Efficacy and Improved Resistance Potential of a Cofactor-Independent InhA Inhibitor of Mycobacterium tuberculosis in the C3HeB/FeJ Mouse Model

ABSTRACT AN12855 is a direct, cofactor-independent inhibitor of InhA in Mycobacterium tuberculosis. In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.

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Evaluation of a Mouse Model of Necrotic Granuloma Formation Using C3HeB/FeJ Mice for Testing of Drugs against Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00217-12 ◽

2012 ◽

Vol 56 (6) ◽

pp. 3181-3195 ◽

Cited By ~ 117

Author(s):

Emily R. Driver ◽

Gavin J. Ryan ◽

Donald R. Hoff ◽

Scott M. Irwin ◽

Randall J. Basaraba ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Therapy ◽

Mouse Model ◽

Bacterial Load ◽

Lung Lesions ◽

Content Type ◽

Liquefactive Necrosis ◽

Progression Of Disease ◽

Staining Techniques ◽

Late Stages

ABSTRACTPersistence ofMycobacterium tuberculosisremains a significant challenge for the effective treatment of tuberculosis in humans. In animals that develop necrotic lung lesions following infection withM. tuberculosis, drug-tolerant bacilli are present and persist in an extracellular microenvironment within the necrotic cores. In this study, we examined the efficacy of drug treatment in C3HeB/FeJ (Kramnik) mice that develop lesions with liquefactive necrosis, in comparison to BALB/c mice that develop nonnecrotic lesions following aerosol challenge. To accomplish this, Kramnik and BALB/c mice were infected by aerosol withM. tuberculosisand treated for 7 to 8 weeks with monotherapy using drugs with different modes of action. The efficacy of drug therapy was quantified by enumeration of bacterial load. The progression of disease and location and distribution of bacilli within lesions were visualized using various staining techniques. In the late stages of infection, Kramnik mice developed fibrous encapsulated lung lesions with central liquefactive necrosis containing abundant extracellular bacilli, whereas BALB/c mice formed nonnecrotic lesions with primarily intracellular bacilli. Necrotic lesions in Kramnik mice showed evidence of hypoxia by pimonidazole staining. Kramnik mice were significantly more refractory to drug therapy, especially for pyrazinamide. Metronidazole showed no bactericidal activity in either model. There were significantly higher numbers of drug-resistant colonies isolated from the Kramnik mice compared to BALB/c mice. These results suggest that the Kramnik mouse model will be a valuable model to test antituberculosis drugs, especially against bacilli that persist within necrotic lesions.

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Mycobacterium tuberculosis Lsr2 Is a Global Transcriptional Regulator Required for Adaptation to Changing Oxygen Levels and Virulence

mBio ◽

10.1128/mbio.01106-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 39

Author(s):

I. L. Bartek ◽

L. K. Woolhiser ◽

A. D. Baughn ◽

R. J. Basaraba ◽

W. R. Jacobs ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mouse Model ◽

Oxygen Tension ◽

Reactive Oxygen ◽

Transcriptional Regulator ◽

Oxygen Availability ◽

World Population ◽

Lung Pathology ◽

Content Type ◽

Oxygen Levels

ABSTRACTTo survive a dynamic host environment,Mycobacterium tuberculosismust endure a series of challenges, from reactive oxygen and nitrogen stress to drastic shifts in oxygen availability. The mycobacterial Lsr2 protein has been implicated in reactive oxygen defense via direct protection of DNA. To examine the role of Lsr2 in pathogenesis and physiology ofM. tuberculosis, we generated a strain deleted forlsr2. Analysis of theM. tuberculosisΔlsr2strain demonstrated that Lsr2 is not required for DNA protection, as this strain was equally susceptible as the wild type to DNA-damaging agents. Thelsr2mutant did display severe growth defects under normoxic and hyperoxic conditions, but it was not required for growth under low-oxygen conditions. However, it was also required for adaptation to anaerobiosis. The defect in anaerobic adaptation led to a marked decrease in viability during anaerobiosis, as well as a lag in recovery from it. Gene expression profiling of the Δlsr2mutant under aerobic and anaerobic conditions in conjunction with published DNA binding-site data indicates that Lsr2 is a global transcriptional regulator controlling adaptation to changing oxygen levels. The Δlsr2strain was capable of establishing an early infection in the BALB/c mouse model; however, it was severely defective in persisting in the lungs and caused no discernible lung pathology. These findings demonstrateM. tuberculosisLsr2 is a global transcriptional regulator required for control of genes involved in adaptation to extremes in oxygen availability and is required for persistent infection.IMPORTANCEM. tuberculosiscauses nearly two million deaths per year and infects nearly one-third of the world population. The success of this aerobic pathogen is due in part to its ability to successfully adapt to constantly changing oxygen availability throughout the infectious cycle, from the high oxygen tension during aerosol transmission to anaerobiosis within necrotic lesions. An understanding of howM. tuberculosiscopes with these changes in oxygen tension is critical for its eventual eradication. Using a mutation inlsr2, we demonstrate that the Lsr2 protein present in all mycobacteria is a global transcriptional regulator in control of genes required for adaptation to changes in oxygen levels.M. tuberculosislackinglsr2was unable to adapt to both high and very low levels of oxygen and was defective in long-term anaerobic survival. Lsr2 was also required for disease pathology and for chronic infection in a mouse model of TB.

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Investigation of Elimination Rate, Persistent Subpopulation Removal, and Relapse Rates of Mycobacterium tuberculosis by Using Combinations of First-Line Drugs in a Modified Cornell Mouse Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02548-15 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4778-4785 ◽

Cited By ~ 10

Author(s):

Yanmin Hu ◽

Henry Pertinez ◽

Fatima Ortega-Muro ◽

Laura Alameda-Martin ◽

Yingjun Liu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mouse Model ◽

Culture Filtrate ◽

Control Method ◽

Elimination Rate ◽

Therapeutic Effects ◽

Content Type ◽

Solid Agar ◽

Main Components ◽

Relapse Rates

ABSTRACTCurrently, the most effective tuberculosis control method involves case finding and 6 months of chemotherapy. There is a need to improve our understanding about drug interactions, combination activities, and the ability to remove persistent bacteria using the current regimens, particularly in relation to relapse. We aimed to investigate the therapeutic effects of three main components, rifampin (RMP), isoniazid (INH), and pyrazinamide (PZA), in current drug regimens using a modified version of the Cornell mouse model. We evaluated the posttreatment levels of persistentMycobacterium tuberculosisin the organs of mice using culture filtrate derived fromM. tuberculosisstrain H37Rv. When RMP was combined with INH, PZA, or INH-PZA, significant additive activities were observed compared to each of the single-drug treatments. However, the combination of INH and PZA showed a less significant additive effect than either of the drugs used on their own. Apparent culture negativity of mouse organs was achieved at 14 weeks of treatment with RMP-INH, RMP-PZA, and RMP-INH-PZA, but not with INH-PZA, when conventional tests, namely, culture on solid agar and in liquid broth, indicated that the organs were negative for bacteria. The relapse rates for RMP-containing regimens were not significantly different from a 100% relapse rate at the numbers of mice examined in this study. In parallel, we examined the organs for the presence of culture filtrate-dependent persistent bacilli after 14 weeks of treatment. Culture filtrate treatment of the organs revealed persistentM. tuberculosis. Modeling of mycobacterial elimination rates and evaluation of culture filtrate-dependent organisms showed promise as surrogate methods for efficient factorial evaluation of drug combinations in tuberculosis in mouse models and should be further evaluated against relapse. The presence of culture filtrate-dependent persistentM. tuberculosisis the likely cause of disease relapse in this modified Cornell mouse model.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01341-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 6873-6881 ◽

Cited By ~ 13

Author(s):

Kathryn Winglee ◽

Shichun Lun ◽

Marco Pieroni ◽

Alan Kozikowski ◽

William Bishai

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Efflux Pump ◽

Transcriptional Regulator ◽

Cross Resistance ◽

Loss Of Function ◽

Strong Activity ◽

Content Type ◽

Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

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A Novel 6-Benzyl Ether Benzoxaborole Is Active against Mycobacterium tuberculosisIn Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01205-17 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 1

Author(s):

Nipul Patel ◽

Theresa O'Malley ◽

Yong-Kang Zhang ◽

Yi Xia ◽

Bjorn Sunde ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Liquid Medium ◽

Inhibitory Concentration ◽

Solid Medium ◽

Eukaryotic Cells ◽

Intracellular Bacteria ◽

Benzyl Ether ◽

Content Type ◽

Resistant Mutants

ABSTRACT We identified a novel 6-benzyl ether benzoxaborole with potent activity against Mycobacterium tuberculosis. The compound had an MIC of 2 μM in liquid medium. The compound was also able to prevent growth on solid medium at 0.8 μM and was active against intracellular bacteria (50% inhibitory concentration [IC50] = 3.6 μM) without cytotoxicity against eukaryotic cells (IC50 > 100 μM). We isolated resistant mutants (MIC ≥ 100 μM), which had mutations in Rv1683, Rv3068c, and Rv0047c.

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Characterization of T Cells Specific for CFP-10 and ESAT-6 in Mycobacterium tuberculosis-Infected Mauritian Cynomolgus Macaques

Infection and Immunity ◽

10.1128/iai.01009-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 5

Author(s):

Amy Ellis ◽

Alexis Balgeman ◽

Mark Rodgers ◽

Cassaundra Updike ◽

Jaime Tomko ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

Mhc Class Ii ◽

Nonhuman Primates ◽

Content Type ◽

Host Immune Responses ◽

Cell Responses ◽

Cynomolgus Macaques ◽

Mhc Haplotype

ABSTRACT Nonhuman primates can be used to study host immune responses to Mycobacterium tuberculosis. Mauritian cynomolgus macaques (MCMs) are a unique group of animals that have limited major histocompatibility complex (MHC) genetic diversity, such that MHC-identical animals can be infected with M. tuberculosis. Two MCMs homozygous for the relatively common M1 MHC haplotype were bronchoscopically infected with 41 CFU of the M. tuberculosis Erdman strain. Four other MCMs, which had at least one copy of the M1 MHC haplotype, were infected with a lower dose of 3 CFU M. tuberculosis. All animals mounted similar T-cell responses to CFP-10 and ESAT-6. Two epitopes in CFP-10 were characterized, and the MHC class II alleles restricting them were determined. A third epitope in CFP-10 was identified but exhibited promiscuous restriction. The CFP-10 and ESAT-6 antigenic regions targeted by T cells in MCMs were comparable to those seen in cases of human M. tuberculosis infection. Our data lay the foundation for generating tetrameric molecules to study epitope-specific CD4 T cells in M. tuberculosis-infected MCMs, which may guide future testing of tuberculosis vaccines in nonhuman primates.

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Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00165-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 18

Author(s):

Zhaojing Zong ◽

Wei Jing ◽

Jin Shi ◽

Shu'an Wen ◽

Tingting Zhang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Novel Mutation ◽

Multidrug Resistant ◽

23S Rrna ◽

Drug Resistant ◽

Content Type ◽

Extensively Drug Resistant ◽

Significant Difference ◽

Mdr Tb

ABSTRACT Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis. In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

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Monkey Models of Tuberculosis: Lessons Learned

Infection and Immunity ◽

10.1128/iai.02850-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 852-862 ◽

Cited By ~ 45

Author(s):

Juliet C. Peña ◽

Wen-Zhe Ho

Keyword(s):

Mycobacterium Tuberculosis ◽

Animal Models ◽

Nonhuman Primates ◽

Lessons Learned ◽

Preclinical Testing ◽

Content Type ◽

Cynomolgus Macaques ◽

Drug Regimens

The use of animal models has been invaluable for studying the pathogenesis ofMycobacterium tuberculosisinfection, as well as for testing the efficacy of vaccines and drug regimens for tuberculosis. Among the applied animal models, nonhuman primates, particularly macaques, share the greatest anatomical and physiological similarities with humans. As such, macaque models have been used for investigating tuberculosis pathogenesis and preclinical testing of drugs and vaccines. This review focuses on published major studies which illustrate how the rhesus and cynomolgus macaques have enriched and may continue to advance the field of global tuberculosis research.

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Discordance between Xpert MTB/RIF Assay and Bactec MGIT 960 Culture System for Detection of Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Country with a Low Tuberculosis (TB) Incidence

Journal of Clinical Microbiology ◽

10.1128/jcm.03412-14 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1351-1354 ◽

Cited By ~ 17

Author(s):

Eiman Mokaddas ◽

Suhail Ahmad ◽

Hanaa S. Eldeen ◽

Noura Al-Mutairi

Keyword(s):

Mycobacterium Tuberculosis ◽

Culture System ◽

Content Type ◽

Rifampin Resistance ◽

Xpert Assay

Among 452 samples that were positive by the Xpert MTB/RIF (Xpert) assay and MGIT 960 system (MGIT), 440 and 10Mycobacterium tuberculosissamples were detected as rifampin susceptible and rifampin resistant, respectively. Two isolates that were rifampin susceptible by the MGIT system were rifampin resistant by the Xpert assay.rpoBsequencing identified a silent (CTG521TTG) mutation in one isolate and a missense (GAC516TAC) mutation in another. The detection of rifampin resistance is imperfect with both the Xpert assay and MGIT system. Any discordant rifampin resistance results should be confirmed by sequencing of therpoBgene.

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