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Background  The accumulation of thick stagnant mucus provides a suitable environment for the growth of  Pseudomonas aeruginosa  and  Staphylococcus aureus  within the lung alveoli of cystic fibrosis (CF) patients. These infections cause significant lung damage, leading to respiratory failure and death. In an artificial mucin containing medium ASM+,  P. aeruginosa  forms structures that resemble typical biofilms but are not attached to any surface. We refer to these structures as biofilm like structures (BLS). Using ASM+ in a static microtiter plate culture system, we examined the roles of mucin, extracellular DNA, environmental oxygen (EO 2 ), and quorum sensing (QS) in the development of biofilm-like structures (BLS) by  P. aeruginosa ; and the effect of EO 2  and  P. aeruginosa  on  S. aureus  BLS.    Results  Under 20% EO 2 ,  P. aeruginosa  strain PAO1 produced BLS that resemble typical biofilms but are confined to the ASM+ and not attached to the surface. Levels of mucin and extracellular DNA within the ASM+ were optimized to produce robust well developed BLS. At 10% EO 2 , PAO1 produced thicker, more developed BLS, while under 0% EO 2 , BLS production was diminished. In contrast, the  S. aureus  strain AH133 produced well-developed BLS only under 20% EO 2 . In PAO1, loss of the QS system genes  rhlI  and  rhlR  affected the formation of BLS in ASM+ in terms of both structure and architecture. Whether co-inoculated into ASM+ with AH133, or added to established AH133 BLS, PAO1 eliminated AH133 within 48–56 h.    Conclusions  The thick, viscous ASM+, which contains mucin and extracellular DNA levels similar to those found in the CF lung, supports the formation of biofilm-like structures similar to the aggregates described within CF airways. Alterations in environmental conditions or in the QS genes of  P. aeruginosa , as occurs naturally during the progression of CF lung infection, affect the architecture and quantitative structural features of these BLS. Thus, ASM+ provides an  in vitro  medium in which the effect of changing levels of substances produced by the host and the bacteria can be analyzed to determine the effect on such structures and on the susceptibility of the bacteria within the BLS to various treatments.

Characterization of biofilm-like structures formed by Pseudomonas aeruginosa in a synthetic mucus medium