For this study, 64 clinical isolates of pulmonary origin (sputum and bronchoalveolar lavage [BAL]), were collected from suspected tuberculosis patients who referred to regional TB reference laboratories in Khuzestan province of Iran, from September 2019 to December 2020. The preliminary proposal of the study was approved by the Institutional Ethics and Review Board (Code: IR.AJUMS.REC. 1398. 013) of the Ahvaz Jundishapur University of Medical Sciences, Iran, and the necessary permission for sample collection was granted.

The isolates were cultured on Lowenstein-Jensen (LJ) medium, and incubated at 37 °C. The LJ tubes were examined daily for 30 days to obtain observable colonies. For phenotypic identification of the NTM isolates, standard conventional and biochemical tests, including growth rate, pigment production, acid-fast staining, urease, iron uptake, Tween 80 hydrolysis, heat-stable catalase (pH 7, 68 °C), arylsulfatase, niacin test, and tellurite reduction, according to the standard guidelines provided by the Centers for Disease Control and Prevention (CDC) were performed (Kent 1985). All materials for biochemical tests were purchased from Sigma-Aldrich Corporation (Sigma, St. Louis, MO, USA).

For extraction of DNA, the simple boiling method was used as described earlier (Blackwood et al. 2000). In brief, a loopful of bacteria grown on the LJ medium was suspended in one ml of sterile distilled H2O containing 5 mm glass beads and vortexed for 2 min for mechanical breakage, and then boiled for 10 min. The quality and quantity of each extracted DNA were checked and measured with the aid of a NanoDrop (ND-1000, Thermo Scientific, DE, USA). The supernatant comprising DNA was kept at −20 °C until use.

For NTM species identification, rpoB gene-based PCR was applied. Amplification was performed as a 50 µl reaction mixture, comprising 10 × PCR buffer, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphate (dNTPs); 0.2 µM of each primer of mycoF and mycoR primers, as listed in Table 1 (Adekambi et al. 2003), 2.5 U Taq polymerase (AMPLIQON, Denmark), and 5 µl of template DNA (10 ng). The PCR program was carried out in a thermocycler (Eppendorf 6333, Hamburg, Germany) as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 62 °C for 45 s, and extension at 72 °C for 40 s, with a final extension at 72 °C for 5 min.

About 620-bp and 640-bp fragments of the argH and cya genes were amplified using sets of primers of argH (argininosuccinate lyase), and cya (adenylate cyclase), as shown in Table 1 (Macheras et al. 2009). A reaction mixture of 50 µl containing 5 µl of 10 × PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM of each primer, 2.5 U of Taq polymerase, and 5 µl of template DNA (10 ng) was prepared. Amplification was done with the following cycling program: initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 40 s, annealing at 59 °C (argH)/ 60 °C (cya) for 45 s, and extension at 72 °C for 40 s, with a final extension at 72 °C for 5 min.

The PCR products were separated by electrophoresis on a 1.2% agarose gel and stained with the SYBR® Safe DNA Gel Stain (Thermo Fisher Scientific), and by using a gel documentation system (Uvidoc, Jencons Scientific Inc., Cambridge, UK), the DNA bands were visualized (Lee et al. 2012). The size of PCR products were determined using a 100-bp molecular size marker.

The amplified PCR products for each isolate, were purified with the DNA Extraction and purification Kit (Bio Basic, Canada), according to the manufacturer’s instructions. An ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was utilized to determine the sequences of the products.

The generated sequences of rpoB, argH, and cya for tested isolates were separately analyzed. The GeneBank™ database was employed to analyze and blast the data at the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The result of each sequence of NTM isolates was aligned, and a comparison was made with several existing sequences of mycobacterial species in this database. The sequences were aligned using the Clustal W algorithm in the MEGA 11 software. By using the neighbor-joining method, phylogenetic trees were constructed and verified by the maximum likelihood method with 1,000 bootstrap replications. The phylogenetic tree was constructed with MTB and Nocardia asteroids as outgroups (Tamura et al. 2021; Saitou and Nei 1987).