In vitro reconstitution of the polymicrobial community associated with CF airway infections

The airways of persons with cystic fibrosis (CF) provide a nutritionally rich environment that is prone to colonisation by a diverse and dynamic community of microbes (including both bacteria and fungi). Traditionally, Pseudomonas aeruginosa (PA) was thought to be the dominant pathogen associated with CF airway infections. However, it is becoming increasingly clear that interspecies interactions play a crucial role in modulating the physiology and gene expression profile(s) of the protagonists, and also impact on the response to therapeutic intervention. However, there are currently no suitable experimental models that permit long-term successful co-cultivation of PA with other CF-associated pathogens. Simply mixing bacterial or fungal species together and hoping for the best is not a recipe for success, since PA rapidly outcompetes most other species when grown in batch culture. In this work, we rectify this by describing a “3Rs-compliant” continuous-flow in vitro co-culture model. Using our model, it is possible to maintain remarkably stable steady-state co-cultures of major CF-associated pathogens (PA, Staphylococcus aureus and Candida albicans). Our findings reveal that even numerically minor (0.1%, by cell number) species can have a profound impact on quorum sensing and virulence factor production by PA. Furthermore, we show that complete polymicrobial communities derived from CF sputum can be inoculated directly into the model, thus enabling the recapitulation of the entire microbiome associated with CF airway infections to be studied under physiologically relevant conditions.