A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms

Pathogenic bacteria form biofilms during infection, and polymicrobial biofilms are the most frequent manifestation. Biofilm attachment, maturation, and/or antibiotic sensitivity are mainly evaluated with microtiter plate assays, in which bacteria are stained to enable the quantification of the biomass by optical absorbance or fluorescence emission. However, using these methods to distinguish different species in dual-species or polymicrobial biofilms is currently impossible. Colony-forming unit counts from homogenized dual-species biofilms on selective agar medium allow species differentiation but are time-consuming for a high-throughput screening. Thus, reliable, feasible, and fast methods are urgently needed to study the behavior of polymicrobial and dual-species communities. This study shows that Pseudomonas aeruginosa and Burkholderia cenocepacia strains expressing specific fluorescent or bioluminescent proteins permit the more efficient study of dual-species biofilms compared to other methods that rely on measuring the total biomass. Combining fluorescence and bioluminescence measurements allows an independent analysis of the different microbial species within the biofilm, indicating the degree of presence of each one over time during a dual-species biofilm growth. The quantitative strategies developed in this work are reproducible and recommended for dual-species biofilm studies with high-throughput microtiter plate approaches using strains that can constitutively express fluorescent or bioluminescent proteins.