Pseudomonas aeruginosa and Candida albicans both accumulate greater biomass in dual species biofilms under flow

Spatially structured communities of microbes – biofilms – are widespread in nature, and biofilm-dwelling microbes often respond to their environments in ways that are different from their planktonic counterparts. Further, most natural biofilms are multi-species mixtures of microorganisms; the ecology of intra- and inter-species interactions in these consortia, and the resulting effects on total community properties, are often not well understood. A common site of polymicrobial biofilm infections is the lungs of patients with cystic fibrosis (CF). CF is a genetic disorder in humans that leads to colonization of the lungs by a variety of microorganisms, including Pseudomonas aeruginosa and Candida albicans. These opportunistic pathogens are frequently co-isolated from infected lungs, in addition to other infection sites including urinary and intravenous catheters. To study how these microbes behave together in biofilms, we developed a modified artificial sputum medium that is optically clear for use with microfluidic culture. In addition, we engineered strains with optimized fluorescent protein expression constructs allowing for single-cell resolution confocal microscopy. Using these tools and recently developed methods for spatial analysis of 3-D image data, we found that both P. aeruginosa and C. albicans display increased biovolume accumulation in multi-species biofilms relative to single-species biofilms. This pattern did not occur in planktonic co-culture and was thus specific to the biofilm environment. Interestingly, introduction of P. aeruginosa supernatants over dual-species biofilms strongly reduced C. albicans biovolume. This suggests that products that accumulate in batch culture were still inhibitory to C. albicans under a flow regime, but that they their de novo production in mixed species biofilms was not sufficient to inhibit C. albicans biofilm accumulation. Altogether our results indicate a critical impact of flow environment for the outcome of polymicrobial interactions and the need for high-resolution analysis of such communities in future work.