(247e) A High Throughput Screening Platform for Antibiotic Drug Discovery

We describe an ultra-high throughput screening platform enabling discovery and/or engineering of antibacterial biomolecules. Individual recombinant expression hosts and bacterial pathogens are co-encapsulated within hydrogel-in-oil emulsions, hosts secreting bactericidal agents kill adjacent bacterial targets, and the corresponding gel microdroplets are selectively labeled using a fluorogenic viability probe. Importantly, the hydrogel matrix serves to physically link active expression hosts with cognate killed pathogens, and the fluorescent dye provides an easily detected phenotype. We have successfully employed both bulk emulsification and microfluidics technology to generate picoliter-scale gel microdroplets that are size-compatible with conventional flow cytometry. Using fluorescence activated cell sorting, libraries can be screened at rates exceeding 3,000 microdroplets per second. We have easily achieved throughputs of more than 5 million clones per day, demonstrating the capacity to outstrip conventional screening methods by orders of magnitude. In proof-of-concept experiments using a 10,000-fold excess of negative control cells, we efficiently selected yeast engineered to secrete lysostaphin, a powerful anti-Staphylococcal enzyme. As a practical extension of this work, we constructed a small metagenomic library derived from the native plasmids of Staphylococcus simulans, Staphylococcus arlettae, and Staphylococcus equorum, each a natural environmental competitor of Staphylococcus aureus. In this case we utilized an E. coli expression host and screened the library against a methicillin resistant S. aureus strain. In a matter of just 4 days, we isolated the native lysostaphin gene, which is naturally encoded on the native pACK1 plasmid of S. simulans. Our results establish the practical utility of the screening platform, and we anticipate that the accessible nature of our methods will enable others seeking to identify and engineer the next generation of antibacterial biomolecules.