Energy expenditure and cellular activity underlie antibiotic tolerance of <i>Pseudomonas aeruginosa</i>

Persisters are a subpopulation of bacterial cells that survive a lethal dose of antibiotic. The failure to treat infections has been linked to the presence of these drug-tolerant cells. The recalcitrant, and often incurable, infection of cystic fibrosis airways by <i>Pseudomonas aeruginosa</i> is attributed to persisters found within aggregate biofilms that contain stationary cells. In all bacteria studied, the fraction of persisters is the highest in stationary populations. However, the level of persisters is unusually low in stationary phase <i>P. aeruginosa</i>, which is unexpected, given the recalcitrance to antibiotic therapy. Here, we set out to investigate the mechanism of <i>P. aeruginosa</i> antibiotic susceptibility in stationary phase. We find that <i>P. aeruginosa</i> is highly active in stationary phase, based on its rate of translation, and this correlates with increased killing compared to <i>Escherichia coli</i>. Growth within alginate beads improves <i>P. aeruginosa</i> stationary cell tolerance to antibiotic treatment, likely due to a subpopulation of low-translating cells. Secondary messenger cyclic diguanylate (c-di-GMP) regulates biofilm formation, and we find that a population of low-translating persisters also appeared in planktonically growing cells when c-di-GMP production is induced. This phenotype only occurred when exopolysaccharide production was intact, and we propose that the energetic demand of EPS overproduction induces dormancy. The level of energy expenditure appears to determine persistence in <i>P. aeruginosa. In vivo</i>-like growth conditions and a shift to a biofilm lifestyle, mediated by high c-di-GMP production, increase antibiotic tolerance by generating low-energy, dormant persisters.IMPORTANCERecalcitrant bacterial infections are a continued burden on the healthcare system. Antibiotic treatment failure, especially for chronic infections, can be attributed to persisters, a subpopulation of dormant cells that survive a lethal dose of drug. The infection of cystic fibrosis (CF) airways by <i>Pseudomonas aeruginosa</i> is often incurable to treatment by multiple classes of antibiotics due to the presence of persisters. <i>P. aeruginosa</i>, however, is highly susceptible to antibiotic killing <i>in vitro</i>, in apparent contradiction of its drug tolerance during infection. Here, we show that <i>P. aeruginosa</i> susceptibility to antibiotic killing is due to its continued protein synthesis and cellular activity even with entrance into stationary phase. Furthermore, we identify that the greater energetic demand of biofilm growth generates a larger fraction of low-translating persisters and increases antibiotic tolerance. These findings improve our understanding of <i>P. aeruginosa</i> antibiotic tolerance during CF infection and will aid the development of better treatment regimens.