Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

Abstract

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.

Keywords: Pseudomonas aeruginosa; bacteriophage; cystic fibrosis; experimental evolution; mobile genetic element.

Fig. S1.

Bacterial-free phage dynamics through time when grown in ASM. (A) Colony-forming units per milliliter in each population, separated by treatment. (B) Total free phage (plaque-forming units per milliliter) in phage-treated populations. (C) Relative densities of free virions for each of the LES phages in end point populations from the phage treatment, where relative density is calculated as log₁₀ (copies per microliter + 1), as determined by qPCR. Boxplots represent the median and interquartile range.

Fig. S2.

Lysogen dynamics over time in populations evolving with phages. Each population is represented by a single area plot denoting the frequency of bacterial isolates lysogenic for each specified combination of LES prophages through time.

Fig. 1.

Fitness response to selection in populations evolving with and without phages. Data points represent the mean ± SE fitness calculated as selection rate for populations evolved with (filled symbols) or without (open symbols) phages in competition against either ancestral PAO1 or an isogenic phage-resistant competitor, PAO1ΔpilA.

Fig. 2.

Genetic loci under positive selection as indicated by parallel genomic evolution in populations evolving with and without phages. Each concentric circle corresponds to a replicate population in either the control (without phages) or treatment group (with phages), as indicated. Positions around each concentric circle, starting at the 12 o'clock position and in a clockwise direction, correspond to positions around the published P. aeruginosa PAO1 single circular chromosome. The smaller circles around each concentric circle indicate the positions of variants in each replicate that were observed in an ORF under positive selection, i.e., mutated in parallel in at least one other replicate. Only variants in fha1 were at precisely the same position and were likely to be homologous. Variants are also listed in Table S6.

Fig. 3.

The evolution of resistance to phages and pilus-dependent twitching motility traits. (A) Boxplot of phage resistance in end point populations. The thick horizontal line denotes the median frequency of isolates in a population resistant to one or more LES phages for each treatment. Asterisks denote outliers and narrow horizontal lines denote the upper and lower quartiles. (B) Frequency of bacterial isolates in each population through time displaying impaired twitching motility in the control (gray circles, solid line) and phage treatment (black triangles, dotted line). (C) Allele frequency dynamics of LESφ4 integrated into fimU and pilV for populations P7 and P11, respectively, and loss of twitching motility in these populations. Closed black circles and open white diamonds represent populations P7 and P11, respectively, solid gray lines denote loss of twitching motility data, and dashed black lines denote allele frequency data.

Fig. S3.

Fitness of QS-deficient mutants (PAO1ΔlasR) relative to WT PAO1 in the presence and absence of temperate phages in ASM. We find no effect of phage presence on the relative fitness of PAO1ΔlasR (two-sample t test, t₁₀ = −0.7989, P = 0.44), which was fitter than WT in both the presence [one-sample t test (alt = 0), t₅ = 5.0331, P < 0.01] and absence [one-sample t test (alt = 0), t₅ = 6.7065, P = 0.001] of phages. r = 0, fitness of PAO1ΔlasR = PAO1; r > 0, fitness of PAO1ΔlasR > PAO1. Data are means (n = 6) ± SEM.