Rapid Methods for Testing Inhibitors of Mycobacterial Growth

Abstract

Considering the increased concerns with controlling infectious epidemics such as tuberculosis, a global concerted effort (WHO) is now dead-lined to tackle the emergence of extensive drug resistance through identifying a novel line of therapeutics which will on the one hand shorten the course of treatment and on the other is also expected to be effective against the emerging resistant strains. Major problems with the preclinical drug screening against the uniquely slow-growing pathogen Mycobacterium tuberculosis are either found expensive, time-consuming, or require a highly complex laboratory setup. A rapid and convenient, although relatively inexpensive, method requiring very little consumption of inhibitors within a simple microbiology setup for antimycobacterial screening is thus timely. The spot-culture growth inhibition assay aims to test the biological activity of a number of newly discovered natural products and thousands of novel chemicals synthesized on the basis of basic structural and molecular biology studies. Many different classes of novel chemical entities are now independently prepared around the world by distinguished chemists on the chemical behavior of the group of molecules. To serve the purpose of antimycobacterials screening, we aim to describe a method in this chapter performed in a six-well plate format. This method can also be extended accurately to a 96-well plate format according to the necessity of the project. In addition to evaluating a range of prospective drug candidates, this method would also contribute to elucidate substrates for many putative endogenous pathways through comparing the chemical inhibition with the corresponding genetic modification.

Antitubercular specific activity of ibuprofen and the other 2-arylpropanoic acids using the HT-SPOTi whole-cell phenotypic assay

“…The spot culture growth inhibition (SPOTi) assay method has been enhanced in various studies using 6-well, 24-well and 48-well plates for screening growth inhibitors against mycobacteria. [4][5][6][7] Of immediate importance, the methodology has also been independently employed for drug susceptibility testing (DST) of clinical isolates in a TB reference laboratory in the Netherlands. 8 There is currently a renewed interest in re-purposing old drugs especially for treating infectious diseases, 9 saving time, effort and expenses from a new drug development because of the existing knowledge of their pharmacological (PK/PD) profiles, toxicity and safety.…”

“…Antimycobacterial, cytotoxic and anti-infective activities of commercially available 2-arylpropanoic acids(1)(2)(3)(4)(5)(6)(7)(8)(9) MIC values against Mycobacterium (mg/L)…”

Development of a rapid, reliable and quantitative method — “SPOTi” for testing antifungal efficacy

“…The methodology, with required modification from previously published method (Guzman et al, 2013;Evangelopoulos and Bhakta, 2010;Gupta and Bhakta, 2012), is schematically shown in Fig. 1.…”

“…We originally developed and previously reported a highthroughput, solid agar-based assay conducted in multi-well (six-well to ninety-six well) plates to test inhibitors of bacterial growth and viability, known as the SPOTi assay (Evangelopoulos and Bhakta, 2010;Gupta and Bhakta, 2012;Guzman et al, 2013). The latter has been independently used in a clinical setting for antibacterial drug susceptibility testing (Hall et al, 2012).…”

An Integration of Interdisciplinary Translational Research in Anti-TB Drug Discovery: Out of the University Research Laboratories to Combat Mycobacterium tuberculosis

“…Phagocytosis and survival of the TB pathogen through interaction of the host and bacterial cell receptors inside the macrophage environment are two key consequences. In the spot culture growth inhibition (SPOTi) assay [33], a different physiology of the mycobacteria is represented as a biofilm on the surface of solid media mimicking the in vivo growth of the pathogen which is typically seen in histopathogical M. tuberculosis lesions. By exploiting the characteristics of M. aurum and by the use of the HT-SPOTi assay, Gupta and Bhakta recently established an integrated surrogate drug screening model for intracellular screening of inhibitors [32].…”