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The coexistence between species that occurs in some infections remains hard to achieve  in vitro  since bacterial fitness differences eventually lead to a single organism dominating the mixed culture.  Pseudomonas aeruginosa  and  Staphylococcus aureus  are major pathogens found growing together in biofilms in disease-affected lungs or wounds. Herein, we tested and analyzed different culture media, additives and environmental conditions to support  P. aeruginosa  and  S. aureus  coexistence  in vitro . We have unraveled the potential of DMEM to support the growth of these two organisms in mature cocultured biofilms (three days old) in an environment that dampens the pH rise. Our conditions use equal initial inoculation ratios of both strains and allow the stable formation of separate  S. aureus  microcolonies that grow embedded in a  P. aeruginosa  biofilm, as well as  S. aureus  biofilm overgrowth when bovine serum albumin is added to the system. Remarkably, we also found that  S. aureus  survival is strictly dependent on a well-characterized phenomenon of oxygen stratification present in the coculture biofilm. An analysis of differential tolerance to gentamicin and ciprofloxacin treatment, depending on whether  P. aeruginosa  and  S. aureus  were growing in mono- or coculture biofilms, was used to validate our  in vitro  coculture conditions.

Optimal environmental and culture conditions allow the in vitro coexistence of Pseudomonas aeruginosa and Staphylococcus aureus in stable biofilms