Milieu matters: An in vitro wound milieu to recapitulate key features of, and probe new insights into, mixed-species bacterial biofilms.

Bacterial biofilms are a major cause of delayed wound healing. Consequently, the study of wound biofilms, particularly in host-relevant conditions, has gained importance. Most in vitro studies employ refined laboratory media to study biofilms, representing conditions that are not relevant to the infection state. To mimic the wound milieu, in vitro biofilm studies often incorporate serum or plasma in growth conditions, or employ clot or matrix-based biofilm models. While incorporating serum or plasma alone is a minimalistic approach, the more complex in vitro wound models are technically demanding, and poorly compatible with standard biofilm assays. Based on previous reports of clinical wound fluid composition, we have developed an in vitro wound milieu (IVWM) that includes, in addition to serum (to recapitulate wound fluid), matrix elements and biochemical factors. With Luria-Bertani broth and Fetal Bovine Serum (FBS) for comparison, the IVWM was used to study planktonic growth, biofilm features, and interspecies interactions, of common wound pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. We demonstrate that the IVWM recapitulates widely reported in vivo biofilm features such as biomass formation, metabolic activity, increased antibiotic tolerance, 3D structure, and interspecies interactions for monospecies and mixed-species biofilms. Further, the IVWM is simple to formulate, uses laboratory-grade components, and is compatible with standard biofilm assays. Given this, it holds potential as a tractable approach to study wound biofilms under host-relevant conditions.