ThePseudomonas aeruginosaWsp pathway undergoes positive evolutionary selection during chronic infection

Pathogens experience pressure in an infection to adapt, with selection favoring mutants that persist. Pseudomonas aeruginosa commonly adapts by evolving mutants with hyper-biofilm production that evade clearance. Despite our understanding of the adaptive phenotypes, studying their emergence and dynamics in an infection has proven challenging. Here we used a porcine full-thickness burn wound model of chronic infection to study how mixed strains of P. aeruginosa adaptively evolve. Wounds were infected with six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days post-infection. Rugose small-colony variants (RSCVs) were detected at 3-d and persisted, with the majority evolved from PA14-1. Whole genome sequencing of PA14-1 RSCVs revealed driver mutations exclusively in the wsp pathway. RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) present in the inoculum. The rapid rise of RSCVs to detectable frequencies is evidence of positive selection of the Wsp chemosensory system and suggests that RSCVs may arise earlier in an infection than originally appreciated, to facilitate infection. Given the prevalence of RSCVs in chronic infections, we predict that RSCVs may be a common, early adaptation during infections.