Heterogeneity of ROS levels in antibiotic-exposed mycobacterial subpopulations confers differential susceptibility

Nair, Rashmi Ravindran; Sharan, Deepti; Sebastian, Jees; Swaminath, Sharmada; Ajitkumar, Parthasarathi

doi:10.1099/mic.0.000797

Cited by 17 publications

(34 citation statements)

References 70 publications

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“…Both Mtb and Msm SCs were found to be significantly more susceptible to anti-tuberculosis drugs, rifampicin (RIF), and isoniazid (INH), and to H 2 O 2 and acidified sodium nitrite (Vijay et al, 2017). Further, the RIF/INH-exposed SCs showed significantly elevated levels of ROS, superoxide and hydroxyl radical, unlike the NCs (Nair et al, 2019). These observations alluded to the possibility that the SCs might be inherently predisposed to produce higher levels of ROS than the NCs in the native antibiotics-unexposed condition.…”

Section: Introductionmentioning

confidence: 89%

“…Redox biosensor Mrx1-rogfp2 was used to determine the redox status of Msm SCF and NCF cells. The stable Msm integrant cells, which carry single copy of pAKMN2/ hsp60-Mrx1-rogfp2 integrated in the genome at the mycobacteriophage L5 att site (Nair et al, 2019), were cultured and SCF and NCF cells were isolated using Percoll density gradient. The Mrx1-rogfp2 integrated SCF and NCF samples were taken for flow cytometry analyses, with at least 10000 cells gated from each sample.…”

Section: Methodsmentioning

confidence: 99%

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A Minor Subpopulation of Mycobacteria Inherently Produces High Levels of Reactive Oxygen Species That Generate Antibiotic Resisters at High Frequency From Itself and Enhance Resister Generation From Its Major Kin Subpopulation

2019

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Antibiotic-exposed bacteria produce elevated levels of reactive oxygen species (ROS), to which either they succumb or get mutated genome-wide to generate antibiotic resisters. We recently showed that mycobacterial cultures contained two subpopulations, short-sized cells (SCs; ∼10%) and normal/long-sized cells (NCs; ∼90%). The SCs were significantly more antibiotic-susceptible than the NCs. It implied that the SCs might naturally be predisposed to generate significantly higher levels of ROS than the NCs. This in turn could make the SCs more susceptible to antibiotics or generate more resisters as compared to the NCs. Investigation into this possibility showed that the SCs in the actively growing mid-log phase culture naturally generated significantly high levels of superoxide, as compared to the equivalent NCs, due to the naturally high expression of a specific NADH oxidase in the SCs. This caused labile Fe 2+ leaching from 4Fe-4S proteins and elevated H 2 O 2 formation through superoxide dismutation. Thus, the SCs of both Mycobacterium smegmatis and Mycobacterium tuberculosis inherently contained significantly higher levels of H 2 O 2 and labile Fe 2+ than the NCs. This in turn produced significantly higher levels of hydroxyl radical through Fenton reaction, promoting enhanced antibiotic resister generation from the SCs than from the NCs. The SCs, when mixed back with the NCs, at their natural proportion in the actively growing mid-log phase culture, enhanced antibiotic resister generation from the NCs, to a level equivalent to that from the unfractionated whole culture. The enhanced antibiotic resister generation from the NCs in the reconstituted SCs-NCs natural mixture was found to be due to the high levels of H 2 O 2 secreted by the SCs. Thus, the present work unveils and documents the metabolic designs of two mycobacterial subpopulations where one subpopulation produces high ROS levels, despite higher susceptibility, to generate significantly higher number of antibiotic resisters from itself and to enhance resister generation from its kin subpopulation. These findings show the existence of an inherent natural mechanism in both the non-pathogenic and pathogenic mycobacteria to generate antibiotic resisters. The presence of the SCs and the NCs in the pulmonary tuberculosis patients’ sputum, reported by us earlier, alludes to the clinical significance of the study.

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Section: Introductionmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

A Minor Subpopulation of Mycobacteria Inherently Produces High Levels of Reactive Oxygen Species That Generate Antibiotic Resisters at High Frequency From Itself and Enhance Resister Generation From Its Major Kin Subpopulation

2019

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“…Spatial and temporal differences in drug distribution and the kinetics of accumulation of drugs in specific lesion compartments may create local windows of monotherapy that increase the risk of the emergence of drug-resistance [17,37]. This is in keeping with the knowledge that genetically resistant mutants of Mtb may emerge from the persistence phase of some TB drugs, due to hydroxyl radical-mediated genome-wide random mutagenesis [38][39][40]. In this view, drug combinations should contain complementary drugs preferentially distributing in lesions in which their most vulnerable target population resides [17].…”

Section: Complexity Of Tb Granulomasmentioning

confidence: 72%

“…Interestingly, RIF-resistant or MXF resistant mutants carrying mutations in rpoB or gyrA genes emerged at high frequency from the persistent phase of Mtb cells exposed to RIF for prolonged periods. These cells carried elevated levels of the hydroxyl radical, which inflicted genome-wide mutations facilitating resistance to the same, or another, antibiotic [38,39]. In consideration of the long TB therapy, these observations may have clinical significance in the emergence of drug-resistant mutants if local monotherapy occurs in patients who do not correctly take multi-drug TB therapy.…”

Section: Phenotypic Drug-resistance Of Mtbmentioning

confidence: 99%

Drug-Resistant Tuberculosis 2020: Where We Stand

2020

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The control of tuberculosis (TB) is hampered by the emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains, defined as resistant to at least isoniazid and rifampin, the two bactericidal drugs essential for the treatment of the disease. Due to the worldwide estimate of almost half a million incident cases of MDR/rifampin-resistant TB, it is important to continuously update the knowledge on the mechanisms involved in the development of this phenomenon. Clinical, biological and microbiological reasons account for the generation of resistance, including: (i) nonadherence of patients to their therapy, and/or errors of physicians in therapy management, (ii) complexity and poor vascularization of granulomatous lesions, which obstruct drug distribution to some sites, resulting in resistance development, (iii) intrinsic drug resistance of tubercle bacilli, (iv) formation of non-replicating, drug-tolerant bacilli inside the granulomas, (v) development of mutations in Mtb genes, which are the most important molecular mechanisms of resistance. This review provides a comprehensive overview of these issues, and releases up-dated information on the therapeutic strategies recently endorsed and recommended by the World Health Organization to facilitate the clinical and microbiological management of drug-resistant TB at the global level, with attention also to the most recent diagnostic methods.

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“…However, ROS-generating antimicrobials may increase the efficiency of the free radical biosynthesis produced by macrophages during the oxidative burst, which may facilitate the phagocytosis of the pathogen [ 46 ]. Several antituberculosis drugs have been tested as promising ROS-generating antimicrobials against M. tuberculosis , and the majority of them have produced an oxidative shift during infection, especially clofazimine [ 46 ], but also rifampicin and isoniazid [ 47 ]. The combination of several ROS-generating compounds could be a new solution against intracellular M. tuberculosis as it was demonstrated for other intracellular pathogens [ 44 , 45 , 48 ].…”

Section: Repurposing Anti-infectives Against M Tuberculmentioning

confidence: 99%

Novel Treatments against Mycobacterium tuberculosis Based on Drug Repurposing

et al. 2020

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Tuberculosis is the leading cause of death, worldwide, due to a bacterial pathogen. This respiratory disease is caused by the intracellular pathogen Mycobacterium tuberculosis and produces 1.5 million deaths every year. The incidence of tuberculosis has decreased during the last decade, but the emergence of MultiDrug-Resistant (MDR-TB) and Extensively Drug-Resistant (XDR-TB) strains of M. tuberculosis is generating a new health alarm. Therefore, the development of novel therapies based on repurposed drugs against MDR-TB and XDR-TB have recently gathered significant interest. Recent evidence, focused on the role of host molecular factors on M. tuberculosis intracellular survival, allowed the identification of new host-directed therapies. Interestingly, the mechanism of action of many of these therapies is linked to the activation of autophagy (e.g., nitazoxanide or imatinib) and other well-known molecular pathways such as apoptosis (e.g., cisplatin and calycopterin). Here, we review the latest developments on the identification of novel antimicrobials against tuberculosis (including avermectins, eltrombopag, or fluvastatin), new host-targeting therapies (e.g., corticoids, fosfamatinib or carfilzomib) and the host molecular factors required for a mycobacterial infection that could be promising targets for future drug development.

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Heterogeneity of ROS levels in antibiotic-exposed mycobacterial subpopulations confers differential susceptibility

Cited by 17 publications

References 70 publications

A Minor Subpopulation of Mycobacteria Inherently Produces High Levels of Reactive Oxygen Species That Generate Antibiotic Resisters at High Frequency From Itself and Enhance Resister Generation From Its Major Kin Subpopulation

A Minor Subpopulation of Mycobacteria Inherently Produces High Levels of Reactive Oxygen Species That Generate Antibiotic Resisters at High Frequency From Itself and Enhance Resister Generation From Its Major Kin Subpopulation

Drug-Resistant Tuberculosis 2020: Where We Stand

Novel Treatments against Mycobacterium tuberculosis Based on Drug Repurposing

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