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Biofilms are structured microbial communities that are the leading cause of numerous chronic infections which are difficult to eradicate. Within the lungs of individuals with cystic fibrosis (CF), Pseudomonas aeruginosa causes persistent biofilm infection that is commonly treated with aminoglycoside antibiotics such as tobramycin. However, sublethal concentrations of this aminoglycoside were previously shown to increase biofilm formation by P. aeruginosa , but the underlying adaptive mechanisms still remain elusive. Herein, we combined confocal laser scanning microscope analyses, proteomics profiling, gene expression assays and phenotypic studies to unravel P. aeruginosa potential adaptive mechanisms in response to tobramycin exposure during biofilm growth. Under this condition, we show that the modified biofilm architecture is related at least in part to increased extracellular DNA (eDNA) release, most likely as a result of biofilm cell death. Furthermore, the activity of quorum sensing (QS) systems was increased, leading to higher production of QS signaling molecules. We also demonstrate upon tobramycin exposure an increase in expression of the PrrF small regulatory RNAs, as well as expression of iron uptake systems. Remarkably, biofilm biovolumes and eDNA relative abundances in pqs and prrF mutant strains decrease in the presence of tobramycin. Overall, our findings offer experimental evidences for a potential adaptive mechanism linking PrrF sRNAs, QS signaling, biofilm cell death, eDNA release, and tobramycin-enhanced biofilm formation in P. aeruginosa . These specific adaptive mechanisms should be considered to improve treatment strategies against P. aeruginosa biofilm establishment in CF patients' lungs., Competing Interests: Competing interestsThe authors declare no competing interests.

Pseudomonas aeruginosa is an important multidrug-resistant human pathogen by dint of its high intrinsic, acquired, and adaptive resistance mechanisms, causing great concern for immune-compromised individuals and public health. Additionally, P. aeruginosa resilience lies in the production of a myriad of virulence factors, which are known to be tightly regulated by the quorum sensing (QS) system. Anti-virulence therapy has been adopted as an innovative alternative approach to circumvent bacterial antibiotic resistance. Since plants are known repositories of natural phytochemicals, herein, we explored the anti-virulence potential of Azorella atacamensis, a medicinal plant from the Taira Atacama community (Calama, Chile), against P. aeruginosa. Interestingly, A. atacamensis extract (AaE) conferred a significant protection for human lung cells and Caenorhabditis elegans nematodes towards P. aeruginosa pathogenicity. The production of key virulence factors was decreased upon AaE exposure without affecting P. aeruginosa growth. In addition, AaE was able to decrease QS-molecules production. Furthermore, metabolite profiling of AaE and its derived fractions achieved by combination of a molecular network and in silico annotation allowed the putative identification of fourteen diterpenoids bearing a mulinane-like skeleton. Remarkably, this unique interesting group of diterpenoids seems to be responsible for the interference with virulence factors as well as on the perturbation of membrane homeostasis of P. aeruginosa. Hence, there was a significant increase in membrane stiffness, which appears to be modulated by the cell wall stress response ECFσ SigX, an extracytoplasmic function sigma factor involved in membrane homeostasis as well as P. aeruginosa virulence. [ABSTRACT FROM AUTHOR]