Genetic diversity assessment of the Mycobacterium tuberculosis isolates of patients with tuberculosis in Karaj, Iran

to


Background
Tuberculosis (TB) is a worldwide health threat globally.According to the World Health Organization (WHO) report, there were 10.0 million new TB cases (5.6 million men, 3.2 million women, and 1.2 million children), which resulted in 1.4 million deaths in 2020 [1].Despite an auspicious decline in the incidence of TB in Iran from 36 cases per 100,000 people in 1990 to 17 cases per 100,000 people in 2010 [2], TB endures being an important public health threat among Iranians [3].
TB is a complicated and polymorphic disease [4,5].Various combination therapy is available to treat TB [6].However, there is an emergence of multidrug-resistant TB (MDR-TB) [7].Presently, the MDR-TB has a cumulative incidence rate globally.Additionally, MDR-TB is a signi cant public health threat in TB control and prevention [8].Thus, it is essential to use rapid and sensitive TB diagnostic methods and assess the new epidemiological aspects of TB in an area with a high incidence rate [9,10].
Genotyping of M. tuberculosis strains using IS6110 restriction fragment length polymorphism (IS6110-RFLP) is considered a practical approach in TB molecular epidemiology [11,12].Such genotyping helps TB control by allowing the unpredicted outbreaks identi cation and laboratory cross-contamination, and it helps discriminate exogenous reinfection from endogenous reactivation, in that way permitting an understanding of the strain's genetic diversity [13].
According to the high incidence rate of TB among Iranians and uncertain status of the genotyping patterns of isolates in some area of Iran, the present survey was aimed to assess the molecular features of the M. tuberculosis isolates from the sputum samples of patients referred to the Alborz tuberculosis center using the RFLP method.

Ethical issues
Written informed consent was taken from volunteer patients or their parents.

Samples and preparation
Twenty sputum samples were taken from randomly selected 20 pulmonary TB-suspected outpatients.
These were visiting patients to the university hospitals of Alborz from December 2012 to October 2013.
Patients' demographic data were collected, and their consent through questionnaires lled out at the clinic when specimens were taken.Samples were immediately transferred to Razi Vaccine & Serum Research Institute, Karaj, Iran, for further analysis.Samples were subjected to Petroff's protocol for digestion/decontamination.

M. tuberculosis culture
Processed samples were used to inoculate slopes of plain (glycerinated) and Lowenstein-Jensen medium (LJ) supplemented with sodium pyruvate and glycerol (Merck, Germany).Cultures were incubated at 37°C for 12 weeks.The weekly inspection was performed to found any bacterial growth.The acid-fast staining was used for the microscopic assessment of cultures with visual features of M. tuberculosis complex (MTC) [14].All principles were performed according to the instructions of the Department of Tuberculin & Mallein, Razi Vaccine & Serum Research Institute, Karaj, Iran.Identi cation of M. tuberculosis isolates was performed using the PCR ampli cation of the 16S rRNA gene and IS6110 marker [15,16].

DNA extraction and quality assessment
For DNA extraction, 3 loopful colonies of M. tuberculosis were scraped, transferred to a microtube that contained 400µl Tris-ethylenediaminetetraacetic acid (EDTA) buffer (pH 8) inactivated by heating using a water bath (80°C for 30 min).Then, 50µl of 10mg/mL lysozyme was added, vortexed, and incubated overnight (37°C).Sodium dodecyl sulfate (SDS)/proteinase K (70µl of 10% [w/v] SDS and 5µl of 10mg/mL of proteinase K) were added to tubes vortexed and incubated at 65°C for 10 min.One-hundred microliters of 5M NaCl and N-cetyl-N,N, N-trimethylammonium bromide (CTAB)-NaCl solution (4.1g of NaCl and 10 g of CTAB per 100mL) was added and incubated at 65°C for 10 min.An equal volume of chloroform: isoamyl alcohol (24:1v/v) was added to the mixture.After centrifugation for 5 min, 0.6 volumes of isopropanol were added to the supernatant liquid to precipitate the DNA.After incubation for 20 min at − 20°C and centrifugation for 15 min at 12,000 × g and 4°C, the pellet was washed once with 70% (v/v) ethanol, and the air-dried pellet was dissolved in 20µL of Tris-EDTA buffer [17].Purity (A260/A280) and extracted DNA concentration were then checked (NanoDrop, Thermo Scienti c, Waltham, MA, USA).The DNA's truth was assessed on a 2% agarose gel stained with ethidium bromide (0.5 µg/mL) (Thermo Fisher Scienti c, St. Leon-Rot, Germany) [18,19].

Restriction fragment length polymorphism (RFLP) analysis
DNA ngerprinting was performed by a standardized procedure described previously ().Brie y, chromosomal DNA (2-3.5 µg) was restricted with PvuII at 37°C overnight.The restriction fragments were separated overnight by 1% (w/v) agarose gel electrophoresis in 1× Tris-borate-EDTA buffer.The fragments were transferred onto positively charged nylon membranes using the capillary method.RFLP analysis was performed using an internationally standardized procedure as described previously [20].The IS6110 ngerprint patterns were analyzed using GelCompar II software version 6.5 (Applied Maths, Sint-Martens-Latem, Belgium).

Results
Patients of the present study consisted of 6 females and 14 males who had Iranian citizenship.According to the patients' les, all sputum samples were received from the patients.All M. tuberculosis isolates and the standard control (M.tuberculosis H37Rv strain) were examined for genomic ngerprinting and polymorphism in Alborz province.Due to technical reasons, including the quality of the genomic material extracted from the sample, DNA extracted from 20 isolates was suitable for this test.In this study, 17 isolates were enzymatically digested by Pvu II enzyme and assessed in the RFLP. Figure 1 shows the RFLP pattern of M. tuberculosis isolates using the Pvu II enzyme.
Visual comparison of high molecular weight bands (2027 to 23130 bp) and using Gel Pro software (analysis based on the number and size of bands), from 20 strains digested with Pvu II enzyme, after RFLP and hybridization with the probe IS6110 and detection, 16 different genetic types were obtained (Fig. 2), which were named HPIS1 to HPIS12 and identi ed, and the number of unique genotypes in this method was determined to be 16.
The number of copies of IS6110 in each isolate ranged from 0 to 12.However, most isolates (66%) had copy numbers between 6 and 12. On average, each isolate had 6.9 copies of IS6110.9 strains containing 10 to 12 copies of IS6110 5 strains containing 6 to 10 copies, and 2 strains had copies less than 6.No copy of IS6110 was found among the 4 strains.No relationship was found between gender and copy numbers.
The present study showed 17 different genetic types in the M. tuberculosis strains isolated from patients in Karaj, Iran, according to the RFLP-IS6110 ndings.This result showed the remarkable genetic diversity of this pathogen in Alborz province, Iran.Increasing the number of isolates in future supplementary researches is likely to increase the observed polymorphism even higher than current values.The active cases of tuberculosis in this province are probably due to the infection's reactivation, and the recent transmission has a much lesser role.In the present study, a large percentage of isolates had copy numbers between 6 and 12, which the average number of copies of IS6110 marker was similar to the results of the study of Doroudchi et al. (2000) [33].In many studies worldwide [34][35][36], some M. tuberculosis isolates did not have the IS6110 component.In the present study, 4 isolates did not have a copy of IS6110 marker.In another study in Vietnam [37], RFLP-IS6110 and Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) techniques were used to assess the genetic characters of 2664 isolates.There was a great variety of isolates in both techniques resulting in the recurrence of latent tuberculosis infection in Vietnam.Additionally, many isolates from Vietnam did not contain any copy of the IS6110 marker, which was less resistant to antibiotics, especially streptomycin.Various researches reported that the IS6110 is the most common genetic marker for identifying and genetic identi cation of M. tuberculosis with a high resolution for DNA ngerprinting of isolated strains in the study population [38,39].Therefore, this fragment can be the basis for future molecular epidemiological studies on TB.The high genetic diversity of IS6110 has been reported in previous Iranian studies [40][41][42][43][44].The high genetic diversity of IS6110 in different strains of M. tuberculosis probably indicates that most people with different origins have been infected.As a result, the infection's reactivation has played a more signi cant role in spreading tuberculosis in Iran.Although the disease's activation due to patients' infection in the years before the onset of the disease has been introduced as an important factor explaining this epidemiological nding, the de nitive causes or reasons for this condition remain unknown.
Due to the limited number of samples studied in the present study, it is impossible to comment with certainty on mycobacteria other than the M. tuberculosis complex without the IS6110 sequence in Alborz province, Iran.So, in this case, more study is needed.Regarding the diagnosis of atypical mycobacteria in Iran, only one report was published in 2009, which showed that 43 out of 371 samples (11.5%) were diagnosed as non-tuberculous mycobacteria [45].

Conclusion
In conclusion, high genetic diversity was found amongst the M. tuberculosis strains isolated from patients in Alborz province, Iran, according to the IS6110-RFLP technique.A total of 17 strains were identi ed in RFLP.This molecular technique is acknowledged as a rst-line assay in the molecular epidemiology assessment of M. tuberculosis.Findings revealed that M. tuberculosis is the leading cause of tuberculosis in the region.Given the patients' age and the genetic diversity of the strains, the assumption of hemoplasia is raised.In the end, this research can be considered as a good background for future studies in the region, comparing the obtained results with each other and better analysis of the epidemiological situation of tuberculosis in Alborz province, Iran. Figures The relevant authorities issued ethical approval for this retrospective genotyping work at the Alborz University of Medical