An investigation of how temperate bacteriophage affect the mode of evolution in biofilms of pathogens such as Pseudomonas aeruginosa. Temperate phages and evolution in pathogen biofilms

Temperate phages are an important driver of genomic diversification in bacterial pathogens1. Phage-derived sequences are more common in pathogenic than non-pathogenic taxa2, while the majority of genetic divergence between certain virulent and avirulent strains is attributable to prophage elements3. Host-associated microbial communities exhibit high abundances4 and rates of mobilisation of temperate phages5, highlighting the potential for these mobile elements to facilitate rapid within-host evolution of bacterial pathogens. However, the role of temperate phages in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of 3 temperate phages isolated from cystic fibrosis (CF) lung infections (LESɸ2, ɸ3 and ɸ4)6. Populations grew as free-floating biofilms in artificial sputum medium (ASM)7, mimicking sputum of CF lungs where P. aeruginosa is the most common and clinically important pathogen and undergoes extensive evolutionary adaptation8 and diversification9,10 during chronic infection. Bacterial populations both with and without phages adapted to the biofilm environment, however, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. We observed more highly parallel evolution in populations with phages than populations without phages, targeting motility-associated genes and regulators of quorum sensing systems controlling cell-cell communication and virulence. Moreover, prophage integration increased the supply of parallel positively selected mutations. Temperate phages are therefore likely to facilitate within host adaptive evolution of bacterial pathogens.