Bacterial cell surface characterization by phage display coupled to high-throughput sequencing

The remarkable capacity of bacteria to adapt in response to selective pressures fuels the growth of antimicrobial resistance. _P. aeruginosa_ is a case in point: already intrinsically resistant to many antibiotics, it establishes decades-long chronic infections during which it evolves to survive antimicrobial treatment and evade host defenses. Many such adaptive changes occur on the _P. aeruginosa_ cell surface but our technologies for directly identifying such adaptive phenotypes are limited. Here we combined phage display with high-throughput DNA sequencing to create a high throughput, highly multiplexed technology for surveying bacterial cell surfaces. We performed phage display panning on hundreds of pairs of bacterial genotypes and used sequencing data to analyze the dynamics of the phage display selection process. We showed that these datasets capture important biological information about the surfaces of the cells under study. We discovered dozens of nanobodies that recognize key _P. aeruginosa_ virulence factors, including determinants of antimicrobial resistance, in their native conformations on live cells; these antibodies have numerous potential applications in diagnostics and therapeutics. We propose that "Phage-seq" enables a new paradigm for studying the bacterial cell surface by identifying and profiling many surface features in parallel. The techniques we have developed also enable interrogation of bacterial adaptations to immune pressure and antimicrobial treatment.