In the lung, a thick and dense mucus builds up over the pulmonary epithelium, converting it into the perfect niche for bacterial colonization and growth 9, 10. CF is a lethal genetic disease characterized by the production of abnormal secretions in different organs 8. Regarding human pathogenesis, cystic fibrosis (CF) is a model example of how bacterial interactions within biofilms can modulate the outcome of the disease, thus playing a critical role in the patient’s wellbeing 7. Therefore, there is an intrinsic effect of the surrounding environment on the way microbes behave and establish their connections 3. Clusters or aggregates perfectly arranged and determined by the different bacteria that are able to grow concurrently are more likely to occur during the establishment and persistence of the infection. For instance, Pseudomonas aeruginosa, in addition to forming large mushroom-shaped biofilm structures in vitro, can behave differently and form different bacterial arrangements in vivo. The way bacteria are distributed and interact with each other or with the host fluctuates depending on the environment 5, 6. Polymicrobial biofilms can develop greater antimicrobial resistance than single-species biofilms 4. Although they have been historically investigated as monoculture events, some infection-associated biofilms are currently recognized to be mainly polymicrobial and involve synergistic interactions that often worsen the disease outcome 2, 3. Most chronic infections occur due to the inherent capacity of some bacterial pathogens to grow in biofilms 1. aureus were growing in mono- or coculture biofilms, was used to validate our in vitro coculture conditions. An analysis of differential tolerance to gentamicin and ciprofloxacin treatment, depending on whether P. aureus survival is strictly dependent on a well-characterized phenomenon of oxygen stratification present in the coculture biofilm. aureus biofilm overgrowth when bovine serum albumin is added to the system. aureus microcolonies that grow embedded in a P. Our conditions use equal initial inoculation ratios of both strains and allow the stable formation of separate S. 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. Herein, we tested and analyzed different culture media, additives and environmental conditions to support P. Pseudomonas aeruginosa and Staphylococcus aureus are major pathogens found growing together in biofilms in disease-affected lungs or wounds. 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.
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