The prevalence and structure of Staphylococcus aureus and Staphylococcus epidermidis within multispecies biofilms were found to depend sensitively on physical environment and antibiotic dosage. Although these species commonly infect similar sites, such as orthopedic implants, little is known about their behavior in multispecies communities, particularly in response to treatment. This research establishes that S. aureus is much more prevalent than S. epidermidis when simultaneously seeded and grown under unstressed conditions (pH 7, 37°C) in both laboratory and clinical strains. In multispecies communities, S. epidermidis is capable of growing a more confluent biofilm when the addition of S. aureus is delayed 4 to 6 h during 18 h of growth. Different vancomycin dosages generate various behaviors: S. epidermidis is more prevalent at a dose of 1.0 μg/ml vancomycin, but reduced growth of both species occurs at 1.9 μg/ml vancomycin. This variability is consistent with the different MICs of S. aureus and S. epidermidis Growth at higher temperature (45°C) results in an environment where S. aureus forms porous biofilms. This porosity allows S. epidermidis to colonize more of the surface, resulting in detectable S. epidermidis biomass. Variations in pH result in increased prevalence of S. epidermidis at low pH (pH 5 and 6), while S. aureus remains dominant at high pH (pH 8 and 9). This work establishes the structural variability of multispecies staphylococcal biofilms as they undergo physical and antimicrobial treatments. It provides a basis for understanding the structure of these communities at infection sites and how treatments disrupt their multispecies behaviors.IMPORTANCEStaphylococcus aureus and Staphylococcus epidermidis are two species of bacteria that are commonly responsible for biofilm infections on medical devices. Biofilms are structured communities of bacteria surrounded by polysaccharides, proteins, and DNA; bacteria are more resistant to antimicrobials as part of a biofilm than as individual cells. This work investigates the structure and prevalence of these two organisms when grown together in multispecies biofilms and shows shifts in the behavior of the polymicrobial community when grown in various concentrations of vancomycin (an antibiotic commonly used to treat staphylococcal infections), in a high-temperature environment (a condition previously shown to lead to cell disruption and death), and at low and high pH (a change that has been previously shown to soften the mechanical properties of staphylococcal biofilms). These shifts in community structure demonstrate the effect such treatments may have on multispecies staphylococcal infections.

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