Global transcriptome responses including small RNAs during mixed‐species interactions with methicillin‐resistant Staphylococcus aureus and Pseudomonas aeruginosa

Pseudomonas aeruginosa and Staphylococcus aureus mixed‐species biofilm infections are more resilient to biocide attacks compared to their single‐species counterparts. Therefore, this study used an in vitro model recapitulating bacterial burdens seen in in vivo infections to investigate the interactions of P. aeruginosa and S. aureus in biofilms. RNA sequencing ( RNA ‐seq) was utilized to identify the entire genomic response, both open reading frames ( ORF s) and small RNA s ( sRNA s), of each species. Using competitive indexes, transposon mutants validated uncharacterized PA 1595 of P. aeruginosa and Panton–Valentine leukocidin ORF s of S. aureus are required for competitive success. Assessing spent media on biofilm development determined that the effects of these ORF s are not solely mediated by mechanisms of secretion. Unlike PA 1595, leukocidin ( lukS‐ PV ) mutants of S. aureus lack a competitive advantage through contact‐mediated mechanisms demonstrated by cross‐hatch assays. RNA ‐seq results suggested that during planktonic mixed‐species growth there is a robust genomic response or active combat from both pathogens until a state of equilibrium is reached during the maturation of a biofilm. In mixed‐species biofilms, P. aeruginosa differentially expressed only 0.3% of its genome, with most ORF s necessary for growth and biofilm development, whereas S. aureus modulated approximately 5% of its genome, with ORF s suggestive of a phenotype of increased virulence and metabolic quiescence. Specific expression of characterized sRNA s aligned with the genomic response to presumably coordinate the adaptive changes necessary for this homeostatic mixed‐species biofilm and sRNA s may provide viable foci for the design of future therapeutics.