Cystic fibrosis (CF) is the most common life-threatening genetic disease among Caucasians. CF patients suffer from chronic lung infections due to the presence of thick mucus, caused by cftr gene dysfunction. The two most commonly found bacteria in the mucus of CF patients are Staphylococcus aureus and Pseudomonas aeruginosa. It is well known that early-infecting P. aeruginosa strains produce anti-staphylococcal compounds and inhibit S. aureus growth. More recently, it has been shown that late-infecting P. aeruginosa strains develop commensal-like/coexistence interaction with S. aureus. The aim of this study was to decipher the impact of P. aeruginosa strains on S. aureus. RNA sequencing analysis showed 77 genes were specifically dysregulated in the context of competition and 140 genes in the context of coexistence in the presence of P. aeruginosa. In coexistence, genes encoding virulence factors and proteins involved in carbohydrates, lipids, nucleotides and amino acids metabolism were downregulated. On the contrary, several transporter family encoding genes were upregulated. In particular, several antibiotic pumps belonging to the Nor family were upregulated: tet38, norA and norC, leading to an increase in antibiotic resistance of S. aureus when exposed to tetracycline and ciprofloxacin and an enhanced internalization rate within epithelial pulmonary cells. This study shows that coexistence with P. aeruginosa affects the S. aureus transcriptome and virulence.

In the context of infection, Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated, particularly in cystic fibrosis (CF) patients. Within lungs, the two pathogens exhibit a range of competitive and coexisting interactions. In the present study, we explored the impact of S. aureus on P. aeruginosa physiology in the context of coexistence. Transcriptomic analyses showed that S. aureus affects significantly and specifically the expression of numerous genes involved in P. aeruginosa carbon and amino acid metabolism. In particular, 65% of the strains presented an important overexpression of the genes involved in the acetoin catabolic (aco) pathway. We demonstrated that acetoin is (i) produced by clinical S. aureus strains, (ii) detected in sputa from CF patients and (iii) involved in P. aeruginosa's aco system induction. Furthermore, acetoin is catabolized by P. aeruginosa, a metabolic way that improves the survival of both pathogens by providing a new carbon source for P. aeruginosa and avoiding toxic accumulation of acetoin on S. aureus. Due to its beneficial effects on both bacteria, acetoin catabolism could testify to the establishment of trophic cooperation between S. aureus and P. aeruginosa in the CF lung environment, promoting their persistence. 4 (16). Several independent studies thus observed this coexistence state between S. aureus and P. aeruginosa isolated from chronic infections (10, 15, 17, 18). Briaud et al. recently demonstrated that this interaction pattern appears to be more frequent than expected. Indeed, among the quarter of CF patients co-infected by both pathogens, 65% were infected by a coexisting S. aureus-P. aeruginosa pair (18, 19). Recent studies show that coexistence between P. aeruginosa and S. aureus could promote their persistence throughout establishment of cooperative interaction. In these conditions, coexisting bacteria demonstrated an increased tolerance to antibiotics: to tobramycin and tetracycline for S. aureus and to gentamicin for P. aeruginosa; this appeared to be related to the induction of small colony variants (15, 17, 18, 20). However, the effects of coexistence on bacterial general physiology, and not only virulence-associated traits, have not been explored yet. Despite its significance in infectious ecosystem, coexistence between P. aeruginosa and S. aureus remains thus poorly understood. Using global and targeted transcriptomic approaches, we evaluated the impact of S. aureus presence on P. aeruginosa gene expression on a set of clinical pairs of strains isolated from CF coinfected patients. Coexistence with S. aureus induced the overexpression of many genes involved in utilization of alternative carbon sources in P. aeruginosa, such as amino acids and acetoin. Acetoin was shown to be produced by clinical S. aureus isolates in vitro and in CF sputum, and catabolized by P. aeruginosa. The beneficial effects of acetoin catabolism on both bacteria during their interaction highlight a trophic cooperation between P. aeruginosa and S. aureus in CF lung in...