AbstractTraditional antibacterial screens rely on growing bacteria in nutrient-replete conditions which are not representative of the natural environment or sites of infection. Instead, screening in more physiologically relevant conditions may reveal novel activity for existing antibiotics. Here, we screened a panel of antibiotics reported to lack activity against the opportunistic Gram-negative bacterium,Pseudomonas aeruginosa,under low-nutrient conditions, and discovered that the glycopeptide vancomycin inhibited growth ofP. aeruginosaat low micromolar concentrations through its canonical mechanism of action, disruption of peptidoglycan cross-linking. Spontaneous vancomycin-resistant mutants had activating mutations in the sensor kinase of the two-component CpxSR system, which induced cross-resistance to almost all classes of ý-lactams, including the siderophore antibiotic cefiderocol. Other mutations that conferred vancomycin resistance mapped to WapR, an α-1,3-rhamnosyltransferase involved in lipopolysaccharide core biosynthesis. A WapR P164T mutant had a modified LPS profile compared to wild type that was accompanied by increased susceptibility to select bacteriophages. We conclude that screening in nutrient-limited conditions can reveal novel activity for existing antibiotics and lead to discovery of new and impactful resistance mechanisms.For Table of Contents Use Only
