Antibiofilm activity of South African plant extracts against Mycobacterium spp. and their mechanism of action using Mycothiol reductase

Abstract

Tuberculosis (TB) is an infectious disease, mostly affecting lungs or abdominal area. The causative agent of TB is a bacterium called Mycobacterium tuberculosis; it is the second leading cause of death in the world. Plants have been considered as a possible additive to current treatment, due to resistance of M. tuberculosis to commercially available drugs. Twenty South African medicinal plants ethanol extracts, tradititonally used to treat TB related symptoms were evaluated for their antituberculosis activity. Twenty South African medicinal plant extracts were evaluated for in vitro antimycobacterial activities using the Microtiter Alamar Blue Assay and cytotoxicity with XTT (2, 3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay. Mycobacterium tuberculosis H37Rv strain was used as the test organism. Cytotoxicity test was done on human macrophages (U937 cell line). Of all the 20 extracts 7 showed activities against Mycobacterium with minimum inhibitory concentrations ranging from 125-31.25 μg/ml. Ficus sur had a selectivity index of 3 followed by Salvia africana-lutea and Sphedamnocarpus pruriens with selectivity indexes of 2. The plant extracts were further tested for their activity against Glutathione disulfide reductase (Human analogue) and Mycothiol disulfide reductase (Mycobacterium analogue). The inhibitory activity of the plant extracts was determined using a DTNB-coupled Glutathione/Mycothiol disulfide reductase assay. Typha minima showed a potential inhibitory activity against Mycothione reductase (Mtr) with an effective concentration at which 50% activity is inhibited (EC50) of 47.89±47.5μg/ml and had less inhibitory activity against Glutathione reductase (Gtr) with an EC50 of 813.5±3.21μg/ml. The plant extracts were screened to evaluate whether they had antimicrobial and antibiofilm formation activity, utilizing Mycobacterium smegmatis as the test organism, which is a genetic homologue to Mycobacterium tuberculosis. No significant antimicrobial activity was observed from the plant extracts. Based on a visual analysis the plant extracts; Sphedamnocarpus pruriens (N), Salvia africana-lutea (L), Withania somnifera (R) showed considerable antibiofilm activity as compared to Ciproflaxicin (positive control) displayed a better inhibitory activity and was validated by determining quantitavely using crystal violet absorption. The effective concentration (EC50) was determined and Leonotis leonurus L. (K), Salvia africana-lutea (L), and Sphedamnocarpus pruriens (N) showed potential biofilm formation inhibition at concentration 45.55±0.2475μg/ml, 100±56.83μg/ml and 61.39±60.59μg/ml respectively when compared to Ciproflaxicin, which inhibited at a concentration of 1.9802μg/ml.