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.

Bacterial persisters are phenotypic variants with extraordinary tolerances toward antibiotics. Persister survival has been attributed to inhibition of essential cell functions during antibiotic stress, followed by reversal of the process and resumption of growth upon removal of the antibiotic. Metabolism plays a critical role in this process, since it participates in the entry, maintenance, and exit from the persister phenotype. Here, we review the experimental evidence that demonstrates the importance of metabolism to persistence, highlight the successes and potential of targeting metabolism in the search for anti-persister therapies, and discuss the current methods and challenges to understand persister physiology.

Persisters are phenotypic variants present within isogenic bacterial populations that exhibit extreme tolerance toward antibiotic stress. We previously elucidated a mechanistic pathway by which Escherichia coli persisters to ofloxacin form in response to a carbon source transition. Here, we examine how persisters to ampicillin form from the same metabolic stress and identify the shared and unique elements of the persister formation pathways. We discovered that diauxie-dependent formation of ampicillin persisters required RelA and that loss of clpA, ssrA, or smpB eliminated persister formation through relaxation of the stringent response. Further, we found that tolerance to ampicillin was achieved through broad inhibition of peptidoglycan biosynthesis, as evidenced by the formation of persisters to antibiotics that target enzymes in different areas of that biosynthetic pathway. Interestingly, ppGpp was required for formation of both ampicillin and ofloxacin persisters, and we demonstrated that higher synthesis of the alarmone was needed to increase persisters to ampicillin compared to ofloxacin. Further, we found trans-translation and DksA to be common mediators of both pathways, whereas ClpA was unique for ampicillin persisters and nucleoid-associated proteins were unique for ofloxacin persisters. These results highlight the need to consider an antibiotic's mode of action when analyzing persister formation, demonstrate that individual stresses can produce persister heterogeneity, and emphasize the importance of identifying each respective pathway to identify common mediators that possess the most therapeutic potential to combat persisters.

Chronic and recurrent infections have been attributed to persisters in biofilms, and despite this importance, the mechanisms of persister formation in biofilms remain unclear. The plethora of biofilm characteristics that could give rise to persisters, including slower growth, quorum signaling, oxidative stress, and nutrient heterogeneity, have complicated efforts to delineate formation pathways that generate persisters during biofilm development. Here we sought to specifically determine whether nutrient transitions, which are a common metabolic stress encountered within surface-attached communities, stimulate persister formation in biofilms and if so, to then identify the pathway. To accomplish this, we established an experimental methodology where nutrient availability to biofilm cells could be controlled exogenously, and then used that method to discover that diauxic carbon source transitions stimulated persister formation in Escherichia coli biofilms. Previously, we found that carbon source transitions stimulate persister formation in planktonic E. coli cultures, through a pathway that involved ppGpp and nucleoid-associated proteins, and therefore, tested the functionality of that pathway in biofilms. Biofilm persister formation was also found to be dependent on ppGpp and nucleoid-associated proteins, but the importance of specific proteins and enzymes between biofilm and planktonic lifestyles was significantly different. Data presented here support the increasingly appreciated role of ppGpp as a central mediator of bacterial persistence and demonstrate that nutrient transitions can be a source of persisters in biofilms.

BackgroundHard water is associated with atopic dermatitis (eczema). We wanted to determine if a baby cleanser and its individual components altered free ionized calcium (Ca2+) in a simulated hard water baby bath. For these studies, an in vitro determination of free Ca2+ in a simulated hard water baby bath, and an in vivo exploratory study of free Ca2+ absorption into skin from hard water were performed.MethodsFree Ca2+ was measured with an ion-sensitive electrode in vitro in hard water (100–500 ppm, Ca2+) before and after addition of the cleanser and/or its components. In an exploratory study, absorption of Ca2+ into skin from hard water was determined in three female participants (aged 21–29 years).ResultsAt an in-use dilution of 1%, the test cleanser reduced free Ca2+ from ~500 ppm to <200 ppm; a 10% in-use dilution bound virtually all free Ca2+. The anionic surfactant component contributed the most to this effect. In the exploratory in vivo study, we measured a reduction of ~15% in free Ca2+ from simulated hard water over 10 minutes.ConclusionBaby cleansers can bind free Ca2+ and reduce the effective water hardness of bath water. Reducing the amount of free Ca2+ in the water will reduce the availability of the ion for binding to the skin. Altering or reducing free Ca2+ concentrations in bath water may be an important parameter in creating the ideal baby bath.