Pseudomonas aeruginosa biofilm-deficient mutants undergo parallel evolution during chronic infection.

UNLABELLED: Pseudomonas aeruginosa readily adapts to infection by acquiring stable and heritable mutations. Previously, we discovered that the first adaptations in a porcine wound model were rugose small-colony variants (RSCVs) caused by mutations in the wsp operon. These mutants overproduce Pel and Psl biofilm exopolysaccharides that improve defense against host responses. To identify other mechanisms of host adaptation that lead to hyperbiofilm phenotypes, we created a mutant with an activated wsp pathway but unable to produce these exopolysaccharides (PAO1ΔwspFΔpelAΔpslBCD). Porcine wounds were infected with this mutant, and biopsies were sampled at days 7, 14, and 35. Colony variants were isolated from the wound, peaking at approximately 5% of the total P. aeruginosa population, and whole genome sequencing revealed that these variants had acquired mutations in genes in lipopolysaccharide and type IV pili biosynthesis, with wzy and pilU genes being most commonly targeted. PAO1 pilU mutants were associated with a hyperbiofilm phenotype that outcompeted the parental strain, and PAO1 wzy mutants were associated with a hyperbiofilm phenotype and increased tolerance to host antimicrobial products. We further identified that several variants had acquired large genome deletions that spanned up to 320 consecutive genes and other variants with high copy numbers of Pf6 filamentous phage. Together our results suggest that the hyperbiofilm phenotype is adaptive in chronic infections and that P. aeruginosa has redundant and diverse pathways to generate this phenotype.

IMPORTANCE

We demonstrate that in a porcine full-thickness thermal injury wound model, a Pseudomonas aeruginosa mutant deficient in biofilm formation undergoes adaptive evolution by acquiring mutations that alter the outer membrane, either type IV pili (T4P) or lipopolysaccharide (LPS) mutations, that restores the deficient biofilm phenotype. We also observe a striking degree of mutational parallelism, at both the biosynthetic pathway and gene level, indicating the strong selective pressures experienced by these pathways during chronic wound infection.