Rapid and strain-specific resistance evolution of Staphylococcus aureus against inhibitory molecules secreted by Pseudomonas aeruginosa

Pseudomonas aeruginosa and Staphylococcus aureus frequently occur together in polymicrobial infections, and there is evidence that their interactions negatively affect disease outcome in patients. At the molecular level, interactions between the two bacterial species are well-described, with P. aeruginosa usually being the dominant species suppressing S. aureus through a variety of inhibitory molecules. However, in chronic infections the two species interact over prolonged periods of time, and S. aureus might be able to evolve resistance against inhibitory molecules deployed by P. aeruginosa. Here, we used experimental evolution to test this hypothesis by exposing three different S. aureus strains (Cowan I, 6850, JE2) to the growth-inhibitory supernatant of P. aeruginosa PAO1 over 30 days. Prior to evolution, we found that S. aureus strains were inhibited by secreted compounds regulatorily controlled by the PQS quorum-sensing system of P. aeruginosa. Following evolution, S. aureus strains were no longer inhibited: we observed that phenotypic adaptations were strain-specific and involved the up-regulation of virulence traits, such as staphyloxanthin production and the formation of small colony variants. At the genetic level, mutations in membrane transporters were the most frequent evolutionary targets. Our work indicates that adaptations of S. aureus to co-infecting pathogens occurs rapidly and involves both virulence traits and membrane transporters involved in drug resistance. Thus, pathogen evolution could promote species co-existence, complicate treatment options and therefore worsen disease outcome.