Characterization of novel mycobacteriophages with a focus on phage Pisa4 and its interaction with human innate immune cells.

Four mycobacteriophages Pisa1, Pisa4, Florence1 and Florence4, taxonomically classified as new species, were isolated using Mycobacterium smegmatis mc2155 as a host strain. Genomic analysis revealed dsDNA genomes devoid of virulence or antibiotic-resistance genes. All encoded lysogeny-associated elements, but Pisa4 lacked the integrase gene and showed a partial immunity repressor gene, suggesting a potentially impaired lysogenic cycle. Phages exhibited siphoviral morphology, short replication cycles (≤30 min) and burst sizes of 50-150 PFUs/bacterial cell. All phages remained active between pH 4-12 and up to 45 °C. Given its potentially impaired lysogeny, Pisa4 was considered a promising candidate for antibacterial applications; therefore, its interaction with THP-1 macrophages was investigated. The phage was internalized in THP-1 and co-localized with its host both after co-incubation with bacteria prior to infection and upon post-infection administration. However, phage replication was restricted to the co-incubation condition. Importantly, Pisa4 did not trigger neutrophil extracellular trap release and elicited minimal cytokine responses in peripheral blood mononuclear cells, with no TNF-α or IFN-γ induction. In contrast, a significant increase in IL-6 production was observed at high phage concentrations, which may be influenced by co-purified bacterial components. Finally, Pisa1 and Pisa4 infected 12 and 8 out of the 48 Mycobacterium abscessus clinical isolates tested, respectively, while Florence phages were inactive. Collectively, these results highlight that Pisa phages, particularly Pisa4, are promising candidates for future therapeutic development.