Temperate phages both mediate and drive adaptive evolution in pathogen biofilms.

Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote within-host evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as free-floating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts.

Evolutionary diversification of Pseudomonas aeruginosa in an artificial sputum model.

BACKGROUND: During chronic lung infections of cystic fibrosis patients Pseudomonas aeruginosa populations undergo extensive evolutionary diversification. However, the selective drivers of this evolutionary process are poorly understood. To test the effects of temperate phages on diversification in P. aeruginosa biofilms we experimentally evolved populations of P. aeruginosa for approximately 240 generations in artificial sputum medium with or without a community of three temperate phages. RESULTS: Analysis of end-point populations using a suite of phenotypic tests revealed extensive phenotypic diversification within populations, but no significant differences between the populations evolved with or without phages. The most common phenotypic variant observed was loss of all three types of motility (swimming, swarming and twitching) and resistance to all three phages. Despite the absence of selective pressure, some members of the population evolved antibiotic resistance. The frequency of antibiotic resistant isolates varied according to population and the antibiotic tested. However, resistance to ceftazidime and tazobactam-piperacillin was observed more frequently than resistance to other antibiotics, and was associated with higher prevelence of isolates exhibiting a hypermutable phenotype and increased beta-lactamase production. CONCLUSIONS: We observed considerable within-population phenotypic diversity in P. aeruginosa populations evolving in the artificial sputum medium biofilm model. Replicate populations evolved both in the presence and absence of phages converged upon similar sets of phenotypes. The evolved phenotypes, including antimicrobial resistance, were similar to those observed amongst clinical isolates from cystic fibrosis infections.

The role of temperate bacteriophages in bacterial infection.

Bacteriophages are viruses that infect bacteria. There are an estimated 10(31) phage on the planet, making them the most abundant form of life. We are rapidly approaching the centenary of their identification, and yet still have only a limited understanding of their role in the ecology and evolution of bacterial populations. Temperate prophage carriage is often associated with increased bacterial virulence. The rise in use of technologies, such as genome sequencing and transcriptomics, has highlighted more subtle ways in which prophages contribute to pathogenicity. This review discusses the current knowledge of the multifaceted effects that phage can exert on their hosts and how this may contribute to bacterial adaptation during infection.

Temperate phages enhance pathogen fitness in chronic lung infection.

The Liverpool Epidemic Strain (LES) is a polylysogenic, transmissible strain of Pseudomonas aeruginosa, capable of superinfecting existing P. aeruginosa respiratory infections in individuals with cystic fibrosis (CF). The LES phages are highly active in the CF lung and may have a role in the competitiveness of the LES in vivo. In this study, we tested this by competing isogenic PAO1 strains that differed only by the presence or absence of LES prophages in a rat model of chronic lung infection. Lysogens invaded phage-susceptible populations, both in head-to-head competition and when invading from rare, in the spatially structured, heterogeneous lung environment. Appreciable densities of free phages in lung tissue confirmed active phage lysis in vivo. Moreover, we observed lysogenic conversion of the phage-susceptible competitor. These results suggest that temperate phages may have an important role in the competitiveness of the LES in chronic lung infection by acting as anti-competitor weapons.

Lytic activity by temperate phages of Pseudomonas aeruginosa in long-term cystic fibrosis chronic lung infections.

Pseudomonas aeruginosa is the most common bacterial pathogen infecting the lungs of cystic fibrosis (CF) patients. The transmissible Liverpool epidemic strain (LES) harbours multiple inducible prophages (LESϕ2; LESϕ3; LESϕ4; LESϕ5; and LESϕ6), some of which are known to confer a competitive advantage in an in vivo rat model of chronic lung infection. We used quantitative PCR (Q-PCR) to measure the density and dynamics of all five LES phages in the sputa of 10 LES-infected CF patients over a period of 2 years. In all patients, the densities of free-LES phages were positively correlated with the densities of P. aeruginosa, and total free-phage densities consistently exceeded bacterial host densities 10-100-fold. Further, we observed a negative correlation between the phage-to-bacterium ratio and bacterial density, suggesting a role for lysis by temperate phages in regulation of the bacterial population densities. In 9/10 patients, LESϕ2 and LESϕ4 were the most abundant free phages, which reflects the differential in vitro induction properties of the phages. These data indicate that temperate phages of P. aeruginosa retain lytic activity after prolonged periods of chronic infection in the CF lung, and suggest that temperate phage lysis may contribute to regulation of P. aeruginosa density in vivo.