Multiplexing antibiotic screening assay in droplet microfluidics.

Environmental samples contain complex microbial communities hiding a treasure trove of active compounds. However, screening for active natural products from environmental samples is challenging due to inefficient cultivation techniques and a lack of proper screening platforms. For empowering antibiotic screening assays from complex microbial communities, we have developed and optimized a droplet-based platform with multiplexing capability. A cultivation strategy for bacteria in picoliter droplets was combined with phenotypic screening using multiple whole-cell reporter species. A mixture of two different fluorescently labelled reporter strains, one Gram-positive and one Gram-negative, was picoinjected to each of millions of picoliter cultures, which were screened for inhibiting activity based on the independent survival signals of each reporter species. Proof-of-concept experiments demonstrate efficient detection, selection, and recovery of a model Streptomyces strain from a synthetic mixture according to the specific inhibition of the reporter strains by the produced antibiotic. Subsequently, the established platform was successfully applied to screen environmental microbial communities from soil samples. This approach showcases multiplexing capabilities for screening assays in microfluidic droplets in order to simultaneously screen for new bioactive compounds with various inhibition profiles.