Unveiling co-infection in cystic fibrosis airways: transcriptomic analysis of Pseudomonas aeruginosa and Staphylococcus aureus dual-species biofilms

Abstract

Cystic fibrosis (CF) is a common heritable genetic disorder caused by a defect in the cystic fibrosis conductance regulator gene, resulting in several complications in the human body (Kreda et al., 2012). So far, the pathological changes in the lungs are best studied due to the high mortality rates linked to poor lung function and the recurrent development of severe biofilm-related infections (Flume et al., 2009; Ciofu et al., 2015). Staphylococcus aureus and Pseudomonas aeruginosa are the most prevalent pathogens that colonize structurally abnormal airways such as those diagnosed with CF and other chronic obstructive lung diseases (Lyczak et al., 2002; Hubert et al., 2013). Although these bacteria seem to succeed with one another, CF patients acquire coinciding P. aeruginosa and S. aureus pulmonary infections, being co-infection usually associated with decreased lung function and increased frequency of pulmonary exacerbations (Limoli et al., 2016). Furthermore, P. aeruginosa and S. aureus pathogens adopt a biofilm mode of growth, which contributes to high tolerance to antibiotic treatment (Schobert and Jahn, 2010) and the recalcitrant nature of these chronic co-infections (Burmølle et al., 2006; Lopes et al., 2012), leading to significant patient morbidity and mortality (Cox et al., 2010). Interactions between P. aeruginosa and S. aureus have been widely studied, and it is commonly admitted that P. aeruginosa outcompetes S. aureus, perhaps outcompeting S. aureus for limited nutrients (Mashburn et al., 2005) or producing antistaphylococcal compounds (DeLeon et al., 2014; Fugère et al., 2014), having S. aureus a minimal contribution to the overall course of the CF-associated biofilm infections (Bragonzi et al., 2012; Filkins et al., 2015). However, P. aeruginosa and S. aureus have been identified in the same lobe of CF lungs (Hogan et al., 2016; Wakeman et al., 2016) and are frequently diagnosed (Limoli et al., 2016; Zolin et al., 2019) as co-infecting species in CF patients. Moreover, P. aeruginosa strains isolated from early infection outcompete S. aureus, while strains isolated from chronic infection are less aggressive and can be co-cultivated with S. aureus (Frydenlund Michelsen et al., 2016; Limoli et al., 2017), suggesting that these pathogens can interact in vivo. In a previous study, we showed that S. aureus can grow and coexist with P. aeruginosa under dualspecies biofilm conditions (Magalhães et al., 2021). Following up on these findings, and acknowledging that the molecular mechanisms behind these interactions are largely unknown, the purpose of the present study was, therefore, to identify the major transcriptomic features of P. aeruginosaS. aureus dual-species biofilms, using high-throughput RNA-sequencing (RNA-seq). Herein, we described the full transcriptome of P. aeruginosa and S. aureus single- and dual-species biofilms and used a data analysis approach based on direct and functional gene interactions, namely gene set enrichment. These results will be invaluable for future functional studies involving P. aeruginosaS. aureus interactions.