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


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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    ABSTRACT: The aminoglycoside antibiotic hygromycin B (Hyg) inhibits prokaryotic, chloroplast and mitochondrial protein synthesis. Because of the toxic effect of Hyg on plant cells, the HPT gene, encoding hygromycin phosphotransferase, has become one of the most widely used selectable markers in plant transformation. Yet the mechanism behind Hyg-induced cell lethality in plants is not clearly understood. In this study, we aimed to decipher this mechanism. With Hyg treatment, rice calli exhibited cell death, and rice seedlings showed severe growth defects, leaf chlorosis and leaf shrinkage. Rice seedlings also exhibited severe lipid peroxidation and protein carbonylation, for oxidative stress damage at the cellular level. The production of reactive oxygen species such as O 2·−, H2O2 and OH· was greatly induced in rice seedlings under Hyg stress, and pre-treatment with ascorbate increased resistance to Hyg-induced toxicity indicating the existence of oxidative stress. Overexpression of mitochondrial Alternative oxidase1a gene without HPT selection marker in rice enhanced tolerance to Hyg and attenuated the degradation of protein content, whereas the rice plastidial glutathione reductase 3 mutant showed increased sensitivity to Hyg. These results demonstrate that Hyg-induced cell lethality in rice is not only due to the inhibition of protein synthesis but also mediated by oxidative stress.
    No preview · Article · Sep 2015 · Plant Molecular Biology
    • "All rights reserved. resistance profile, as well as recently described inhibitory activity against mycobacterial persisters [15] [16] [17] [18]. To address this issue, this study was undertaken with the primary objectives of determining: (i) the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of clofazimine against actively growing planktonic and slowly replicating biofilmproducing mycobacteria as well as non-replicating biofilmencased organisms; and (ii) the inhibitory effects of clofazimine both on biofilm formation and the structural resilience of preformed biofilm. "
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    ABSTRACT: Mycobacteria form lipid-rich biofilms that restrict the efficacy of antimicrobial chemotherapy, possibly necessitating the use of lipophilic antibiotics. In the current study, the activity of one such agent, clofazimine, against Mycobacterium tuberculosis and Mycobacterium smegmatis planktonic cells and biofilms was investigated. Minimum inhibitory concentrations (MICs) of clofazimine were determined for planktonic cultures, whilst minimum bactericidal concentrations (MBCs) were determined for planktonic, biofilm-producing and biofilm-encased organisms using standard bacteriological procedures. The effects of clofazimine on biofilm formation and the stability of pre-formed biofilm were measured using a crystal violet-based spectrophotometric procedure. In the case of M. smegmatis, clofazimine was found to be active against planktonic phase (MICs and MBCs of 2.5 mg/L and >20 mg/L, respectively) and biofilm-producing organisms (MBC of 2.5 mg/L); clofazimine demonstrated greater activity against M. tuberculosis, corresponding values of 0.06, 5 and 0.3 mg/L. Although clofazimine inhibited biofilm production both by M. tuberculosis and M. smegmatis (P < 0.05 at ≥0.07 mg/L and ≥0.3 mg/L, respectively) and appeared to reduce the pre-formed M. tuberculosis biofilm, addition of antimicrobial agent to pre-existing biofilm matrices failed to kill biofilm-encased organisms. In conclusion, clofazimine is more effective against M. tuberculosis than against M. smegmatis, exhibiting bactericidal activity both for actively growing and slowly replicating bacilli but not for non-replicating organisms of both species.
    No preview · Article · Dec 2014 · Journal of Global Antimicrobial Resistance
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    • "Furthermore, it remains to be seen whether the use of mannitol is effective in patients. A related approach to enhancing the effectiveness of the existing antibiotics in killing persisters is to increase ROS production.93 Recently, it has been shown that silver, which produces ROS, enhanced the activity of vancomycin, improving the treatment of bacterial infections in mice.94 "
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    ABSTRACT: Persisters are a small fraction of quiescent bacterial cells that survive lethal antibiotics or stresses but can regrow under appropriate conditions. Persisters underlie persistent and latent infections and post-treatment relapse, posing significant challenges for the treatment of many bacterial infections. The current definition of persisters has drawbacks, and a Yin–Yang model is proposed to describe the heterogeneous nature of persisters that have to be defined in highly specific conditions. Despite their discovery more than 70 years ago, the mechanisms of persisters are poorly understood. Recent studies have identified a number of genes and pathways that shed light on the mechanisms of persister formation or survival. These include toxin–antitoxin modules, stringent response, DNA repair or protection, phosphate metabolism, alternative energy production, efflux, anti-oxidative defense and macromolecule degradation. More sensitive single-cell techniques are required for a better understanding of persister mechanisms. Studies of bacterial persisters have parallels in other microbes (fungi, parasites, viruses) and cancer stem cells in terms of mechanisms and treatment approaches. New drugs and vaccines targeting persisters are critical for improved treatment of persistent infections and perhaps cancers. Novel treatment strategies for persisters and persistent infections are discussed.
    Full-text · Article · Jan 2014 · Emerging Microbes and Infections
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