Antagonism between bacteriophages and macrophages decreases efficacy of a phage cocktail and increases bacteriophage resistance

Castledine, M.; Szczutkowska, Z.; Matthews, A.; Walsh, S. K.; Lewis, R.; Kay, S.; Willment, J. A.; Brown, G. D.; Buckling, A.

doi:10.1101/2025.05.16.654446

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bioRxiv · 10.1101/2025.05.16.654446

Antagonism between bacteriophages and macrophages decreases efficacy of a phage cocktail and increases bacteriophage resistance

Castledine, M.·Szczutkowska, Z.·Matthews, A.·Walsh, S. K.·Lewis, R.·Kay, S.·Willment, J. A.·Brown, G. D.·Buckling, A.

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Abstract

Phage therapy, the use of viruses that infect bacteria (bacteriophages), is a promising complement to antibiotics during the antimicrobial resistance crisis, but treatment success is very variable. Evolution of bacterial resistance to bacteriophages and bacteriophage counter-resistance (coevolution) during therapy may explain some of this variation, the dynamics of which may be affected by interactions with the patients immune system. Here, we examine how a pathogenic bacterium, Pseudomonas aeruginosa coevolves with two clinically relevant bacteriophages (14-1 and PNM) when in the presence of macrophages (RAW 264.7 cell line). We show macrophages reduced the rate by which bacteria were killed by bacteriophages, likely by reducing bacteria-bacteriophage contact rates. Over evolutionary time-scales, macrophages increased the proportion of bacteriophage resistant bacteria compared to where macrophages were absent. These differences in resistance rates were likely driven by the early advantage in density offered by macrophages to bacteria, and exclusion of PNM from the bacteriophage cocktail which otherwise increased in frequency in the absence of macrophages. Consequently, macrophages significantly altered the short- and long-term efficacy of a bacteriophage cocktail. In line with a growing body of work, our results suggest that the patients immune system can reduce the efficacy of phage therapy, potentially driving variable outcomes in therapy success in patients. Significance statementPhage therapy, the use of viruses that infect bacteria (bacteriophages), is a promising complement to antibiotics during the antimicrobial resistance crisis. However, treatment success is very variable. One, often overlooked, variable is the immune system and how this influences bacteriophage efficacy, and how bacteria evolve resistance to bacteriophages. We find that macrophages reduce the rate by which bacteria are killed by bacteriophages. Resistance to bacteriophages also increased in the presence of macrophages, showing macrophages affect the short- and long-term efficacy of phage therapy. These results highlight the importance of the immune system in phage therapy, and the need for more research in this area.

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Castledine, M., Szczutkowska, Z., Matthews, A., Walsh, S. K., Lewis, R., Kay, S., Willment, J. A., Brown, G. D., Buckling, A.. 2025-05-21. Antagonism between bacteriophages and macrophages decreases efficacy of a phage cocktail and increases bacteriophage resistance. https://doi.org/10.1101/2025.05.16.654446

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