Co-infection with Streptococcus and Rothia spp. drives prophage dynamics in Pseudomonas aeruginosa in an artificial sputum model.

Chronic bacterial lung infections with Pseudomonas aeruginosa are common in people with cystic fibrosis (CF), but interactions with other members of the CF lung community, such as Streptococcus and Rothia spp., are not well understood. The aim of this study was to determine the impact of other species on P. aeruginosa using an artificial sputum medium (ASM) model. P. aeruginosa LESB58, representing a CF strain, was inoculated into ASM either alone or in co-culture with Streptococcus anginosus, Streptococcus vestibularis, Rothia mucilaginosa, or Rothia dentocariosa. Gene expression was assessed using RNAseq at 72 and 96 h timepoints. Free phage numbers assessed by plaque assay and qPCR. Differential expression of P. aeruginosa genes, including those related to virulence factors such as prophage, type III secretion, and exopolysaccharides, was seen in co-culture conditions. At 72 h, there was lower expression of prophage genes under co-culture conditions, and this corresponded with lower free phage. During co-culture, there were reduced levels of a D3112-like transposable phage, previously shown to drive CF-like adaptations in an experimental evolution model, and increased Pf1-like filamentous phage, associated with altered biofilm formation and immune responses. Gene expression changes during co-culture with Streptococcus and Rothia spp. were identified, affecting key P. aeruginosa virulence factors, including phage. Further work indicated changes to free phage levels of two important prophages with potential to affect P. aeruginosa adaptation, antibiotic resistance, and host immune response in the CF lung. This study emphasizes the importance of understanding the influence of lung commensals on focal pathogens.