Resazurin Microtiter Assay Plate method for detection of susceptibility of multidrug resistant Mycobacterium tuberculosis to second-line anti-tuberculous drugs

Mycobacterium tuberculosis infection causes high rates of morbidity and mortality. Host-directed therapy may enhance the immune response, reduce tissue damage and shorten treatment duration. The inflammasome is integral to innate immune responses but over-activation has been described in tuberculosis (TB) pathology and TB-immune reconstitution syndrome. Here we explore how clinical isolates differentially activate the inflammasome and how inflammasome inhibition can lead to enhanced bacterial clearance. Wild-type, Nlrp3 −/− /Aim2 −/− , Casp1/11 −/− and Asc −/− murine bonemarrow derived macrophages (BMDMs) were infected with laboratory strain M. tuberculosis H37Rv or clinical isolates from various lineages. Inflammasome activation and bacterial numbers were measured, and pharmacological inhibition of NLRP3 was achieved using MCC950. Clinical isolates of M. tuberculosis differed in their ability to activate inflammasomes. Beijing isolates had contrasting effects on IL-1β and caspase-1 activation, but all clinical isolates induced lower IL-1β release than H37Rv. Our studies suggest the involvement of NLRP3, AIM2 and an additional unknown sensor in IL-1β maturation. Pharmacological blockade of NLRP3 with MCC950 reduced bacterial survival, and combined treatment with the antimycobacterial drug rifampicin enhanced the effect. Modulating the inflammasome is an attractive adjunct to current anti-mycobacterial therapy that warrants further investigation. Although significant strides have been made in reducing the disease burden attributed to TB, it remains a considerable cause of mortality. Current figures estimate that 1.6 million people died from TB in 2017 1. Progress has been impeded by the rise in drug resistance and the HIV-epidemic. However, it is the ability of the bacterium to modulate its metabolism within the host environment 2 , enter non-replicating persistence 3 and form biofilms 4 in order to facilitate its own survival that leads to treatment difficulties 2. Treatment of M. tuberculosis necessitates a prolonged multi-drug regimen 5. Anti-microbials target actively replicating bacteria, but the intracellular population is composed of a mixed phenotype, requiring extended therapy to eradicate those bacterial populations that transiently and stochastically leave the slowly replicating state to enter an actively replicating state 5. However, the extended treatment is associated with non-compliance and selection of resistant mutations. To identify alternative anti-mycobacterial therapies efforts have been directed at altering the host immune response through host-directed therapy (HDT), which is to be used as an adjunct to standard quadruple therapy. Deregulated host immune responses may be counter-productive to bacterial killing and lead to tissue destruction, such that half of TB-survivors have some degree of persisting lung damage following successful microbiological cure 6. Thus, the host response may be manipulated in two ways; firstly by augmenting bacterial killing and secondly by rebalancing the inflamm...

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“…The change from blue resazurin to pink implied bacterial growth. The MIC was the lowest concentration that did not yield a change in colour 61 .…”

Section: Methodsmentioning

confidence: 99%

Genetic and pharmacological inhibition of inflammasomes reduces the survival of Mycobacterium tuberculosis strains in macrophages

Subbarao

Sanchez‐Garrido

Krishnan

et al. 2020

Sci Rep

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“…The REMA plate assay was carried out in an identical fashion as depicted earlier [56][57][58] with slight variations. The REMA plate assay was carried out in an identical fashion as depicted earlier [56][57][58] with slight variations.…”

Section: Resazurin Microtiter Assay (Rema) Plate Methods For Determinamentioning

confidence: 99%

Synthesis and molecular docking studies of a new series of bipyrazol-yl-thiazol-ylidene-hydrazinecarbothioamide derivatives as potential antitubercular agents

et al. 2016

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Various substituted 2-(2-(5-(3/4-substituted phenyl)-4-hydroxy-3'-(3/4-substituted phenyl)-1'phenyl-1H,1'H-[3,4'-bipyrazol]-1-yl)thiazol-4(5H)ylidene) hydrazinecarbothioamide derivatives have been synthesized in good yields by an efficient method. The synthesized compounds (6a-r) were evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis (MTCC 300) strain. Compounds 6o (MIC-3.90 µg/mL), 6p (MIC-3.90 µg/mL), and 6q (MIC-7.81 µg/mL), exhibited significant activity against Mycobacterium tuberculosis. The molecular docking studies revealed an interesting binding profile with very high receptor affinity.

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“…michiganensis(Smith 1910) isolated from tomato (Solanum lycopersicum L., Solanaceae), Xanthomonas sp. isolated from bean (Phaseolus vulgaris L., Fabaceae) and Pseudomonas syringae(van Hall 1902) isolated from tomato, the assays were carried out using the doubling dilution in 96-well microtiter plates using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ) 28 and resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) 29 as revelator:…”

Section: Microbial Strainsmentioning

confidence: 99%

Chemical Composition and Antibacterial Activity of Essential Oils From Azorella Spinosa (Apiaceae) Against Wild Phytopathogenic Bacteria

Jara-Bermeo

Peñailillo

San-Martı́n

et al. 2016

J. Chil. Chem. Soc.

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The aim of the present investigation was to appraise variation in the chemical composition and antimicrobial activity against phytopatogenic bacterial of essential oils from Azorella spinosa. Three samples of A. spinosa were collected in different date in the same area. The essential oils were obtained for hydrodestillation using a Clevenger-type apparatus. The chemical composition of essential oils were analysed using Gas chromatography-mass spectrometry showed variation in the yield and composition, the main detected compounds were monoterpenes such as limonene (10.66-12.30%), tricyclene (10.81-16.99%) and sabinene (9.10-11.49%), the most abundant sesquiterpene was γ-gurjenene (5.48-8.47%). The biological activity of essential oils from A. spinosa against wild phytopatogenic microorganisms showed selectivity over Pseudomonas syringae. This is the first report of the essential oil composition of A. spinosa, and confirms that natural products obtained from aromatic plants represent an important source of bioactive molecules that could be used as new alternatives for the treatment of phytopatogenic bacteria infections.

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Resazurin Microtiter Assay Plate method for detection of susceptibility of multidrug resistant Mycobacterium tuberculosis to second-line anti-tuberculous drugs

Cited by 62 publications

References 34 publications

Genetic and pharmacological inhibition of inflammasomes reduces the survival of Mycobacterium tuberculosis strains in macrophages

Genetic and pharmacological inhibition of inflammasomes reduces the survival of Mycobacterium tuberculosis strains in macrophages

Synthesis and molecular docking studies of a new series of bipyrazol-yl-thiazol-ylidene-hydrazinecarbothioamide derivatives as potential antitubercular agents

Chemical Composition and Antibacterial Activity of Essential Oils From Azorella Spinosa (Apiaceae) Against Wild Phytopathogenic Bacteria

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