scholarly journals Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium.

1997 ◽  
Vol 41 (5) ◽  
pp. 1004-1009 ◽  
Author(s):  
L Collins ◽  
S G Franzblau
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
Mycobacterium Avium ◽  
High Throughput Screening ◽  
Antimicrobial Agents ◽  
Alamar Blue Assay ◽  
Alamar Blue ◽  
Mycobacterium Tuberculosis H37rv ◽  
Large Numbers ◽  
Highly Correlated

In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.

scholarly journals High throughput screening of a library based on kinase inhibitor scaffolds against Mycobacterium tuberculosis H37Rv

Tuberculosis ◽  
2012 ◽  
Vol 92 (1) ◽  
pp. 72-83 ◽  
Author(s):  
Robert C. Reynolds ◽  
Subramaniam Ananthan ◽  
Ellen Faaleolea ◽  
Judith V. Hobrath ◽  
Cecil D. Kwong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitor ◽  
Tuberculosis H37rv ◽  
Mycobacterium Tuberculosis H37rv ◽  
Inhibitor Scaffolds

scholarly journals High-throughput screening for inhibitors of Mycobacterium tuberculosis H37Rv

Tuberculosis ◽  
2009 ◽  
Vol 89 (5) ◽  
pp. 334-353 ◽  
Author(s):  
Subramaniam Ananthan ◽  
Ellen R. Faaleolea ◽  
Robert C. Goldman ◽  
Judith V. Hobrath ◽  
Cecil D. Kwong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Tuberculosis H37rv ◽  
Mycobacterium Tuberculosis H37rv

scholarly journals Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis

2007 ◽  
Vol 51 (4) ◽  
pp. 1380-1385 ◽  
Author(s):  
Sang Hyun Cho ◽  
Saradee Warit ◽  
Baojie Wan ◽  
Chang Hwa Hwang ◽  
Guido F. Pauli ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Antimicrobial Agents ◽  
Physiological State ◽  
Recovery Phase ◽  
Low Oxygen ◽  
Anaerobic Conditions ◽  
Recovery Assay

ABSTRACT Screening for new antimicrobial agents is routinely conducted only against actively replicating bacteria. However, it is now widely accepted that a physiological state of nonreplicating persistence (NRP) is responsible for antimicrobial tolerance in many bacterial infections. In tuberculosis, the key to shortening the 6-month regimen lies in targeting this NRP subpopulation. Therefore, a high-throughput, luminescence-based low-oxygen-recovery assay (LORA) was developed to screen antimicrobial agents against NRP Mycobacterium tuberculosis. M. tuberculosis H37Rv containing a plasmid with an acetamidase promoter driving a bacterial luciferase gene was adapted to low oxygen conditions by extended culture in a fermentor with a 0.5 headspace ratio. The MICs of 31 established antimicrobial agents were determined in microplate cultures maintained under anaerobic conditions for 10 days and, for comparative purposes, under aerobic conditions for 7 days. Cultures exposed to drugs under anaerobic conditions followed by 28 h of “recovery” under ambient oxygen produced a luminescent signal that was, for most compounds, proportional to the number of CFU determined prior to the recovery phase. No agents targeting the cell wall were active against NRP M. tuberculosis, whereas drugs hitting other cellular targets had a range of activities. The calculated Z′ factor was in the range of 0.58 to 0.84, indicating the suitability of the use of LORA for high-throughput assays. This LORA is sufficiently robust for use for primary high-throughput screening of compounds against NRP M. tuberculosis.

scholarly journals A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sadaf Kalsum ◽  
Blanka Andersson ◽  
Jyotirmoy Das ◽  
Thomas Schön ◽  
Maria Lerm
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Aggregate Size ◽  
Live Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

scholarly journals Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

2005 ◽  
Vol 10 (7) ◽  
pp. 725-729 ◽  
Author(s):  
Upasana Singh ◽  
Vinita Panchanadikar ◽  
Dhiman Sarkar
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Coli Strain ◽  
Compound Library ◽  
Essential Enzyme ◽  
Assay Protocol

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Kmof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 [.proportional]M, 5 [.proportional]M, and 25 [.proportional]M, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25° C and 37° C. The Z′ factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-[.proportional]l volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.

ChemInform Abstract: Discovery of Novel Inhibitors Targeting the Mycobacterium tuberculosis O-Acetylserine Sulfhydrylase (CysK1) Using Virtual High-Throughput Screening.

ChemInform ◽  
2013 ◽  
Vol 44 (28) ◽  
pp. no-no
Author(s):  
Variam Ullas Jean Kumar ◽  
Oemer Poyraz ◽  
Shalini Saxena ◽  
Robert Schnell ◽  
Perumal Yogeeswari ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals Mycobacterium tuberculosis virulence inhibitors discovered by Mycobacterium marinum high-throughput screening

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hasan Tükenmez ◽  
Isabel Edström ◽  
Ramesh Ummanni ◽  
Stina Berglund Fick ◽  
Charlotta Sundin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mycobacterium Marinum

Patterned superhydrophobic surfaces to process and characterize biomaterials and 3D cell culture

2018 ◽  
Vol 5 (3) ◽  
pp. 379-393 ◽  
Author(s):  
A. I. Neto ◽  
P. A. Levkin ◽  
J. F. Mano
Keyword(s):  
Cell Culture ◽  
High Throughput ◽  
High Throughput Screening ◽  
3D Cell Culture ◽  
Superhydrophobic Surfaces ◽  
Technological Breakthrough ◽  
Large Numbers

Microarrays are a technological breakthrough for high-throughput screening of large numbers of assays.

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