SUMMARY The goal of the Harvard-wide Program on Antibiotic Resistance is to apply innovative advanced technologies to developing new strategies for treating leading causes of multidrug resistant hospital-associated infection caused by staphylococci and enterococci. Although much attention has appropriately been focused on the recent emergence of pan-resistant gram negative bacteria, staphylococci and enterococci remain the leading causes of both morbidity and mortality associated with infection by multidrug resistant ESKAPE pathogens, highlighting the limitation of current therapeutic approaches. This multi-investigator project brings together experts from fields ranging from mathematics and systems biology to infectious disease clinicians, and creates an infrastructure for them to work collaboratively and synergistically, bringing new insights and capabilities to bear in solving the leading public health crisis of antibiotic resistant infection. Important discoveries and advances were made in the previous period of support by pressing the envelope with new technologies, including the most advanced high throughput mutational and comparative genomic analyses yet conducted on these microbes. In the proposed continuation period, these technologies will be combined with the latest advances in microfluidics and single cell time lapse imaging, to reveal new vulnerabilities of these bacteria for targeting by new compounds. The development and use of innovative screens that de-risk targets as well as unveil new opportunities, inform the design of new and more efficacious compounds for treating multidrug resistant staphylococcal and enterococcal infection. Filling critical knowledge gaps related to the various mechanisms by which microbes survive antibiotic challenge, including stochastic entry into alternate growth states and actively reducing drug concentration through efflux, make this program exceptionally comprehensive in its approach. Finally, this project actively shares its results with the community by establishing and promoting information exchange and networking with other academic and industry scientists, and by ensuring important antibiotic discovery knowledge is preserved and transmitted to the next generation of scientists. As a result of internal and external synergies, the impact of the Harvard-wide Program on Antibiotic Resistance is much greater than would otherwise be possible through individual research efforts.