Human airway model reveals host-pathogen co-infection dynamics of Staphylococcus aureus and Pseudomonas aeruginosa

Abstract Polymicrobial airway infections, particularly those involving Staphylococcus aureus and Pseudomonas aeruginosa, are a hallmark of severe chronic respiratory diseases such as those in people with cystic fibrosis (pwCF). Yet, the mechanisms enabling their co-existence remain poorly understood. Using a human air-liquid interface (ALI) airway model, we dissected host-pathogen and interspecies dynamics during mono- and co-infections with clinically relevant bacterial pathogens. We found that S. aureus strains exhibited distinct colonization patterns and virulence profiles, with the deletion of the agr quorum sensing locus leading to attenuated epithelial disruption. Additionally, the H1 component of P. aeruginosa’s type VI secretion system was critical for competitive exclusion of S. aureus, its deletion allowing for occasional co-localization of both species. Importantly, the order of co-infection strongly influenced pathogen dominance, with prior colonization of ALI models with S. aureus enabled coexistence with P. aeruginosa, thereby mimicking clinical observations in CF. Our results reveal that strain-specific traits and secretion systems influence microbial competition and persistence in the airway and underscore the importance of physiologically relevant models for studying chronic infections. Moreover, they provide the foundation for exploring therapeutic strategies for polymicrobial infections that account for the colonization sequence and microbial evolution for treatment design.