Temperate bacteriophages shape intraspecies competition during Pseudomonas aeruginosa Infection

Temperate bacteriophages are increasingly recognized as important drivers of microbial evolution. However, their role in mediating competition among members of the same species during infection remains poorly defined. Pseudomonas aeruginosa (Pa), an opportunistic pathogen associated with acute and chronic infections, frequently exhibits clonal dominance within infected individuals, suggesting the existence of effective mechanisms for excluding conspecific competitors. Here, we investigate the contribution of temperate bacteriophages to intraspecies competition among clinical Pa isolates. Screening of filtered cell-free supernatants from a diverse panel of clinical strains revealed that cell-independent antagonism is widespread, with most strains inhibiting the growth of at least one other Pa isolate. Susceptibility was strongly associated with twitching motility, a type IV pili-dependent phenotype, which also serves as the primary receptor for most Pa bacteriophages. Plaque assays confirmed the presence of infectious phage particles with strain-specific host ranges. Exposure of susceptible strains to infective phages resulted in the emergence of phage-induced small colony variants (pSCVs) that retained and propagated the acquired phages and associated inhibitory capacity. Whole-genome sequencing revealed extensive prophage diversity and mosaicism across clinical isolates, supporting a model in which poly-lysogeny expands competitive potential. Using isogenic pSCV systems and murine wound infection models, we demonstrate that acquisition of specific temperate phages confers a competitive advantage both in vitro and in vivo. These findings establish temperate bacteriophages as dynamic ecological weapons that shape intraspecies competition providing a mechanistic basis for clonal dominance during Pa infection and highlighting the broader role of prophages in structuring microbial populations.