ZnO Nanoparticles and Rifampicin Synergistically Damage the Membrane of Mycobacteria

The emergence of drug resistance in tuberculosis requires alternative strategies to combat the resistance. To this end, we have synthesized zinc oxide nanoparticles (ZnO NPs) of 11 nm diameter and evaluated its effect against mycobacteria alone, as well as in combination with the first-line anti-TB drug, rifampicin. These ZnO NPs themselves are not inhibitory against the wild-type (WT) Mycobacterium smegmatis, up to a concentration as high as 256 μg/mL. However, a subinhibitory concentration of only 32 μg/mL of ZnO NPs in combination with rifampicin, showed a 4-fold reduction in the minimum inhibitory concentration of rifampicin, against WT M. smegmatis. Thus, the fractional inhibitory concentration index for the combination was found to be 0.375, which proves the effect to be synergistic. This is explained by high membrane permeability observed in presence of the combination of ZnO NPs and rifampicin, which increased with time. High membrane permeability, in turn, increased rifampicin uptake in cells, which subsequently enhanced the bactericidal effect of the drug. The resultant morphology of the bacteria, from Cryo-SEM, showed membrane damage, following treatment with a combination of ZnO NPs and rifampicin. However, this combination showed very less reactive oxygen species (ROS) production, compared to ZnO NPs alone. Hence, we can conclude that membrane damage is the dominant mechanism behind synergism of the combination, rather than that of ROS generation. This combination also proved to be successful against our laboratory-generated, drug resistant strains of M. smegmatis, as well as against vaccine strain- M. bovis BCG, which proves the general applicability of this synergism for different strains. We hence propose that our developed ZnO NPs and drug combination can be promising for overcoming mycobacterial drug resistance.