Genetic and spatial determinants of Staphylococcus aureus and Pseudomonas aeruginosa competition in colony biofilms

Abstract: Staphylococcus aureus and Pseudomonas aeruginosa frequently coexist in chronic infections, including cystic fibrosis and non-healing wounds, where their interactions within biofilms complicate treatment and worsen patient outcomes. Understanding of the spatial and genetic factors governing their co-existence remains limited. Here, we developed a colony biofilm model to examine how intrinsic and extrinsic factors alter interspecies competition. Through transposon mutant screens and spatial analyses, we identified six transposon mutants that significantly altered S. aureus (htrA1, proC, SAUSA300_0847) and P. aeruginosa (pqsA, pilG, flgK) co-existence and distribution within colony biofilms. We found that spatial arrangement of microcolonies, driven by initial cell positioning, motility, and metabolic flexibility, was a variable determining S. aureus fitness. This work demonstrates that competitive outcomes within S. aureus and P. aeruginosa colony biofilms are driven by general physiological adaptations to spatial and metabolic constraints rather than specialized antagonistic mechanisms. Content: Majority of data volume is raw and processed microscopy images. Remainder is code and spreadsheets with values used to generate figures in the main paper. Check the README for more details.