The influence of nutrient medium on the reproduction of bacteriophages active against clinically significant microorganisms

Valeriia  Poniatovska; Vadym  Poniatovskyi; Volodymyr  Shyrobokov

doi:10.32345/USMYJ.2(154).2025.249-256

Valeriia Poniatovska Assistant of the Department of Microbiology and Parasitology with Basics of Immunology of the Bogomolets National Medical University, 01601, Kyiv, Ukraine. https://orcid.org/0000-0001-6087-5755
Vadym Poniatovskyi Assistant of the Department of Microbiology and Parasitology with Basics of Immunology of the Bogomolets National Medical University, 01601, Kyiv, Ukraine. https://orcid.org/0000-0002-1503-3935
Volodymyr Shyrobokov Academician of the NAS and the NAMS of Ukraine, Head of the Department. of Microbiology and Parasitology with the Basics of Immunology of the Bogomolets National Medical University, 01601, Kyiv, Ukraine. https://orcid.org/0000-0003-0882-148X

DOI: https://doi.org/10.32345/USMYJ.2(154).2025.249-256

Keywords: Bacteriophages; Reproduction; Phage Therapy; Biotechnology; Microbiology; phage-bacterial infection, nutrient agar, plaques

Abstract

іmproving bacteriophage cultivation methods, optimizing nutrient media composition, and implementing modern production technologies will significantly increase the yield and quality of phage preparations. Enhancing production efficiency is a crucial step toward expanding the potential of modern phage therapy, raising the ability to combat antibiotic-resistant microorganisms, and stimulating the development of innovative biotechnological solutions. One of the key factors in improving the efficiency of phage production is the selection of optimal cultivation parameters. Changes in the chemical composition of the medium can either slow down or accelerate the growth rate of the bacterial culture, thereby indirectly affecting phage replication.

To determine the impact of nutrient media type on bacteriophage reproduction, five different bacteriological media and nine bacteriophages active against nine species of clinically significant microorganisms were used. The effect of media composition was assessed based on the number and size of plaque formations. Optimal replication of all nine bacteriophages was observed on Nutrient Agar No 1, which contained peptic digest of animal tissue, meat extract, and yeast extract. Additionally, good phage reproduction was noted on Mueller-Hinton Agar and Nutrient Medium. The least suitable medium for phage cultivation was Tryptic Soy Agar, where bacteriophage replication was the lowest. Improving cultivation methods and optimizing the composition of nutrient media for bacteriophage reproduction are crucial steps toward enhancing the efficiency of their production and application in phage therapy.

References

Batinovic S, Wassef F, Knowler SA, Rice DTF, Stanton CR, Rose J, et al. Bacteriophages in natural and artificial environments. Pathogens. 2019 Jul 12;8(3):100. doi:10.3390/pathogens8030100.

Pallavali R, Shin D, Choi J. Phage-based biocontrol of antibiotic-resistant bacterium isolated from livestock wastewater treatment plant. Water. 2023 Apr 21;15(8):1616. doi:10.3390/w15081616.

Hyla K, Dusza I, Skaradzińska A. Recent advances in the application of bacteriophages against common foodborne pathogens. Antibiotics. 2022 Nov 2;11(11):1536. doi:10.3390/antibiotics11111536.

Zhang J, He X. Living bacteriophage engineering for functional material synthesis, bioanalytical sensing and disease theranostics. Targets. 2024 Jul 8;2(3):157–85. doi:10.3390/targets2030010.

García R, Latz S, Romero J, Higuera G, García K, Bastías R. Bacteriophage production models: An overview. Frontiers in Microbiology. 2019 Jun 4;10. doi:10.3389/fmicb.2019.01187.

Goldman E, Green LH. Practical Handbook of Microbiology. Boca Raton: CRC Press; 2023. Plaque assay for bacteriophage, p 93–97.

Gratia A. Des relations numériques entre bactéries lysogènes et particules de bactériophages. Annales de l'Institut Pasteur. 1936; 57:652-676.

Hershey AD, Kalmanson G, Bronfenbrenner J. Quantitative methods in the study of the phage-antiphage reaction. The Journal of Immunology. 1943 May 1;46(5):267–79. doi:10.4049/jimmunol.46.5.267.

Kleczkowski A, Kleczkowski J. The ability of single phage particles to form plaques and to multiply in liquid cultures. Journal of General Microbiology. 1951 May 1;5(2):346–56. doi:10.1099/00221287-5-2-346.

Abedon ST, Yin J. Bacteriophage plaques: Theory and analysis. Methods in Molecular Biology. 2009;161–74. doi:10.1007/978-1-60327-164-6_17.

Warner CM, Barker N, Lee S-W, Perkins EJ. M13 bacteriophage production for large-scale applications. Bioprocess and Biosystems Engineering. 2014 Apr 13;37(10):2067–72. doi:10.1007/s00449-014-1184-7.

Grieco S-HH, Wong AY, Dunbar WS, MacGillivray RT, Curtis SB. Optimization of fermentation parameters in phage production using response surface methodology. Journal of Industrial Microbiology and Biotechnology. 2012 Oct 1;39(10):1515–22. doi:10.1007/s10295-012-1148-3.

Abedon ST, Culler RR. Optimizing bacteriophage plaque fecundity. Journal of Theoretical Biology. 2007 Dec;249(3):582–92. doi:10.1016/j.jtbi.2007.08.006.

Santos SB, Carvalho CM, Sillankorva S, Nicolau A, Ferreira EC, Azeredo J. The use of antibiotics to improve phage detection and enumeration by the double-layer agar technique. BMC Microbiology. 2009 Jul 23;9(1). doi:10.1186/1471-2180-9-148.

Taj MK, Yunlin W, Taj I, Hassani TM, Samreen Z et al. Various culture media effect on T4 phage lysis and production. Int J Innov Appl Stud. 2013; 4(3):543–546.

Ramesh N, Archana L, Madurantakam Royam M, Manohar P, Eniyan K. Effect of various bacteriological media on the plaque morphology of Staphylococcus and vibrio phages. Access Microbiology. 2019 Jun 1;1(4). doi:10.1099/acmi.0.000036.