Detrimental and beneficial interactions between co-colonizing bacteria may influence the course of infections. In cystic fibrosis (CF) airways, Staphylococcus aureus prevails in childhood, whereas Pseudomonas aeruginosa progressively predominates thereafter. While a range of interactions has been identified, it is unclear if these represent specific adaptations or correlated responses to other aspects of the environment. Here, we investigate how P. aeruginosa adapts to S. aureus by evolving P. aeruginosa in the presence and absence of S. aureus. P. aeruginosa populations that evolved for 150 generations were sequenced and compared to the ancestor strain. Mutations in the Wsp signaling system were identified in both treatments and likely occurred because of low oxygen availability. Despite showing increased killing activity, wsp mutants were less fit in the presence of S. aureus. In contrast, mutations in lipopolysaccharide (LPS) biosynthesis occurred exclusively in co-cultures with S. aureus and conferred a fitness gain in its presence. Moreover, they increased resistance towards beta-lactam antibiotics. Strikingly, both mutations in wsp and LPS genes are observed in clinical isolates from CF-patients. Our results suggest that P. aeruginosa LPS mutations are a direct consequence of S. aureus imposed selection in vitro.

Summary Efficient delivery of toxic compounds to bacterial competitors is essential during interspecies microbial warfare. Rhamnolipids (RLPs) are glycolipids produced by Pseudomonas and Burkholderia species involved in solubilization and uptake of environmental aliphatic hydrocarbons and perform as biosurfactants for swarming motility. Here, we show that RLPs produced by Pseudomonas aeruginosa associate to form micelles. Using high-resolution microscopy, we found that RLP micelles serve as carriers for self-produced toxic compounds, which they deliver to Staphylococcus aureus cells, thereby enhancing and accelerating S. aureus killing. RLPs also potentiated the activity of lincosamide antibiotics, suggesting that RLP micelles may transport not only self-produced but also heterologous compounds to target competing bacterial species

Summary Interspecies bacterial competition may occur via cell‐associated or secreted determinants and is key to successful niche colonization. We previously evolved Pseudomonas aeruginosa in the presence of Staphylococcus aureus and identified mutations in the Wsp surface‐sensing signalling system. Surprisingly, a ΔwspF mutant, characterized by increased c‐di‐GMP levels and biofilm formation capacity, showed potent killing activity towards S. aureus in its culture supernatant. Here, we used an unbiased metabolomic analysis of culture supernatants to identify rhamnolipids, alkyl quinoline N‐oxides and two siderophores as members of four chemical clusters, which were more abundant in the ΔwspF mutant supernatants. Killing activities were quorum‐sensing controlled but independent of c‐di‐GMP levels. Based on the metabolomic analysis, we formulated a synthetic cocktail of four compounds, showing broad‐spectrum anti‐bacterial killing, including both Gram‐positive and Gram‐negative bacteria. The combination of quorum‐sensing‐controlled killing and Wsp‐system mediated biofilm formation endows P. aeruginosa with capacities essential for niche establishment and host colonization.