Current antibiotics are increasingly ineffective due to rising resistance, and antibiotic discovery campaigns frequently fail. One key factor in effective antibiotic discovery is knowing whether a target’s function is essential in a bacterial species. We present a general paradigm for comprehensively identifying the core essential genome of clinical pathogens to identify candidate drug targets. We applied the paradigm to Pseudomonas aeruginosa, a priority antibiotic-resistant pathogen, by performing genome-wide genetic selection studies across a diverse set of clinical isolates and infection-relevant growth conditions (serum, sputum, and urine). We identified 321 core essential genes that constitute a high-priority list of candidate targets for drug discovery. The strategy should be applicable to define the core essential genome for most clinical pathogens.

Abstract

Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. While numerous factors contributed to the disappointing yield, one factor was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both the genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains of Pseudomonas aeruginosa on five different media and developed a statistical model, FiTnEss, to classify genes as essential versus nonessential across all strain–medium combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of four strains was typically sufficient in P. aeruginosa to converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.