The airways of patients with cystic fibrosis are colonized with diverse bacterial communities that change dynamically during pediatric years and early adulthood. Staphylococcus aureus is the most prevalent pathogen during early childhood, but during late teens and early adulthood, a shift in microbial composition occurs leading to Pseudomonas aeruginosa community predominance in ϳ50% of adults. We developed a robust dual-bacterial in vitro coculture system of P. aeruginosa and S. aureus on monolayers of human bronchial epithelial cells homozygous for the ⌬F508 cystic fibrosis transmembrane conductance regulator (CFTR) mutation to better model the mechanisms of this interaction. We show that P. aeruginosa drives the S. aureus expression profile from that of aerobic respiration to fermentation. This shift is dependent on the production of both 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and siderophores by P. aeruginosa. Furthermore, S. aureus-produced lactate is a carbon source that P. aeruginosa preferentially consumes over medium-supplied glucose. We find that initially S. aureus and P. aeruginosa coexist; however, over extended coculture P. aeruginosa reduces S. aureus viability, also in an HQNO-and P. aeruginosa siderophore-dependent manner. Interestingly, S. aureus small-colony-variant (SCV) genetic mutant strains, which have defects in their electron transport chain, experience reduced killing by P. aeruginosa compared to their wild-type parent strains; thus, SCVs may provide a mechanism for persistence of S. aureus in the presence of P. aeruginosa. We propose that the mechanism of P. aeruginosa-mediated killing of S. aureus is multifactorial, requiring HQNO and P. aeruginosa siderophores as well as additional genetic, environmental, and nutritional factors. IMPORTANCEIn individuals with cystic fibrosis, Staphylococcus aureus is the primary respiratory pathogen during childhood. During adulthood, Pseudomonas aeruginosa predominates and correlates with worse patient outcome. The mechanism(s) by which P. aeruginosa outcompetes or kills S. aureus is not well understood. We describe an in vitro dual-bacterial species coculture system on cystic fibrosis-derived airway cells, which models interactions relevant to patients with cystic fibrosis. Further, we show that molecules produced by P. aeruginosa additively induce a transition of S. aureus metabolism from aerobic respiration to fermentation and eventually lead to loss of S. aureus viability. Elucidating the molecular mechanisms of P. aeruginosa community predominance can provide new therapeutic targets and approaches to impede this microbial community transition and subsequent patient worsening. C omplex polymicrobial communities colonize the airways of cystic fibrosis (CF) patients within the first month of life (1). Culture-independent studies have revealed the simultaneous presence of numerous bacterial taxa, fungi, and viruses in respiratory samples from CF patients at all stages of life (2-8). This abundance of microbes colonizing the respiratory ...