Understanding and Sensitizing Density-Dependent Persistence to Quinolone Antibiotics

Abstract

Physiologic and environmental factors can modulate antibiotic activity and thus pose a significant challenge to antibiotic treatment. The quinolone class of antibiotics, which targets bacterial topoisomerases, fails to kill bacteria that have grown to high density; however, the mechanistic basis for this persistence is unclear. Here, we show that exhaustion of the metabolic inputs that couple carbon catabolism to oxidative phosphorylation is a primary cause of growth phase-dependent persistence to quinolone antibiotics. Supplementation of stationary-phase cultures with glucose and a suitable terminal electron acceptor to stimulate respiratory metabolism is sufficient to sensitize cells to quinolone killing. Using this approach, we successfully sensitize high-density populations of Escherichia coli, Staphylococcus aureus, and Mycobacterium smegmatis to quinolone antibiotics. Our findings link growth-dependent quinolone persistence to discrete impairments in respiratory metabolism and identify a strategy to kill non-dividing bacteria.

Preexisting variation in DNA damage response predicts the fate of single mycobacteria under stress

“…The fate of single cells was independent of both the growth rate and cell age ( Fig 4E and Appendix Fig S4A). Cells cultured in fresh medium produced only 1.5% of survivors, whereas cells cultured in spent medium produced 86% of survivors, which is consistent with reduced CIP efficacy during starvation and stationary phase (Sarathy et al, 2013;Gutierrez et al, 2017). Although cells cultured in spent medium had slower growth rates ( Fig 4E) and greater chromosomal condensation, expressed as an increase in standard deviation of DRAQ5 fluorescence (Appendix Fig S4B), they continued to grow and divide even during CIP exposure, whereas cells cultured in fresh medium continued to elongate without dividing until they arrested (Appendix Fig S4C).…”

“…Fluoroquinolones are a case in point of how complex drug persistence can be. The efficacy of fluoroquinolones in bulk assays is inversely proportional to the bacterial metabolic state and cell density, not merely due to target unavailability and decreased permeability (Sarathy et al, 2013;Gutierrez et al, 2017), but also for the ability of both replicating and non-replicating cells to repair DNA (Volzing and Brynildsen, 2015). In line with this, we observe that single cells cultured in spent medium grow slowly but dynamically respond to CIP by inducing RecA and produce almost 60 times more survivors than cells growing rapidly in fresh medium, an advantage that is lost upon restoration of fresh medium.…”

Multisite phosphorylation regulates phenotypic variability in antibiotic tolerance

“…In the Δ sasA background, we observed a much weaker dependence of antibiotic tolerance on sasA expression. This residual dependence is consistent with previous results that have implicated many global processes in antibiotic tolerance including heterogeneity in growth state 24, 57, 58 and enhanced expression of drug efflux pumps 59, 60 . This is also consistent with the relatively weak correlation in expression between sasA and the constitutive promoter veg (Fig.…”

Contact‐dependent growth inhibition induces high levels of antibiotic‐tolerant persister cells in clonal bacterial populations

“…In support of this hypothesis, CDI‐expressing cells do not exhibit increased persister frequencies at low cell densities (10 7 cfu/ml; Fig A and B). Furthermore, bacteria in dense populations such as biofilms or in stationary phase display increased tolerance to antibiotics compared to exponentially growing planktonic cells (Spoering & Lewis, ; Keren et al , ; Maisonneuve et al , ; Gutierrez et al , ). In addition, repeated exposure to antibiotics rapidly select for mutations that increase the fraction of persisters in stationary phase cultures (Van den Bergh et al , ), suggesting that increasing the fraction of non‐growing cells at high cell densities is a good strategy for survival.…”

“…In contrast to previously identified toxins that contribute to persister formation, CDI only creates non‐growing cells upon cell‐to‐cell contact. Cell‐to‐cell contacts are more common at high cell densities; for example, both stationary phase and in biofilms are conditions where persisters are found frequently (Spoering & Lewis, ; Keren et al , ; Maisonneuve et al , ; Gutierrez et al , ). But in contrast to previously identified systems involved in generation of persisters, CDI functions as a quorum sensing system where an increased fraction of persisters is generated only when there are many cells of the same genotype.…”