Analysis of mono, duo and multispecies biofilms of Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus.

Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis are nosocomial pathogens often associated with hard-to-treat infections due to their ability to form biofilms, a key virulence factor. Within biofilms, microbial interactions modulate virulence and antimicrobial resistance, resulting in therapeutic failures and poor patient outcomes. Biofilms account for nearly 80% of chronic infections, highlighting their persistence and difficulty of eradication. This study evaluated the interactions of P. aeruginosa, E. faecalis, and S. aureus in mono-, duo-, and multi-species cultures, focusing on growth, biofilm formation, and virulence in an in vivo model. In vitro assays assessed bacterial growth (OD620 nm) and mono, duo, and multispecies biofilms through biomass quantification (crystal violet), viable cell counts (CFU/mL), and cell viability (XTT). Additionally, infection assays using Caenorhabditis elegans were used to evaluate the virulence of these pathogens by monitoring host survival. Duo and multispecies biofilms showed greater growth than monospecies, except for S. aureus + E. faecalis. Biofilm biomass was also higher in most combinations, except for S. aureus + P. aeruginosa. E. faecalis predominated in CFU counts of duo- and multispecies biofilms, showing levels similar to S. aureus in biofilm-associated cells and to P. aeruginosa in planktonic cells. XTT results indicated a higher viability of S. aureus cells in the biofilm and P. aeruginosa in the planktonic form. Finally, in vivo infections showed higher mortality in monospecies groups, due to the complexity of interspecies interactions that prioritize survival and persistence within the biofilm over pathogenicity. This study provides, for the first time, the analysis of S. aureus, P. aeruginosa, and E. faecalis in multispecies biofilm and in vivo model infection, advancing the understanding of interspecies dynamics and biofilm-associated infections.