Study Population
The present research was conducted as a case-control study. Case group included isolates of M. tuberculosis that were resistant to EMB at least in terms of phenotype and had mutation in emb306B gene; and the control group was comprised of those types of M. tuberculosis isolates that were susceptible to EMB in phenotype and had no mutation in embB. According to a census carried out by the country's Drug Resistance Surveillance (DRS) program in Western part of the country in a period from 2014 to 2015, only seven samples of antibiotic-resistant M. tuberculosis were identified from all the antibiotic susceptibility tests. The study population included samples sent from Western provinces of Iran to the Mycobacterium Research Center as the reference laboratory, which is located in the city of Kermanshah in Kermanshah province.

Identification of Samples
Initially, sputum samples that were suspected of having TB were sanitized with Modified Petroff Method. The samples were colored using Auramine and Ziehl-Neelsen technique. Löwenstein Jensen (LJ) agar medium (Merck, Germany) was used for culturing the samples and the colonies were studied after incubation at temperature of 37°C for 4 weeks. Suspicious colonies were assessed based on the speed of growth of colonies (slow growing) and by biochemical assays (positive niacin production, positive nitrite reduction, positive urease, positive thiosemicarbazon, sensitive cycloserine 20 µL/mL in LJ, positive oxygen performance, positive pyrazinamide activity, catalase activity at 68°C, negative NaCl tolerance, and pigment production) (8).

Determining the Medicinal Sensitivity of Mycobacterium tuberculosis
A bacterial suspension was added to each of the LJ culture media containing EMB (2 µg/mL) antibiotic, and the results were examined after 28 and 48 days (in case of growth of bacteria in these media, they were considered as resistant isolates, and if no growth was observed, they were considered as sensitive isolates). Concentration rate of EMB was 0.2 µg/mL. The standard M. tuberculosis strain (H37RV) (SinaClon, Iran) was used as quality control of the test of sensitivity to EMB (9).

Identification of embB306 Gene in M. tuberculosis Isolates
DNA was extracted using CTAB (Cetyltrimethylammonium Bromide) after deactivation at 80°C for 1 h. A piece including 863 base-pairs (bp) of embB306 gene was reduplicated by PCR to determine the presence of embB306 gene in isolates sensitive and resistant to EMB. Oligonucleotides included embB306-F (5’-CGACGCCGTGGTGATATTCG-3’) and embB306-R (5’-CCACGCTGGGAATTCGCTTG-3) (SinaClon, Iran). The final PCR mixture (final volume of 50 µL) consisted of 0.2 µmolar of every primer, 25.1 units of Taq-polymerase enzyme (SinaClon, Iran), 0.2 mmolar dNTP, and 1 mmolar of 10X buffer. PCR reaction was conducted as follows: Primary denaturation for 5 min at 95°C, secondary denaturation at 94°C for 30 s, annealing phase at 70°C for 40 s, extension at 95°C for 72 s, in 30 cycles. The final extension phase was carried out at 72°C for 10 m. The embB gene sequence was determined by means of sequence analysis (Gene Faravaran, Iran) and analysis of created point mutations in sequence of embB306 gene was done through comparing them with the sequence of embB gene sequence derived from the standard strain of M. tuberculosis H37RV provided by GeneBank (http://www.ncbi.nlm.nih.gov/genbank) using BLASTn algorithm (http://blast.nbci.nlm.nih.gov). Presence of mutations of embB gene and point mutations were examined in sensitive and resistant strains and finally, the relationship between embB306 gene mutation and resistance to EMB was analyzed (10).

Statistical Analysis
Frequency and percentages were determined by means of Fischer’s test and SPSS 16 (SPSS Inc., Chicago, IL., USA), which were used to compare mutations in both resistant and nonresistant groups to EMB (P≤0.05).

Discussion

In the present study, the maximum rate of identified mutations was found in embB306 gene, which belonged to the case group (EMB-resistant strains), while no mutation was found in the strains sensitive to this medication. In a study conducted in Iran, Derakhshani Nezhad et al. (2012) showed that among 106 M. tuberculosis samples, 33.9% were resistant to EMB using the proportion method. Using Allele-specific PCR (ASPCR), 27.6% of strains were EMB resistant. This method showed that mutation in the first base was 61.5% (Met→Val or Il) and it was 38.5% in the third base (Met→ Il) (11). In the present research, six case samples out of 50 strains had mutation in the embB306 region. Determination of possible mutations with the help of PCR-sequencing is the main technique for mutation analysis in resistance-related genes. Although this technique is a time-consuming and burdensome method, it has high efficiency in terms of sensitivity and property. An accurate laboratory diagnosis of TB is the most important step in diagnosis of the disease. One of the reasons for differences in various studies is the application of different techniques with varying degrees of differentiation and sensitivity, which may lead to different results (8). In the study conducted by Bakula et al. (2013), who used PCR-sequencing, out of 50 studied strains (33 EMB-sensitive strains and 17 EMB-resistant strains), six types of mutations were found in 27 (54%) samples. Mutation in embB gene was observed among 13 (76.5%) EMB-resistant strains and 14 (42.4%) EMB-sensitive strains (1). Unlike Bakula’s study, the current work showed no mutation in EMB-sensitive strains; and among the seven EMB-resistant samples, all of them had mutation in the embB306 region. A strong relationship was observed between embB306 mutants and resistance to EMB. The study’s results showed that mutation in embB306 gene is an important indicator for detection of resistance to EMB. In conclusion, it was demonstrated that embB306 mutation is a common polymorphism in the strains resistant to EMB M. tuberculosis. Considering that only isolates resistant to EMB were studied in this work, further evaluation of a number of EMB-susceptible and resistant isolates from different geographic regions is required to explicate the role of these mutations in resistance to EMB. In addition, even though under normal conditions, definitive diagnosis of resistance to a drug requires about 42 days, it can be diagnosed in only 6-7 days by means of molecular techniques. Quick diagnosis of drug resistance may prevent wrong administration of drugs and it also leads to employing the proper therapeutic diet that prevents the spread of multiple drug-resistant (MDR) strains, which is a large issue (12,13). Using phenotypic susceptibility testing, Khosravi et al. showed that resistance to emb was 3.25% in Iran. Rate of mutation in embB was 10% at codon 306 Met→Val (12). It has been found that mutations in embB306 of embB gene very often occur among other codons. High detection rates of mutation have been reported in codon embB306 among EMB-resistant M. tuberculosis isolates in various countries (1). Therefore, in this study, embB306 was investigated among other genes by PCR-Sequencing. PCR is a rapid method with high discriminatory capabilities, and it is easy to use while being obtainable at a low cost (14,15). Moreover, limitations of this research are as follows: The small number of antibiotic-resistant specimens, especially samples with resistance to EMB in the studied area, the high cost of molecular testing equipment and tools, slow growth of bacteria in a specific culture medium and special incubation conditions, and due to the high contamination level of M. tuberculosis, sampling and DNA extraction should be performed under conditions of high immunity and level 3 safety. Given that embB gene mutations, especially those located in ERDR, are considered as "hot-spots", they have been repeatedly investigated in studies concerned with EMB-resistance in M. tuberculosis cases. Therefore, the PCR-Sequencing method is a precise method for determining these hot-spots. Moreover, mutation rates in first and third bases were very similar to each other. In addition, it was observed that embB306 mutations were present only in isolates that were resistant to EMB and had a significantly higher proportion of embB306 mutants.

Conclusion

The majority of EMB-resistant isolates showed embB306 mutation. No embB306 mutant was detected among the susceptible strains. The current study demonstrated that mutation in embB306 can be considered as an indicator of resistance to EMB in isolates of M. tuberculosis, and most of the found mutations were located in this codon. However, mutations in embB306 are not the main reason for resistance to EMB in isolates; other mechanisms of resistance in M. tuberculosis should be considered as well.

 

Acknowledgements

This paper is part of Mr. Bahman Mohammadi’s (MSc of medical microbiology) project under the code 1394/261, approved by the Research Committee of Kurdistan University of Medical Sciences, who supported this project financially. We would like to send our appreciation to those who showed special attention to this work. We would also like to thank and appreciate Ms. Sara Atashi and Mr. Galini for their fabulous technical assistance.

 

Conflicts of Interest

Authors declared no conflict of interest.