Study design and bacterial isolates
The multidrug resistance strain of Mtb isolated from a 55-year-old man who was proven tuberculosis by imaging, clinical findings, histological and cytological observations, previously. The patient had consumed first-line anti-tubercular treatment for 6 months but the symptoms of TB had reappeared about 7 months after completing the treatment. Spectrum sample of patients was cultured in LJ medium for 28 days. The susceptibility drug testing was done by the indirect proportion method to determining of MDR-Mtb .
In this study, MgONPs (average diameter of 20 nm) and ZnONPs (average diameter of 4 nm) were purchased from Tehran University of Medical Sciences (Tehran, Iran). Before each experiment, MgONPs and ZnONPs were sterilized through heating in an oven at 200 °C for one hour.
Antibiotic susceptibility test
To determine the minimum inhibition concentration (MICs) of NPs, the microplate Alamar blue (MABA) assay is used . Two hundred microliter (200 µl) of sterile deionized water added to outer-perimeter wells of 96-well microplates. Then, 100 ml of 7H9 GC broth was added to each well. 100 µl of MgONPs (12.5 µg.ml -1), ZnONPs (30 µg.ml -1) and MgONPs-ZnONPs (42.5 µg.ml -1) solutions were added to each well, and the serial dilutions were done. A hundred microliter (100 µl) of H37Rv Mtb (Razi serum and vaccine research institute, IRAN) was added to the wells . The plates incubated at 37°C for 5 days. Fifty microliters (50 µl) of a freshly prepared 1:1 mixture of Alamar blue reagent and 10% Tween 80 added to well and reinsulated at 37°C for 24 hours. The blue color in the well interpreted as no growth and a pink color scored as growth. The MICs was defined as the lowest drug concentration which prevented a color change from blue to pink .
To determine the minimum bactericidal concentration (MBCs) of NPs against MDR-Mtb (Razi serum and vaccine research institute, IRAN), proportion method was also done. 10 ml of melted löwenstein-Jensen (LJ) agar and 10 ml of NPs were poured and subsequently, serial dilution was performed. On the following day, 100 µl of 10-2 and 10-4 McFarland of bacteria were added and then they are incubated at 37 °C. The colony-forming unit (CFU) of bacteria was counted after 28 days. The atomic force microscopy (AFM) (Institute for color science & technology, IRAN) of mixture MgONPs-ZnONPs fulfilled by commercial AFM system (Nanosurf, Switzerland) . The TEM image was prepared at the Institute of Biochemistry and Biophysics (IBB) of Tehran University.
To MTT assay, Vero and HepG2 cell lines (Institute for tuberculosis research, the University of Illinois at Chicago, USA) (1×104 cells per well) were seeded in 96‐well plates containing 100 µl of DMEM, separately . 100 µl of MgONPs, ZnONPs, and MgONPs-ZnONPs were inoculated to each well. Next, 5 mg MTT per ml added. Cell viability calculated using DMSO treated Vero and HepG2 as the 100% viable control and measured (A540 of NPs × treated sample/A540 of control) × 100.
Statistical analyses were prepared by using of Kruskal-Wallis test and Mann–Whitney U test. The statistical significance threshold was resolute as P-value ≤ 0.05.