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Antimicrobial resistance is a significant threat to the treatment of infectious disease. Multiple mechanisms of resistance to different classes of antibiotics have been identified and well-studied. However, these mechanisms are studied with bacteria in isolation, whereas often, infections have a polymicrobial basis. Using a biofilm slide chamber model, we visualized the formation and development of clinical Pseudomonas aeruginosa biofilms in the presence of secreted Staphylococcus aureus exoproducts, two bacteria that commonly co-infect pediatric patients with cystic fibrosis. We showed that, over time, certain isolates of P. aeruginosa can form different biofilm architecture in the presence of S. aureus exoproducts. We further determined that this interaction was dependent on Psl produced by P. aeruginosa and staphylococcal protein A from S. aureus. Importantly, we identified a mechanism of antibiotic resistance to tobramycin that is dependent on the polymicrobial interactions between these two bacteria. This interaction occurred in isolates of P. aeruginosa recovered from children with cystic fibrosis who failed to clear P. aeruginosa following inhaled tobramycin treatment., Co-infection: interactions that increase resistance The interaction between two different bacterial species can enhance the antibiotic resistance of biofilms. Resistance to antimicrobial therapy is usually studied using individual bacterial species, but many infections involve more than one species. Researchers in Canada, led by Trevor Beaudoin at The Hospital for Sick Children in Toronto, studied the effect of products secreted by Staphylococcus aureus bacteria on biofilms of Pseudomonas aeruginosa. They worked with biofilms that had resisted treatment, isolated from patients with cystic fibrosis. The co-infection altered the biofilm structure and increased its resistance to the antibiotic tobramycin. The effect depended on bacterial aggregation stimulated by the interaction between a large carbohydrate from P. aeruginosa and a protein secreted by S. aureus. The research highlights the need to study bacterial interactions to understand and treat infections involving different bacteria.