Optimized alveolar epithelial cell model for chronic Pseudomonas aeruginosa and Staphylococcus aureus coinfections

<i>Pseudomonas aeruginosa</i> is a relevant pathogen in chronic respiratory infections, which are usually associated with biofilm formation, complicating <i>in vitro</i> modeling and effective treatment strategies. While <i>P. aeruginosa</i> can coexist with several microorganisms, its association with <i>Staphylococcus aureus</i> is widespread in cystic fibrosis (CF) patients and other bronchiectasis. Finding a reliable and straightforward <i>in vitro</i> model to study long-term <i>P. aeruginosa</i> infections is extremely hard due to the secretion of highly virulent toxins that compromise the model within less than 10 h. Several optimizations, including the use of bovine serum albumin (BSA) and extracellular matrix proteins, led to enhanced A549 cell viability up to 30 h post-infection. Within this time frame, we developed <i>P. aeruginosa</i> biofilms, explored host-pathogen interactions, and delved deeper into the relationship between <i>P. aeruginosa</i> and <i>S. aureus.</i> Additionally, ciprofloxacin treatment was evaluated, revealing changes and differences in antibiotic susceptibility and underlying significant differences between bacterial strains.