The genetic landscape of antibiotic sensitivity in Staphylococcus aureus

Despite the critical importance of essential genes in bacteria, systems-level investigations of their contribution to antibiotic sensitivity have been limited. Using CRISPR Adaptation-mediated Library Manufacturing (CALM), we generated ultra-dense CRISPR-interference libraries in methicillin-sensitive and -resistant Staphylococcus aureus and quantified global gene fitness across ten antibiotics. This approach revealed a comprehensive set of known and novel biological processes affecting bacterial fitness in antibiotics. Importantly, essential genes dominated the hundreds of significant antibiotic-gene interactions we identified; inhibition of diverse processes, including cell wall synthesis/cell division, DNA replication/DNA recombination, coenzyme A, and riboflavin biosynthesis, sensitized bacteria to antibiotics. Gene similarity network and genetic analysis revealed extensive synergistic genetic interactions among essential genes in these processes, a finding that was further corroborated by small molecule inhibitors. Our detailed drug-gene, gene-gene, and drug-drug profiling uncovers critical functional relationships within the core essential genome and establishes a rational foundation for developing effective antimicrobial combinations.