The Pseudomonas aeruginosa Wsp pathway undergoes positive evolutionary selection during chronic infection

Pathogens experience pressure in an infection to adapt, favoring mutants that persist. Pseudomonas aeruginosa commonly adapts by evolving mutants with hyper-biofilm production that evade host immunity and have increased antimicrobial tolerance. Despite our understanding of these adaptive phenotypes, studying their emergence 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. Burns were infected with six different 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 which led to elevated cyclic-di-GMP and the RSCV phenotype. The rapid rise of these mutants to detectable frequencies is evidence of positive selection of the Wsp chemosensory system and challenges the dogma that RSCVs only evolve following prolonged evolution in chronic infections. Given the prevalence of P. aeruginosa RSCVs and related opportunists in other chronic infections, we predict that RSCVs may be a common, early adaptation to persist during infections.