Cystic Fibrosis Aggregate Biofilm Model to Study Infection-relevant Gene Expression

Standard pre-clinical testing methods for novel antimicrobial therapeutics used to treat chronic lung infections in people with cystic fibrosis do not reflect the environmental conditions of the hostile lung niche. Current reductionist testing conditions can lead to the progression of compounds along a preclinical pipeline without evidence of their activity under cystic fibrosis lung niche-appropriate conditions. Several approaches used to study traditional antimicrobials may not be suitable for antibiotic alternatives, including anti-virulence therapeutics like anti-quorum sensing agents and siderophore inhibitors. This protocol documents an aggregate biofilm model of Pseudomonas aeruginosa to compare resistance and infection-relevant gene expression in single-species and multi-species cultures (Staphylococcus aureus and Candida albicans), examining colony-forming unit (CFU) reductions and changes in gene expression, using algD as an exemplar. The model was optimized for small, static volumes of bacterial cultures to allow the study of novel compounds in the discovery phase of the drug development pipeline, where compound quantities may be limited. Single-species P. aeruginosa biofilms were formed in Synthetic Cystic Fibrosis Medium 2 (SCFM2) for 24 h before treatment with meropenem at different concentrations (1, 16, and 256 µg/mL) for a further 24 h. Polymicrobial biofilms were established by growing Staphylococcus aureus and Candida albicans together in SCFM2, then inoculating with P. aeruginosa for an additional 24 h and treating with meropenem. The lack of a direct connection between compound efficacy measures in pre-clinical testing and clinical trial results has cast doubt on the applicability of current laboratory screening tools. This model allows us to understand the impact of relevant factors on P. aeruginosa gene expression, including genes contributing to resistance and virulence, thereby bridging this gap.