Influence of Nutritional Conditions on Coexistence of <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i> in the Dual‐Species Biofilm

Pseudomonas aeruginosa is a prominent uropathogen associated with biofilm-related urinary tract infections. It can coexist in a biofilm environment with other pathogens, including Staphylococcus aureus, and the availability of metabolites in urine may influence interspecies interactions that can be cooperative or competitive. Here, a dual-species biofilm model consisting of uropathogenic and reference strains of P. aeruginosa and S. aureus was used to understand their coexistence under different nutritional conditions using synthetic urine supplemented with creatinine, glucose, albumin, and haem. A dual-species biofilm was developed using equal initial cell densities of pathogens. Biofilm intensity was quantified by crystal violet staining. Biofilm biomass, growth, ureolysis were estimated and compared with mono-species cultures. P. aeruginosa formed higher biofilm than S. aureus (p < 0.01) under mono-species culture. Overall, dual-species biofilm intensity was significantly higher than mono-species biofilm (p < 0.01), except in albumin, where S. aureus mono-species biofilm was higher. Biofilm biomass enumeration indicates near-equilibrium coexistence of species within the biofilm matrix. The ureolytic activity correlated with growth and biofilm observations. Our study highlights the complex interactions in dual-species biofilms and emphasizes the need for further studies involving diverse uropathogenic strains to assess metabolite influences on biofilm structural dynamics and its effect on antibiotic responses for targeted therapeutics.