Article

Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2012; 109(30):12147-52. DOI: 10.1073/pnas.1203735109
Source: PubMed

ABSTRACT

During Mycobacterium tuberculosis infection, a population of bacteria likely becomes refractory to antibiotic killing in the absence of genotypic resistance, making treatment challenging. We describe an in vitro model capable of yielding a phenotypically antibiotic-tolerant subpopulation of cells, often called persisters, within populations of Mycobacterium smegmatis and M. tuberculosis. We find that persisters are distinct from the larger antibiotic-susceptible population, as a small drop in dissolved oxygen (DO) saturation (20%) allows for their survival in the face of bactericidal antibiotics. In contrast, if high levels of DO are maintained, all cells succumb, sterilizing the culture. With increasing evidence that bactericidal antibiotics induce cell death through the production of reactive oxygen species (ROS), we hypothesized that the drop in DO decreases the concentration of ROS, thereby facilitating persister survival, and maintenance of high DO yields sufficient ROS to kill persisters. Consistent with this hypothesis, the hydroxyl-radical scavenger thiourea, when added to M. smegmatis cultures maintained at high DO levels, rescues the persister population. Conversely, the antibiotic clofazimine, which increases ROS via an NADH-dependent redox cycling pathway, successfully eradicates the persister population. Recent work suggests that environmentally induced antibiotic tolerance of bulk populations may result from enhanced antioxidant capabilities. We now show that the small persister subpopulation within a larger antibiotic-susceptible population also shows differential susceptibility to antibiotic-induced hydroxyl radicals. Furthermore, we show that stimulating ROS production can eradicate persisters, thus providing a potential strategy to managing persistent infections.

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    • "Recent reports have shown that bacteria treated with lethal doses of aminoglycoside antibiotics undergo a common oxidative damage, which contributes in part to the cell death (Dwyer et al. 2007). These antibiotics promote the overproduction of hydroxyl radicals through changes in cellular respiration involving NADH consumption and increased tricarboxylic acid cycle, ultimately leading to oxidative-stress mediated cell death (Dwyer et al. 2007; Kohanski et al. 2007, 2008, 2010; Foti et al. 2012; Grant et al. 2012; Liu et al. 2012; Wang and Zhao 2009). In mammalian cells, bactericidal antibiotics induce mitochondrial dysfunction by disrupting the electron transport chain, which triggers dose-and time-dependent overproduction of reactive oxygen species (ROS), thereby leading to oxidative stress damages (Kalghatgi et al. 2013). "
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    • "However, a major difference is that while the mutation spectrum for Δfpg::hyg was obtained at 50 μg/ml of Rif, that for Δfpg::hygΔarr:: kan has been obtained by using a Rif concentration of merely 5 μg/ml. There is evidence to support that a mechanism that is common for the action of many antibiotics, operates through generation of reactive oxygen species [28, 29] . Thus, it is not surprising that the mutation spectrum for Δfpg was found to be somewhat different when lower concentration of the drug was used in the experiment (for the Δarr::kan derivative). "
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    • "Although somewhat controversial (Keren et al. 2013), one hypothesis states that all bactericidal antibiotics kill bacteria by generating reactive oxygen or nitrogen species (ROS/RNS) (Dwyer et al. 2009 ). Recently, it was shown that a relatively small change (20%) in dissolved oxygen can affect killing of bacterial persiters (Grant et al. 2012), a subpopulation of bacteria in an infection that is phenotypically resistant to killing by most antibiotics but still sensitive, however, to high quantities of radicals. This observation can be critical for killing Mtb in granulomas, which have hypoxic cores (Via et al. 2008 ). "
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