Establishment of a Method To Rapidly Assay Bacterial Persister Metabolism

Abstract

Bacterial persisters exhibit an extraordinary tolerance to antibiotics that is dependent on their metabolic state. Although persister metabolism promises to be a rich source of antipersister strategies, there is relatively little known about the metabolism of these rare and transient phenotypic variants. To address this knowledge gap, we explored the use of several techniques, and we found that only one measured persister metabolism. This assay was based on the phenomenon of metabolite-enabled aminoglycoside killing of persisters, and we used it to characterize the metabolic heterogeneity of different persister populations. From these investigations, we determined that glycerol and glucose are the most ubiquitously used carbon sources by various types of Escherichia coli persisters, suggesting that these metabolites might prove beneficial to deliver in conjunction with aminoglycosides for the treatment of chronic and recurrent infections. In addition, we demonstrated that the persister metabolic assay developed here is amenable to high-throughput screening with the use of phenotype arrays. Persisters are phenotypic variants that are tolerant to extraordinary concentrations of antibiotics. Persisters in biofilms have been hypothesized to underlie the proclivity of biofilm infections to relapse (1), and until recently there was no effective way to eliminate persisters. Two promising studies have demonstrated metabolism-dependent killing of persisters: one that potentiates aminoglycoside (AG) activity by stimulating proton motive force generation in persisters (2), and another that stimulates persister awakening (3). These methods suggest that knowledge of persister metabolic capabilities would expedite the discovery and development of antipersister therapies. Consequently, perturbations to numerous metabolic genes and regulators have been shown to alter persister levels (4-9, 30, 31), further supporting a central role for metabolism in maintenance of this phenotypic state. In addition, persisters have recently been shown to largely adopt a metabolically quiescent state that must be achieved and sustained by coordinated metabolic actions (10). Unfortunately, persister metabolism has not been sufficiently studied, and knowledge of the metabolic activities of these phenotypic variants remains limited. In part, this is due to difficulties associated with isolation of persisters from normally growing cultures, where current techniques provide only modest persister enrichment (11,12).In this study, we sought to measure the metabolic activities of natively generated Escherichia coli persisters, not those obtained synthetically by overexpression of a toxin (13) or treatment with persister-stimulating agents (14). First, we attempted to use the only isolation technique previously described to produce pure persister samples (15). In this method, normally growing bacteria are lysed with ampicillin (AMP), and the remaining cells, considered persisters, are sedimented by centrifugation. However, by using LIVE/DEAD staining, a ...

Impacts of Global Transcriptional Regulators on Persister Metabolism

“…The vast majority of persisters are dormant (7), and within such a state AGs are ineffective (9). In previous work, we discovered that specific metabolites produce AG killing of persisters and that such potentiation is dependent on catabolism of the substrate to generate proton motive force through respiration (9,10). This study demonstrated that persisters are metabolically active, and we have since shown that the assay can be used to measure the metabolism of persisters from antibiotic-treated cultures.…”

“…Notably, the approach circumvents the need to segregate persisters from other cell types, because within antibiotic-treated populations that have reached the second regime of biphasic killing, the only cells that remain culturable are persisters. Therefore, survival data from samples treated with an AG with or without a metabolite can be used to infer persister catabolism (10). Using this assay, we analyzed the impacts of ⌬arcA, ⌬cra, ⌬crp, ⌬dksA, ⌬fnr, ⌬lrp, and ⌬rpoS on the ability of persisters to consume carbon sources and generate proton motive force aerobically.…”

“…Due to their low abundance, transient nature, and similarities to cells of the more highly abundant viable but nonculturable cell (VBNC) phenotype, highly pure persister samples have yet to be isolated (10). In the absence of such methods, we developed an approach to measure metabolic activity in persisters that utilizes the phenomenon of metabolite-enabled aminoglycoside (AG) potentiation (9).…”

“…While persisters largely arise from dormant subpopulations (2,4,7,8), recent studies have demonstrated that they remain metabolically active, with the capacity to catabolize specific carbon sources and generate proton motive force through respiration (9,10). This metabolic activity, specifically, proton motive force generation, enables aminoglycoside (AG) transport into cells that results in killing of persisters, and several enzymes needed for this process have been identified (9,10).…”

“…This metabolic activity, specifically, proton motive force generation, enables aminoglycoside (AG) transport into cells that results in killing of persisters, and several enzymes needed for this process have been identified (9,10). Knowledge of the enzymes and metabolic pathways present in persisters, as well as how they can be altered, could prove useful for the development of antipersister therapies.…”