Role of NaCl and Glutamine on Biofilm Production from Pseudomonas aeruginosa

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen capable of forming antibiotic-resistant biofilms, contributing to persistent infections and treatment failure. Environmental factors such as osmolarity and nutrient availability are known to influence biofilm formation and virulence. In this study, we investigated the effects of NaCl depletion and glutamine supplementation on biofilm production in three <i>P. aeruginosa</i> strains: the laboratory strain ATCC 27853 and two clinical isolates with distinct antibiotic resistance profiles and phenazine production patterns (<i>P. aeruginosa</i> Pr, pyorubrin-producing, and <i>P. aeruginosa</i> Pc, pyocyanin-producing). Bacteria were cultured in standard Luria-Bertani (LB) medium, LB without NaCl, and LB in which yeast extract was replaced by glutamine. For each strain and condition, we assessed growth kinetics, phenazine production, and biofilm formation. Biofilm development was quantified via XTT assays and compared to secondary metabolite profiles. NaCl removal did not substantially affect growth, whereas glutamine supplementation reduced growth, especially in the laboratory strain. Both conditions modulated secondary metabolite production and biofilm formation in a strain-specific manner. In <i>P. aeruginosa</i> ATCC 27853, NaCl depletion significantly increased pyoverdine, pyocyanin, and QS gene expression, while biofilm formation showed significant differences only at 72 h; in contrast, glutamine supplementation affected only pyoverdine. A similar trend was observed in the clinical strain <i>P. aeruginosa</i> Pc, although NaCl depletion did not significantly impact pyoverdine production but already enhanced biofilm formation at 48 h. In <i>P. aeruginosa</i> Pr, only glutamine appeared to alter the considered parameters, increasing pyoverdine production while reducing pyocyanin and biofilm levels, although the absence of NaCl also negatively impacted biofilm formation. These findings highlight the impact of osmotic and nutritional signals on <i>P. aeruginosa</i> virulence traits.