Identification of Mycobacterium spp. Isolates From Suspected Tuberculosis Patients Using Molecular Methods in Zahedan, Iran

1. Introduction
Tuberculosis (TB) is a major health problem worldwide, affecting more than one million people every year. Mycobacterium tuberculosis is the causative agent of TB that is transmitted through airborne particles and develops as a latent infection in most cases. In some cases, the patients experience reactivated disease and dissemination to other organs [1]. Antibiotic resistance and co-infection with COVID-19 or HIV in immunocompromised patients are also considered among the most challenging health issues [1, 2]. According to the World Health Organization’s (WHO) 2023 global TB report, there was a very small increase compared with that in 2022. Most of the cases were observed in 30 countries, which accounted for 87% of the cases. Five countries accounted for 56% of the cases, including India (26%), Indonesia (10%), China (6.8%), the Philippines (6.8%), and Pakistan (6.3%) [3]. Sensitive, specific, and time-saving diagnostic tools are essential for controlling TB. However, the conventional tools for detecting TB are often inaccurate or time-consuming, particularly when distinguishing between active and latent TB infections. Polymerase chain reaction (PCR)-based methods allow for the accurate and rapid identification of mycobacterium species [4]. Iran is located in a critical region in the world due to its vicinity to Afghanistan, Pakistan, Iraq, and countries in the north of Iran. The prevalence of TB is not identical throughout the country. In 2019, the highest prevalence of TB was reported in the provinces of Sistan and Baluchestan and Golestan [5]. Limited data are available on the prevalence of members of the M. tuberculosis complex in the human population of Zahedan. Shakiba et al. [6] reported Mycobacterium bois from suspected patients with TB in Zahedan (unpublished data). The lack of industrial and semi-industrial livestock increases the possibility of bovine TB spreading in this city. Therefore, there is a possibility of bovine TB transmission to humans. Since Mycobacterium bovis is inherently resistant to pyrazinamide (the first-line drug for TB treatment), if M. bovis is not correctly identified, the patient may not receive appropriate and effective treatment. According to recent studies, infections caused by Nontubercolous mycobacteria (NTM) are increasing worldwide, especially in poor and developing countries. The clinical symptoms caused by this group of mycobacteria are indistinguishable from those caused by Mycobacterium tuberculosis. NTM are also resistant to anti-TB drugs, and have a different treatment protocol compared to MTBC. Therefore, the rapid and correct diagnosis of this group of mycobacteria is crucial. Thus, this study aimed to identify the species of Mycobacterium among patients with pulmonary TB in Zahedan. This will provide a clearer understanding of the epidemiological situation regarding the causative agents of this disease. 

2. Materials and Methods

2.1. Ethics approval
All humans involved in the study were handled in accordance with the ethics guidelines and protocols approved by the “Research Ethics Committees” of Razi Vaccine and Serum Research Institute, Iran.

2.2. Patient population and sample collection
In this study, we included 500 patients suspected of having TB who were referred to the regional TB reference laboratory in Zahedan, the center of Sistan and Baluchistan Province, Iran, from 2024 to 2025. A total of 500 specimens (53.9% women and 46.1% men), between the ages of 22-99 years, were studied. The patient population exhibited symptoms of pulmonary TB, including cough, phlegm, hemoptysis, and dyspnea. They were referred to the regional TB reference laboratory in Zahedan from 2024 to 2025, where sputum samples were collected from suspected cases.

2.3. Microbiological process
The samples were cultured as follows in Zahedan. Briefly, they were decontaminated with 3.5 M NaOH for 15 min. After that, the samples were centrifuged and neutralized with HCL (0.1 N). The sediments were cultured in two slope tubes of glycerinated and pyruvate Lowenstein–Jensen (LG) medium under Biosafety Level 3 (BSL3). They were then incubated at 37 °C for 8 weeks. Additionally, the sediment from each sample was stained using the cold Ziehl-Neelsen (ZN) technique. The culture tubes were monitored for bacterial growth. After growth, these samples were transferred to the laboratory of the Microbiology Department at Razi Vaccine & Serum Research Institute, Karaj, Iran.

2.4. DNA extraction
DNA was extracted according to van Soolingen’s method [7]. The samples were kept at -20 °C for further analyses.

2.5. PCR amplification
Briefly, primers were synthesized (Metabion, Germany) and used to amplify a 543 bp fragment of the 16S rRNA gene. Then, PCR targeting IS6110 was carried out for the amplification of the TB complex. The master mix without DNA template and M. tuberculosis strain C were used as negative and positive controls, respectively. Subsequently, region-of-difference (RD) typing (RD1/RD4/RD9/RD12) was performed to differentiate between members of the TB complex. M. tuberculosis strain C, M. bovis AN5, and M. bovis BCG strain were used as positive controls. Distilled water was also used as a negative control. The oxyR PCR-RFLP method was used for final confirmation. Primers and PCR conditions are listed in Table 1. 

2.6. PCR-RFLP of oxyR gene
PCR-RFLP of a 548-bp region of the oxyR gene was carried out according to Sreevatsan et al. [8]. The final volume was 12 µL, including PCR product (6 μL), AluI (2.5 μL) (Thermo Fisher Scientific, Lithuania), restriction enzyme buffer (2 μL), and distilled water (1.5 μL). The mixture was incubated at 37 °C overnight. The products were electrophoresed on an agarose gel for 100 min at 90 V. In this study, M. tuberculosis strain C, M. bovis AN5, and M. bovis BCG strain were used as positive controls, and water without DNA was used as the negative control. 

2.7. Identification of NTM species
Targeting the 16S rRNA, hsp65, and rpoB genes using PCR and sequence analysis was performed to identify NTM. M. tuberculosis strain C and master mix without DNA template were used as positive and negative controls, respectively. The outcomes of the nucleotide sequencing were analyzed using Chromas and Clustal X programs [9], and NCBI BLAST [10] was used to align the analyzed sequences.

2.8. Phylogenetic analysis of the 16S rRNA, hsp65, and rpoB genes
Phylogenetic trees of NTM species were constructed using the Neighbor-Joining method using Molecular Evolutionary Genetics Analysis (MEGA) XI software.

3. Result
Sixty-three isolates grew in two LJG and LJP media after two months. However, LJG was better than LJP. Acid-fast bacilli were observed in all the samples. All purified DNAs had a concentration greater than 100 ng/µL; the 260/280 ratio of all samples was between 1.8 and 2.2. The electrophoresis results of the purified DNAs showed that all DNAs had high quality without any breaks in the electrophoresis gel. In this study, the molecular identification of 63 isolates from pulmonary TB patients was investigated using PCR-16S rRNA. The typical 543 bp amplicons were observed in all isolates, which showed that all isolates belonged to the genus Mycobacterium. In the next stage, PCR targeting IS6110 was performed, and it was determined that 60 isolates out of 63 isolates (95.3%) were members of the MTBC. Based on the RD typing results, it was determined that all the TB complex group isolates were recognized as M. tuberculosis (Figure 1).

Digestion with Alul yielded three fragments of 79, 146, and 236 bp for DNA samples from M. bovis AN5 and M. bovis BCG strain. However, only one band at 230 bp was observed for the DNA sample from M. tuberculosis strain C and all of the isolates. PCR-RFLP on oxyR demonstrated that all of them were recognized as M. tuberculosis, which was confirmed by the result of PCR-RD typing. Three isolates were negative for PCR-IS6110. The nucleotide sequencing results based on 16S rRNA, hsp65, and rpoB genes showed that these three samples (4.7%) were positive for NTM. It should be noted that these three isolates were isolated from three patients with the following characteristics: Patient 1 was a 70-year-old male without underlying diseases. Patient 2 was a 57-year-old male with high blood pressure and diabetes. Patient 3 was a 49-year-old male with asthma. The outcomes of the nucleotide sequencing based on 16S rRNA showed similarity to M. kumamotonensis in one isolate (99.5%) and M. simiae in two isolates (99.6% and 99.8%, respectively). Gene sequence alignment results based on hsp65 showed similarity to M. senuense in one isolate (97.8%) and M. simiae in two isolates (99.3% and 99.1%, respectively). Finally, the outcomes of the nucleotide sequencing based on rpoB showed similarity to M. terrae in one isolate (93.3%) and M. simiae in two isolates (99.1% and 99.4%, respectively). Therefore, the NTM isolates obtained from this study can be divided into two groups in the phylogenetic trees of the 16S rRNA, hsp65, and rpoB genes: One isolate was categorized in the M. terrae complex group (MTC), and two isolates belonged to the M. simiae group (Figures 2, 3, and 4). 

4. Discussion
Different genotyping methods were used to differentiate members of the M. tuberculosis complex, which showed a wide range of discrimination power. In this study, the molecular identification of 63 isolates from pulmonary TB patients in Zahedan was investigated. For this purpose, PCR-16S rRNA was performed, which showed that all isolates belonged to the genus Mycobacterium. In the next stage, PCR-IS6110 was performed, and it was determined that 60 isolates out of 63 isolates (95.3%) were members of the MTBC. Based on the RD typing result, it was determined that all 60 isolates studied were M. tuberculosis, similar to the Torabi study [14]. Warren reported that the multiplex PCR method based on the genomic regions of difference enabled the rapid and accurate differentiation of the M. tuberculosis complex [13]. In this study, we used this method. Finally, the comparative genotyping technique of PCR-RFLP on the oxyR gene was performed. The result showed the belonging of 60 isolates to the M. tuberculosis species, and none of them were infected with M. bovis; this confirmed the result of RD typing. Sreevatsan reported that oxyR PCR-RFLP is useful for differentiating M. bovis and M. bovis BCG from other members of the MTBC [8]. The result of this study showed the good ability of this method for confirmation of RD typing, similar to the previous study [14]. In this city, 60 isolates were M. tuberculosis, and none of the 60 isolates were infected with M. bovis, similar to the Torabi study [12]. However, in the Dehghanpour study, M. bovis was identified in approximately 7.4% of cases in Zanjan Province [15]. Shakiba Mehr also conducted a study on pulmonary TB and reported infection with M. bovis in Zahedan [6] (unpublished data), contrary to this study. It is perhaps due to the improvement of the health level and the use of pasteurized milk and dairy products in this city. On the other hand, due to drought and the subsequent loss of traditional livestock farming, especially in rural areas, and the development of industrial livestock farming, the consumption of local milk and dairy products has decreased. This is very important because M. bovis is considered a health indicator of the community. Based on our results, the other three isolates (4.7%) were recognized as NTM species. Nowadays, non-tuberculous mycobacteria are increasing worldwide, especially in developing countries. Karami-Zarandi et al. (2019) reported 12% of positive cases as NTM [16]. Differentiation of non-tuberculous mycobacteria from the TB group is not possible by conventional methods; therefore, molecular-based methods play a very important role in the differentiation of non-tuberculous mycobacteria. For example, Leidsema reported that the sequencing of 16S rRNA, hsp65, and rpoB genes is a reliable method for identifying these bacteria [17]. In this study, we used the sequencing of 16S rRNA, hsp65, and rpoB genes for identifying non-tuberculous mycobacteria and phylogenetic analysis. Previous studies reported that the geographical distribution of the NTM group varies worldwide [18]. In this study, M. simiae was the most predominant among the NTM group, similar to the Shafipour study [19]. Another study showed that the M. avium complex was the most predominant among NTM species in Asia including, South Korea, Japan, and Taiwan [20]. Firoozeh et al. reported that the prevalence of non-tuberculous mycobacteria has increased in Iran. M. simiae was the most common slow-growing NTM group, similar to our study, while M. fortuitum, M. terrae, and M. gastri were the most common fast-growing NTM species, respectively [21]. In this study, one NTM isolate showed heterogeneity resulting from the sequencing of all three genes. Based on our result, sequencing using the 16S rRNA gene showed higher discriminatory power and percentage similarities (99.5%) than the hsp65 gene (97.8%) and the rpoB gene (93.3%). 16S rRNA gene produces a 1436 bp fragment, which is the largest amplified fragment of the 16S rRNA gene; therefore, the results of the 16S rRNA gene are more valid than those of the two other genes, similar to the Hassansoltan Solaghani study [22]. Lee et al. also reported that the sequencing of 16S rRNA and hsp65 genes is useful for the identification of the MTC, but the discriminatory power of the hsp65 gene is lower compared to 16S rRNA, similar to our study [23]. In this study, one NTM isolate was identified as M. kumamotonensis. Also, the similarity of this isolate to M. senuense and M. terrae was identified through the hsp65 and rpoB genes, respectively. According to studies, M. kumamotonensis and M. senuense are new species of the MTC [24]. M. kumamotonensis has a great similarity to members of the MTC based on the sequencing of 16S rRNA, hsp65, and rpoB genes [25]. M. senuense is also classified in the MTC based on phenotypic characterization [26]. Therefore, one of the three NTM isolates belonged to the MTC, and two of the three NTM isolates were identified as M. simiae by sequencing of 16S rRNA, hsp65, and rpoB genes. Two out of three NTM isolates were isolated from sputum samples of patients with underlying diseases. The risk of infection with NTM in underlying diseases, such as diabetes and immunodeficiency diseases, is greater than in patients without underlying disease [27], similar to our study. Wang reported a relationship between patients with diabetes mellitus and NTM, although there were no significant differences reported among age and gender groups [27]. According to the previous study and our study, M. simiae is the most common slow-growing nontuberculous mycobacterium among NTM species in Iran. Because M. simiae is the only niacin-positive NTM, similar to M. tuberculosis, and it can cause pulmonary infections in both immunocompromised and immunocompetent individuals [28], its differentiation from M. tuberculosis is very vital and will help in the rapid recovery of patients. Another NTM species, M. kumamotonensis was isolated from the sputum sample of a 70-year-old male patient without underlying disease. As mentioned, this bacterium is a new species of the MTC. So far, no report has been announced of its isolation in Iran. Based on our results, M. tuberculosis has a high prevalence in this city, similar to the previous study, due to its proximity to the borders of Afghanistan and Pakistan. Khammarnia reported a significant relationship between non-Iranian nationality and TB in Sistan and Balochistan Province [29]. In this study, 5% of disease cases belonged to the non-Iranian population; therefore, the possibility of disease transmission and circulating strains within non-Iranian populations should be considered one of the important contributing factors in this city. Other factors, such as malnutrition, smoking, tropical climate, population density, and poverty, should also be taken into account. Thus, the high prevalence of circulating M. tuberculosis in the human population of Zahedan highlights the need for screening programs to reduce the number of disease cases. Therefore, further studies on the human population to identify Mycobacterium strains are required in the future.

Acknowledgements
The authors are very grateful to the staff of Microbiology Department.

Compliance with ethical guidelines
This study was approved by the Research Ethics Committees of Razi Vaccine and Serum Research Institute, Karaj, Iran (Code: IR.RVSRI.REC.1403.006).

Data availability
The data that support the findings of this study are available upon request from the corresponding author.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Data acquisition, analysis, interpretation: Fatemeh Alimardani; Writing the original draft: All authors; Review and editing: Nader Mosavari, Soheila Moradi Bidhendi, Ali Reza Salimi Khorashad, and Lida Abdolmohammadi Khiav; Nader Mosavari, Soheila Moradi Bidhendi, Ali Reza Salimi Khorashad, and Lida Abdolmohammadi Khiav; Supervision: Nader Mosavari, Soheila Moradi Bidhendi, and Ali Reza Salimi Khorashad.

Conflict of interest
The authors declared no conflict of interest.

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