scholarly journals Simple and Rapid Method To Determine Antimycobacterial Potency of Compounds by Using Autoluminescent Mycobacterium tuberculosis

2014 ◽  
Vol 58 (10) ◽  
pp. 5801-5808 ◽  
Author(s):  
Sreevalli Sharma ◽  
Ekaterina Gelman ◽  
Chandan Narayan ◽  
Deepa Bhattacharjee ◽  
Vijayashree Achar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Slow Growth ◽  
Gc Content ◽  
Content Type ◽  
Slow Growth Rate ◽  
Drug Candidates ◽  
Luminescence Assay ◽  
Bactericidal Activities ◽  
Time Required

ABSTRACTA major obstacle in the process of discovery of drugs againstMycobacterium tuberculosisis its extremely slow growth rate and long generation time (∼20 to 24 h). Consequently, determination of MICs and minimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-based MIC assay [REMA]) and 1 month (CFU enumeration), respectively. We employed a synthetic luciferase operon optimized for expression in high-GC-content bacteria and adapted it for use in mycobacteria. Using luminescence-based readouts, we were able to determine the MICs and bactericidal activities of approved tuberculosis (TB) drugs, which correlated well with currently used methods. Although luminescence-based readouts have been used previously to determine the MICs and bactericidal activities of approved TB drugs, in this study we adapted this assay to carry out a pilot screen using a library of 1,114 compounds belonging to diverse chemical scaffolds. We found that MICs derived from a 3-day luminescence assay matched well with REMA-based MIC values. To determine the bactericidal potencies of compounds, a 1:10 dilution of the cultures from the MIC plate was carried out on day 7, and the bactericidal concentrations determined based on time to positivity in 2 weeks were found to be comparable with MBC values determined by the conventional CFU approach. Thus, the luminescent mycobacterium-based approach not only is very simple and inexpensive but also allowed us to generate the information in half the time required by conventional methods.

scholarly journals Regrowth of Mycobacterium tuberculosis Populations Exposed to Antibiotic Combinations Is Due to the Presence of Isoniazid and Not Bacterial Growth Rate

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Charlotte L. Hendon-Dunn ◽  
Henry Pertinez ◽  
Alice A. N. Marriott ◽  
Kim A. Hatch ◽  
Jon C. Allnutt ◽  
...  
Keyword(s):  
Growth Rate ◽  
Mycobacterium Tuberculosis ◽  
Time Course ◽  
Bacterial Population ◽  
Slow Growth ◽  
Bacterial Growth Rate ◽  
Content Type ◽  
Slow Growth Rate ◽  
Antibiotic Efficacy ◽  
Fast Growth Rate

ABSTRACT Modulation of the growth rate in Mycobacterium tuberculosis is key to its survival in the host, particularly with regard to its adaptation during chronic infection, when the growth rate is very slow. The resulting physiological changes influence the way in which this pathogen interacts with the host and responds to antibiotics. Therefore, it is important that we understand how the growth rate impacts antibiotic efficacy, particularly with respect to recovery/relapse. This is the first study that has asked how growth rates influence the mycobacterial responses to combinations of the frontline antimycobacterials, isoniazid (INH), rifampin (RIF), and pyrazinamide (PZA), using continuous cultures. The time course profiles of log-transformed total viable counts for cultures, controlled at either a fast growth rate (mean generation time [MGT], 23.1 h) or a slow growth rate (MGT, 69.3 h), were analyzed by the fitting of a mathematical model by nonlinear regression that accounted for the dilution rate in the chemostat and profiled the kill rates and recovery in culture. Using this approach, we show that populations growing more slowly were generally less susceptible to all treatments. We observed a faster kill rate associated with INH than with RIF or PZA and the appearance of regrowth. In line with this observation, regrowth was not observed with RIF exposure, which provided a slower bactericidal response. The sequential additions of RIF and PZA did not eliminate regrowth. We consider here that faster, early bactericidal activity is not what is required for the successful sterilization of M. tuberculosis, but instead, slower elimination of the bacilli followed by reduced recovery of the bacterial population is required.

scholarly journals In VitroCombination Studies of Benzothiazinone Lead Compound BTZ043 against Mycobacterium tuberculosis

2012 ◽  
Vol 56 (11) ◽  
pp. 5790-5793 ◽  
Author(s):  
Benoit Lechartier ◽  
Ruben C. Hartkoorn ◽  
Stewart T. Cole
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inhibitory Concentration ◽  
Bactericidal Effect ◽  
Lead Compound ◽  
Content Type ◽  
Drug Candidates ◽  
New Class ◽  
Novel Targets ◽  
Essential Enzyme ◽  
Strain H37rv

ABSTRACTBenzothiazinones (BTZ) are a new class of drug candidates to combat tuberculosis that inhibit decaprenyl-phosphoribose epimerase (DprE1), an essential enzyme involved in arabinan biosynthesis. Using the checkerboard method and cell viability assays, we have studied the interaction profiles of BTZ043, the current lead compound, with several antituberculosis drugs or drug candidates againstMycobacterium tuberculosisstrain H37Rv, namely, rifampin, isoniazid, ethambutol, TMC207, PA-824, moxifloxacin, meropenem with or without clavulanate, and SQ-109. No antagonism was found between BTZ043 and the tested compounds, and most of the interactions were purely additive. Data from two different approaches clearly indicate that BTZ043 acts synergistically with TMC207, with a fractional inhibitory concentration index of 0.5. TMC207 at a quarter of the MIC (20 ng/ml) used in combination with BTZ043 (1/4 MIC, 0.375 ng/ml) had a stronger bactericidal effect onM. tuberculosisthan TMC207 alone at a concentration of 80 ng/ml. This synergy was not observed when the combination was tested on a BTZ-resistantM. tuberculosismutant, suggesting that DprE1 inhibition is the basis for the interaction. This finding excludes the possibility of synergy occurring through an off-target mechanism. We therefore hypothesize that sub-MICs of BTZ043 weaken the bacterial cell wall and allow improved penetration of TMC207 to its target. Synergy between two new antimycobacterial compounds, such as TMC207 and BTZ043, with novel targets, offers an attractive foundation for a new tuberculosis regimen.

scholarly journals Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315

PLoS ONE ◽  
2015 ◽  
Vol 10 (9) ◽  
pp. e0138253 ◽  
Author(s):  
Rose E. Jeeves ◽  
Alice A. N. Marriott ◽  
Steven T. Pullan ◽  
Kim A. Hatch ◽  
Jon C. Allnutt ◽  
...  
Keyword(s):  
Growth Rate ◽  
Mycobacterium Tuberculosis ◽  
Single Nucleotide Polymorphisms ◽  
Slow Growth ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Slow Growth Rate

scholarly journals Quantitative Analysis of mRNA as a Marker for Viability of Mycobacterium tuberculosis

1999 ◽  
Vol 37 (2) ◽  
pp. 290-295 ◽  
Author(s):  
T. J. Hellyer ◽  
L. E. DesJardin ◽  
G. L. Hehman ◽  
M. D. Cave ◽  
K. D. Eisenach
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Quantitative Analysis ◽  
16S Rrna ◽  
Therapeutic Efficacy ◽  
Rapid Determination ◽  
Drug Susceptibility ◽  
Resistant Strains ◽  
Time Required ◽  
Rrna Sequences

Numerous assays which use conserved DNA or rRNA sequences as targets for amplification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of therapeutic efficacy owing to the persistence of amplifiable nucleic acid beyond the point at which smears and cultures become negative. Semiquantitative analysis of rRNA has been used to reduce the time required for antimicrobial susceptibility testing of Mycobacterium tuberculosis, although growth for up to 5 days in the presence of some drugs is still required to discriminate resistant strains. The purpose of the present study was to determine whether quantitative analysis of M. tuberculosis mRNA could be used to assess bacterial viability and to illustrate the application of this technique to rapid determination of drug susceptibility. Levels of mRNA encoding the 85B protein (α-antigen), IS6110 DNA, and 16S rRNA were compared in parallel cultures of M. tuberculosis that were treated with either no drug, 0.2 μg of isoniazid per ml, or 1 μg of rifampin per ml. Exposure of sensitive strains to isoniazid or rifampin for 24 h reduced the levels of 85B mRNA to <4 and <0.01%, respectively, of those present in control cultures without drug. In contrast, the levels of IS6110 DNA and 16S rRNA did not diminish over the same period. Strains which were resistant to either isoniazid or rifampin demonstrated no reduction in 85B mRNA in the presence of the drug to which they were nonresponsive. Quantitative analysis of 85B mRNA offers a potentially useful tool for the rapid determination of M. tuberculosis drug susceptibility and for the monitoring of therapeutic efficacy.

Prognostic implications of the proliferative potential of low-grade astrocytomas

1988 ◽  
Vol 69 (6) ◽  
pp. 839-842 ◽  
Author(s):  
Takao Hoshino ◽  
Luis A. Rodriguez ◽  
Kyung G. Cho ◽  
Kyu S. Lee ◽  
Charles B. Wilson ◽  
...  
Keyword(s):  
Deoxyribonucleic Acid ◽  
Slow Growth ◽  
S Phase ◽  
Proliferative Potential ◽  
Low Grade ◽  
Content Type ◽  
Slow Growth Rate ◽  
Prognostic Implications ◽  
Scientific Rationale

✓ The proliferative potential of low-grade astrocytomas was estimated in 47 patients. Each patient received an intravenous infusion of bromodeoxyuridine (BUdR), 150 to 200 mg/sq m, at the time of craniotomy to label cells in deoxyribonucleic acid (DNA) synthesis; the percentage of S-phase cells, or BUdR labeling index (LI), of each tumor was determined immunohistochemically. In 29 patients (60%), the tumors had BUdR LI's of less than 1%, indicating a slow growth rate; only three (10%) of these patients died of recurrent tumor during a follow-up period of up to 3½ years. In contrast, of the 18 patients (40%) whose tumors had BUdR LI's of 1% or more, 12 (67%) had a recurrence and nine died during the same follow-up period. These results show that the proliferative potential, as reflected by the BUdR LI, is an important prognostic factor that separates low-grade astrocytomas into two groups and provides a more scientific rationale for selecting treatment for individual patients.

scholarly journals In VitroandIn VivoEfficacy of β-Lactams against Replicating and Slowly Growing/Nonreplicating Mycobacterium tuberculosis

2013 ◽  
Vol 57 (6) ◽  
pp. 2506-2510 ◽  
Author(s):  
Suresh Solapure ◽  
Neela Dinesh ◽  
Radha Shandil ◽  
Vasanthi Ramachandran ◽  
Sreevalli Sharma ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Human Macrophage ◽  
Beta Lactams ◽  
Dosing Regimen ◽  
Content Type ◽  
Infection Models ◽  
Chronic Model ◽  
Amoxicillin Clavulanate ◽  
Bactericidal Activities ◽  
Aerosol Infection

ABSTRACTBeta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity againstMycobacterium tuberculosisbacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicatingM. tuberculosisin broth and nonreplicatingM. tuberculosisunder hypoxia, as well as against streptomycin-starvedM. tuberculosisstrain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicatingM. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killedM. tuberculosisunder hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model.

scholarly journals Rifampin Induces Hydroxyl Radical Formation in Mycobacterium tuberculosis

2014 ◽  
Vol 58 (12) ◽  
pp. 7527-7533 ◽  
Author(s):  
Giovanni Piccaro ◽  
Donatella Pietraforte ◽  
Federico Giannoni ◽  
Alessandro Mustazzolu ◽  
Lanfranco Fattorini
Keyword(s):  
Superoxide Dismutase ◽  
Mycobacterium Tuberculosis ◽  
Paramagnetic Resonance ◽  
Content Type ◽  
Target Interaction ◽  
Metal Chelator ◽  
Diethylene Triamine Pentaacetic Acid ◽  
Metal Catalyzed ◽  
Electron Paramagnetic

ABSTRACTThe antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) ofMycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treatedM. tuberculosiswere tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps.M. tuberculosiskilling by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·−and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·−, with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·−-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part inM. tuberculosiskilling by the drug.

scholarly journals Efflux Pumps of Mycobacterium tuberculosis Play a Significant Role in Antituberculosis Activity of Potential Drug Candidates

2012 ◽  
Vol 56 (5) ◽  
pp. 2643-2651 ◽  
Author(s):  
Meenakshi Balganesh ◽  
Neela Dinesh ◽  
Sreevalli Sharma ◽  
Sanjana Kuruppath ◽  
Anju V. Nair ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Vital Role ◽  
Major Facilitator Superfamily ◽  
Content Type ◽  
Active Efflux ◽  
Drug Candidates ◽  
Small Multidrug Resistance ◽  
Major Facilitator

ABSTRACTActive efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms inMycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil andl-phenylalanyl-l-arginyl-β-naphthylamide (PAβN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded byRv1218c, and the SMR (small multidrug resistance) class, encoded byRv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded byRv0849andRv1258calso mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WTM. tuberculosiscells by these compounds in the presence of either verapamil or PAβN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps ofM. tuberculosisplay a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization ofRv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.

scholarly journals Hydrolysis of Clavulanate by Mycobacterium tuberculosis β-Lactamase BlaC Harboring a Canonical SDN Motif

2015 ◽  
Vol 59 (9) ◽  
pp. 5714-5720 ◽  
Author(s):  
Daria Soroka ◽  
Inès Li de la Sierra-Gallay ◽  
Vincent Dubée ◽  
Sébastien Triboulet ◽  
Herman van Tilbeurgh ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fold Increase ◽  
Mycobacterium Abscessus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Emergence Of Resistance ◽  
Hydrolysis Of ◽  
Sequential Formation

ABSTRACTCombinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. SinceMycobacterium tuberculosisproduces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase BlaMAbofMycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAband BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G132N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 104-fold increase inkcat(0.41 s−1), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G132N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G132N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.

scholarly journals Clinical Significance of Interleukin-2/Gamma Interferon Ratios in Mycobacterium tuberculosis-Specific T-Cell Signatures

2011 ◽  
Vol 18 (8) ◽  
pp. 1395-1396 ◽  
Author(s):  
Franziska Suter-Riniker ◽  
Antonia Berger ◽  
Désirée Mayor ◽  
Pascal Bittel ◽  
Patricia Iseli ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Simultaneous Determination ◽  
Clinical Significance ◽  
Interleukin 2 ◽  
Gamma Interferon ◽  
Content Type ◽  
Ifn Γ

ABSTRACTThe simultaneous determination of interleukin-2 (IL-2) and gamma interferon (IFN-γ) in QuantiFERON-TB test plasma supernatants permitted the detection of shifts inMycobacterium tuberculosis-specific T-cell signatures. A subset of the 84 subjects tested revealed a significantly elevated IL-2/IFN-γ ratio, which may be a marker for the successful elimination ofM. tuberculosisinfection.

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