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

Abstract

A major obstacle in the process of discovery of drugs against Mycobacterium tuberculosis is its extremely slow growth rate and long generation time (ϳ20 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 luminescencebased 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.T uberculosis (TB) continues to be a major threat to public health worldwide, accounting for 1.3 million deaths annually, and is also a leading cause of death among people coinfected with HIV/AIDS. Despite the facts that the estimated number of people falling ill with tuberculosis each year is declining and that TB death rates dropped 45% over the last 2 decades, the rising incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB cases is alarming (1). Only half of MDR-TB cases are successfully treated, reflecting exceptionally high mortality rates. Moreover, 1/10 of MDR TB cases are redefined as XDR TB, as they are resistant to the majority of existing first-and second-line anti-TB drugs. The emergence of MDR and XDR TB strains compromises the existing treatment regimens and has prompted the search for new medicines. Therefore, an efficient, rapid, low-cost, highthroughput assay that would allow us to screen compound libraries and follow their effects on mycobacterial cells in real time would be of tremendous help.The extremely slow growth rates of pathogenic mycobacteria and therefore the long incubation times needed to assay antimycobacterial activity represent major obstacles in drug discovery. Plating of bacterial cultures on specialized mycobacterial agar medium for CFU enumeration continues to be the "gold standard" for evaluating the effectiveness of antituberculosis compounds, despite being very labor-intensive and time-consuming...

Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo

“…Both luciferase and fluorescent protein labeled mycobacterial strains have been developed for TB studies [ 10 , 11 , 13 – 19 , 40 ]. Three of these reporter systems have been applied to in vivo detection of Mtb infection in mice [ 11 , 14 , 18 , 19 ].…”

Optimized Background Regimen for Treatment of Active Tuberculosis with the Next-Generation Benzothiazinone Macozinone (PBTZ169)

“…When mentioned, the interaction between MCZ and the drugs was also assessed using the luminescent H37Rv(pEG200) strain, as previously described (34). A mid-logarithmic-phase culture of H37Rv(pEG200) (OD 600 , approximately 0.5) was diluted in 7H9 complete medium to an OD 600 of 0.001 (approximately 3 ϫ 10 5 CFU/ml).…”

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Isolation of isosativenetriol from endophytic fungus Cochliobolus spp. of Aerva lanata

“…Isosativenetriol exhibits significant antimycobacterial potential with MIC of 25 µg/mL against M. tuberculosis H37Rv using microplate alamar blue assay (Sharma et al, 2014). The results were depicted in Table I.…”