Mechanical properties of Staphylococcus aureus and Pseudomonas aeruginosa dual-species biofilms grown in chronic wound-based models

Abstract

Wound infections become chronic due to biofilm formation by pathogenic bacteria; two such pathogens are Staphylococcus aureus and Pseudomonas aeruginosa. These bacteria are known to form polymicrobial biofilms in wounds, which exhibit increased colonization rates, enhanced chronicity, and greater resistance to treatment. Previously, the impacts of a wound bed environment on the mechanical properties of P. aeruginosa biofilms have been explored, and in this work the role of a wound bed environment in the viscoelasticity and microstructure of polymicrobial biofilms is characterized. We hypothesize that common wound bed proteins mediate interactions between S. aureus and P. aeruginosa to enable the formation of more elastic and stiff biofilms. Growth media with varying protein content as well as additional collagen, a protein associated with a wound extracellular matrix, were utilized to test our hypothesis. Microrheology indicates that both P. aeruginosa and S. aureus form relatively stiffer single-species biofilms in a wound environment with collagen. S. aureus produced stiffer biofilms in the presence of collagen, regardless of other wound proteins, likely due to its interactions with collagen. When both species were grown together in wound-like media, synergistic effects led to stiffer dual-species biofilms compared to their single-species forms. Under all growth conditions, collagen significantly contributed to stiffening P. aeruginosa/S. aureus dual-species biofilms, suggesting that it mediates complex interspecies interactions. High-resolution imaging and analysis revealed that collagen also influenced the microstructures of P. aeruginosa/S. aureus dual-species biofilms. In media containing wound proteins and collagen, S. aureus clusters were larger and exhibited more complex shapes. These results indicate that the wound bed environment not only provides improved antibacterial resistance due to cooperative interactions, but also improved mechanical protection, which impact common treatment methods like debridement.