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
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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[Show abstract] [Hide abstract] 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.
- "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). "
[Show abstract] [Hide abstract] ABSTRACT: Rifampicin (Rif) is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr) in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs). Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase) causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr). The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC) for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil) strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G). Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.
- "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). "
[Show abstract] [Hide abstract] ABSTRACT: The recent accelerated approval for use in extensively drug-resistant and multidrug-resistant-tuberculosis (MDR-TB) of two first-in-class TB drugs, bedaquiline and delamanid, has reinvigorated the TB drug discovery and development field. However, although several promising clinical development programs are ongoing to evaluate new TB drugs and regimens, the number of novel series represented is few. The global early-development pipeline is also woefully thin. To have a chance of achieving the goal of better, shorter, safer TB drug regimens with utility against drug-sensitive and drug-resistant disease, a robust and diverse global TB drug discovery pipeline is key, including innovative approaches that make use of recently acquired knowledge on the biology of TB. Fortunately, drug discovery for TB has resurged in recent years, generating compounds with varying potential for progression into developable leads. In parallel, advances have been made in understanding TB pathogenesis. It is now possible to apply the lessons learned from recent TB hit generation efforts and newly validated TB drug targets to generate the next wave of TB drug leads. Use of currently underexploited sources of chemical matter and lead-optimization strategies may also improve the efficiency of future TB drug discovery. Novel TB drug regimens with shorter treatment durations must target all subpopulations of Mycobacterium tuberculosis existing in an infection, including those responsible for the protracted TB treatment duration. This review summarizes the current TB drug development pipeline and proposes strategies for generating improved hits and leads in the discovery phase that could help achieve this goal. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
- "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 ). "