Antimicrobial activity against Mycobacterium tuberculosis under in vitro lipid-rich dormancy conditions

Bacterial persisters are phenotypic variants that form from the action of stress response pathways triggering toxin-mediated antibiotic tolerance. Although persisters form during normal growth from native stresses, the pathways responsible for this phenomenon remain elusive. Here we have discovered that carbon source transitions stimulate the formation of fluoroquinolone persisters in Escherichia coli. Further, through a combination of genetic, biochemical, and flow cytometric assays in conjunction with a mathematical model, we have reconstructed a molecular-level persister formation pathway from initial stress (glucose exhaustion) to the activation of a metabolic toxin-antitoxin (TA) module (the ppGpp biochemical network) resulting in inhibition of DNA gyrase activity, the primary target of fluoroquinolones. This pathway spans from initial stress to antibiotic target and demonstrates that TA behavior can be exhibited by a metabolite-enzyme interaction (ppGpp-SpoT), in contrast to classical TA systems that involve only protein and/or RNA. Copyright © 2013 Elsevier Inc. All rights reserved.

The antibiotic tolerances of bacterial persisters have been attributed to transient dormancy. While persisters have been observed to be growth inhibited prior to antibiotic exposure, we sought to determine whether such a trait was essential to the phenotype. Furthermore, we sought to provide direct experimental evidence of the persister metabolic state so as to determine whether the common assumption of metabolic inactivity was valid. Using fluorescence-activated cell sorting (FACS), a fluorescent indicator of cell division, a fluorescent measure of metabolic activity, and persistence assays, we found that bacteria that are rapidly growing prior to antibiotic exposure can give rise to persisters and that a lack of replication or low metabolic activity prior to antibiotic treatment simply increases the likelihood that a cell is a persister. Interestingly, a lack of significant growth or metabolic activity does not guarantee persistence, as the majority of even "dormant" subpopulations (>99%) were not persisters. These data suggest that persistence is far more complex than dormancy and point to additional characteristics needed to define the persister phenotype.

Bacterial persistence is a state in which a subpopulation of cells (persisters) survives antibiotic treatment, and has been implicated in the tolerance of clinical infections and the recalcitrance of biofilms. There has been a renewed interest in the role of bacterial persisters in treatment failure in light of a wealth of recent findings. Here we review recent laboratory studies of bacterial persistence. Further, we pose the hypothesis that each bacterial population may contain a diverse collection of persisters and discuss engineering strategies for persister eradication. Copyright © 2011 Elsevier Ltd. All rights reserved.