gu, Y., Huang, A., Wang, J., & Hao, H. (2022). Phage products for fighting antimicrobial resistance. Microorganisms, 10(7), 1324.
Jang, J. Y., Lee, D., Oh, S. Y., & Yoo, H. S. (2025). Co-infections with Bordetella bronchiseptica in canine: A systematic review and meta-analysis. Veterinary Immunology and Immunopathology, 280, 110886.
Jo, S. J., Kwon, J., Kim, S. G., & Lee, S. J. (2023). The biotechnological application of bacteriophages: What to do and where to go in the middle of the post-antibiotic era. Microorganisms, 11(9), 2311.
Kadhim, H. M., Al-Galebi, A. A. S., Al-Hassani, M. K. A., & Gharban, H. A. J. (2025). Molecular and serological incidences of Bordetella bronchiseptica in pet dogs with urinary infections. Open Veterinary Journal, 15(3), 1397–1406.
Kameyama, H., Fujimoto, Y., Tomioka, Y., Yamamoto, S., Suyama, H., Inoue, H., Takahashi, E., & Ono, E. (2022). Pathogenicity of Bordetella bronchiseptica isolated from apparently healthy rabbits in guinea pig, rat, and mouse. The Journal of Veterinary Medical Science, 84(4), 574–581.
Kolchyk, O., Illarionova, T., Buzun, A., Paliy, A., & Palii, A. (2022). Influence of probiotic microorganisms on microbial biofilms in feeds. Scientific Horizons, 25(1), 41–50.
Lavan, R., & Knesl, O. (2015). Prevalence of canine infectious respiratory pathogens in asymptomatic dogs presented at US animal shelters. The Journal of Small Animal Practice, 56(9), 572–576.
Li, C., Tan, L., Ma, Y., Li, Z., Xu, S., Zheng, X., Fang, H., Hong, J., Zhu, Q., Huo, X., Guo, H., & Zhang, W. (2026). A bacteriophage with dual host specificity for canine and porcine Bordetella bronchiseptica: Characterization and biofilm disruption potential. Virology, 613, 110714.
Łobocka, M., Dąbrowska, K., & Górski, A. (2021). Engineered bacteriophage therapeutics: Rationale, challenges and future. BioDrugs, 35(3), 255–280.
Loponte, R., Pagnini, U., Iovane, G., & Pisanelli, G. (2021). Phage therapy in veterinary medicine. Antibiotics, 10(4), 421.
Ma, K., Sun, W., Pan, G., Xu, Y., Shi, Y., & Chen, Y. (2026). Antimicrobial resistance in Bordetella pertussis: A systematic review and meta-analysis. Epidemiology and Infection, 154, e25.
Miguelena Chamorro, B., De Luca, K., Swaminathan, G., Longet, S., Mundt, E., & Paul, S. (2023). Bordetella bronchiseptica and Bordetella pertussis: Similarities and differences in infection, immuno-modulation, and vaccine considerations. Clinical Microbiology Reviews, 36(3), e00164-22.
Mugni, S. L., Ambrosis, N., O Toole, G. A., Sisti, F., & Fernández, J. (2025). Interplay of virulence factors and signaling molecules: albumin and calcium-mediated biofilm regulation in Bordetella bronchiseptica. Journal of Bacteriology, 207(4), e0044524.
Nicholson, T. L., & Shore, S. M. (2024). Comparative analysis of antimicrobial resistance and genetic diversity of Bordetella bronchiseptica isolates obtained from swine within the United States. Frontiers in Microbiology, 15, 1501373.
Nikolich, M. P., & Filippov, A. A. (2020). Bacteriophage therapy: Developments and directions. Antibiotics, 9(3), 135.
Olawade, D. B., Fapohunda, O., Egbon, E., Ebiesuwa, O. A., Usman, S. O., Faronbi, A. O., & Fidelis, S. C. (2024). Phage therapy: A targeted approach to overcoming antibiotic resistance. Microbial Pathogenesis, 197, 107088.
Olszewska, P., Spietelun, M., Syguła, K., Ossowski, A., & Grygorcewicz, B. (2025). Bacteriophages as a modern diagnostic tool: Innovations, applications and challenges. Molecular Biology Reports, 52, 997.
Paliy, A. P. (2018). Dyferentsiyna chutlyvist’ mikobakteriy do khlornykh dezinfektantiv [Differential sensitivity of mycobacterium to chlorine disinfectants]. Mikrobiolohichnyi Zhurnal, 80(2), 104-116 (in Ukrainian).
Paliy, A., Pavlichenko, O., Kasianenko, S., Kovalenko, L., Stockiy, A., & Stotska, O. (2023). Peculiarities of the course of demodicosis in domestic animals in a megalopolis in the east of Ukraine. Regulatory Mechanisms in Biosystems, 14(1), 28–33.
Park, G. Y., Yu, H. J., Son, J. S., Park, S. J., Cha, H. J., & Song, K. S. (2020). Specific bacteriophage of Bordetella bronchiseptica regulates B. bronchiseptica-induced microRNA expression profiles to decrease inflammation in swine nasal turbinate cells. Genes and Genomics, 42(4), 441–447.
Petrovic Fabijan, A., Aleksic Sabo, S., Gavric, D., Doffkay, Z., Rakhely, G., & Knezevic, P. (2021). Are Bordetella bronchiseptica siphoviruses appropriate for phage therapy. Viruses, 13(9), 1732.
Rybolt, L. E., Sabunwala, S., & Greene, J. N. (2022). Zoonotic bacterial respiratory infections associated with cats and dogs: A case series and literature review. Cureus, 14(4), e24414.
Stewart, G. S., Jassim, S. A., Denyer, S. P., Newby, P., Linley, K., & Dhir, V. K. (1998). The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification. Journal of Applied Microbiology, 84(5), 777–783.
Strathdee, S. A., Hatfull, G. F., Mutalik, V. K., & Schooley, R. T. (2023). Phage therapy: From biological mechanisms to future directions. Cell, 186(1), 17–31.
Szymczak, M., Grygorcewicz, B., Karczewska-Golec, J., Decewicz, P., Pankowski, J. A., Országh-Szturo, H., Bącal, P., Dołęgowska, B., & Golec, P. (2020). Characterization of a unique Bordetella bronchiseptica vB_BbrP_BB8 bacteriophage and its application as an antibacterial agent. International Journal of Molecular Sciences, 21(4), 1403.
Tabatabaei, M., & Rohani, H. R. (2022). Identification of Bordetella bronchiseptica in the throat and nose of dogs and cats by PCR. Molecular Biology Research Communications, 11(3), 127–131.
Tang, B., Hu, X., Song, Y., Liu, X., Zhao, G., & Yue, M. (2026). Acquired antimicrobial resistance genes in Bordetella species: A global genomic analysis. Journal of Antimicrobial Chemotherapy, 81(1), dkaf418.
Uchechukwu, C. F., & Shonekan, A. (2024). Current status of clinical trials for phage therapy. Journal of Medical Microbiology, 73(9), 001895.
Venturini, C., Petrovic Fabijan, A., Fajardo Lubian, A., Barbirz, S., & Iredell, J. (2022). Biological foundations of successful bacteriophage therapy. EMBO Molecular Medicine, 14(7), e12435.
Walczak, Ł. J., Kwiatkowska, M., Twarowski, B., Kubacka, M., Paluch, J., & Herbet, M. (2025). Disinfectant-induced bacterial resistance and antibiotic cross-resistance-mechanisms and clinical relevance. Clinical and Experimental Medicine, 26(1), 26.
Yi, L., Fan, H., Yuan, S., Li, R., Wang, H., Quan, Y., Zhang, H., Wang, Y., & Wang, Y. (2024). Antimicrobial resistance and biofilm formation of Bordetella bronchiseptica in Central China, with evidence of a rare heteroresistance strain to gentamicin. Animals, 14(9), 1301.
Zhang, Y., Yang, H., Guo, L., Zhao, M., Wang, F., Song, W., Hua, L., Wang, L., Liang, W., Tang, X., Peng, Z., & Wu, B. (2021). Isolation, antimicrobial resistance phenotypes, and virulence genes of Bordetella bronchiseptica from pigs in China, 2018–2020. Frontiers in Veterinary Science, 8, 672716.
Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons «Attribution» 4.0 License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
