<i>Staphylococcus aureus</i> Biofilm Growth on Cystic Fibrosis Airway Epithelial Cells Is Enhanced during Respiratory Syncytial Virus Coinfection

Abstract

<i>Staphylococcus aureus</i> is a major cause of chronic respiratory infection in patients with cystic fibrosis (CF). We recently showed that <i>Pseudomonas aeruginosa</i> exhibits enhanced biofilm formation during respiratory syncytial virus (RSV) coinfection on human CF airway epithelial cells (AECs). The impact of respiratory viruses on other bacterial pathogens during polymicrobial infections in CF remains largely unknown. To investigate if <i>S. aureus</i> biofilm growth in the CF airways is impacted by virus coinfection, we evaluated <i>S. aureus</i> growth on CF AECs. Initial studies showed an increase in <i>S. aureus</i> growth over 24 h, and microscopy revealed biofilm-like clusters of bacteria on CF AECs. Biofilm growth was enhanced when CF AECs were coinfected with RSV, and this observation was confirmed with <i>S. aureus</i> CF clinical isolates. Apical conditioned medium from RSV-infected cells promoted <i>S. aureus</i> biofilms in the absence of the host epithelium, suggesting that a secreted factor produced during virus infection benefits <i>S. aureus</i> biofilms. Exogenous iron addition did not significantly alter biofilm formation, suggesting that it is not likely the secreted factor. We further characterized <i>S. aureus</i>-RSV coinfection in our model using dual host-pathogen RNA sequencing, allowing us to observe specific contributions of <i>S. aureus</i> and RSV to the host response during coinfection. Using the dual host-pathogen RNA sequencing approach, we observed increased availability of nutrients from the host and upregulation of <i>S. aureus</i> genes involved in growth, protein translation and export, and amino acid metabolism during RSV coinfection.<b>IMPORTANCE</b> The airways of individuals with cystic fibrosis (CF) are commonly chronically infected, and <i>Staphylococcus aureus</i> is the dominant bacterial respiratory pathogen in CF children. CF patients also experience frequent respiratory virus infections, and it has been hypothesized that virus coinfection increases the severity of <i>S. aureus</i> lung infections in CF. We investigated the relationship between <i>S. aureus</i> and the CF airway epithelium and observed that coinfection with respiratory syncytial virus (RSV) enhances <i>S. aureus</i> biofilm growth. However, iron, which was previously found to be a significant factor influencing <i>Pseudomonas aeruginosa</i> biofilms during virus coinfection, plays a minor role in <i>S. aureus</i> coinfections. Transcriptomic analyses provided new insight into how bacterial and viral pathogens alter host defense and suggest potential pathways by which dampening of host responses to one pathogen may favor persistence of another in the CF airways, highlighting complex interactions occurring between bacteria, viruses, and the host during polymicrobial infections.