Bacterial strains and drug susceptibility testing
Thirteen MDR and two RMR clinical isolates were selected, from M. tuberculosis strains collection of the Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran. M. tuberculosis strains were isolated from patients with pulmonary TB, from January 2010 to March 2015. Ethical reviews and informed written consent approval were granted by the Ethical Committee of the Pasteur Institute of Iran (Tehran, Iran). Informed consent was obtained from all of the patients, enrolled in the study. The isolate profiles of drug susceptibility were re-confirmed by the proportional method, using Lowenstein-Jensen medium, supplemented with Isoniazid (INH), 0.2 mg/L; Rifampin (RIF), 40 mg/L; Streptomycin (STR), 4 mg/L; Ethambutol (EMB), 2 mg/L; Kanamycin (KAN), 30 mg/L and Ofloxacin (OFX), 2 mg/L [10]. Bacterial growth on antimicrobial agents-containing media, exceeding 1 % of the number of colonies on antimicrobial agents-free media (control), were considered to be resistant to the antimicrobial agents. All experiments were performed, in accordance with the guidelines, approved by Pasteur Institute International networks.
Determination of MIC
An Alamar blue assay (Thermoscientific, USA) was carried out, as previously described, to determine the MICs of RIF of the 15 clinical isolates [11]. RIF concentrations were 0.001–256 mg/L. All tests were conducted in duplicate. The MIC was defined as the lowest antibiotic concentration that inhibits any color change. Isolates with MICs of RIF <1 mg/L were defined, as being susceptible. For next-step analysis, the bacteria, growing in sub MIC concentration were selected.
RNA extraction and reverse transcription
All M. tuberculosis strains were sub-cultured in 7H9 medium, supplemented with Albumin Dextrose Catalase (ADC), in the absence of rifampin (control strains) and presence of sub-MIC of rifampin (MIC = 128 mg/L), and collected after four weeks for RNA extraction. Total bacterial RNA was isolated, using PREP-NA DNA/RNA extraction kit (DNA technology, Russia). The quality and integrity of the total RNA was assessed, using a nanophotometer (Thermoscientific, USA) and agarose gel electrophoresis. After DNase I treatment, RNA (1.5 μg) was reverse transcribed, according to the manufacturer’s recommendations (PrimeScript First Strand cDNA Synthesis Kit, Takara, Japan), and the thermal cycling conditions were as follows: 25 °C for 10 min, 42 °C for 60 min, and 85 °C for 5 min. The cDNAs were maintained at −20 °C, until further use.
Quantification of gene expression using real-time quantitative PCR
The primers for rpf (A-E) and 16S rRNA were described, previously [12]. The assay was performed, using a SYBR Premix Ex Taq™ II kit (Takara, Japan) in a LightCyler 96 thermocycler (Roche, Germany). Briefly, 10 μL qPCR mix, 0.8 pmol each primer, 10 ng cDNA, and RNase-free water were mixed, with a final volume of 20 μ L. The thermal cycling conditions were as follows: 95 °C for 1 min, then 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, and the last step consisted of a melting curve analysis (65–97 °C).
The fold change in the gene expression under rifampin stress in all isolates was calculated by 2-ΔΔCT method [13]. 16S rRNA is a housekeeping gene that is expressed at a stable level in the isolates and can be used, as an internal invariant control. For each assay, the results were compared with corresponding M. tuberculosis control growth, in the absence of the rifampin, as the calibrator. Each assay was performed in triplicate, for each gene.
Statistical analysis
Two-tailed t-test was used for data analysis. All calculations were performed, using SPSS version twenty-two (SPSS Inc., Chicago, IL, USA). Lightcycler 96 software was used for expression data analysis. A p value of <0.05 was considered statistically significant.
