Trends, Patterns, and Demographic Dynamics of Drug Resistance in <i>Mycobacterium tuberculosis</i> and Nontuberculous: A Multi-Year Analysis at the National Tuberculosis Reference Laboratory

Background and Objective: Drug-resistant (DR) mycobacterial infections present escalating threats in Iran, where facility-specific transmission dynamics and demographic disparities remain poorly characterized. This study evaluated the epidemiology of DR Mycobacterium tuberculosis and pulmonary nontuberculous mycobacterial disease over a 9-year period (2016–2024). Materials and Methods: This retrospective study analyzed 21,700 molecular and culture-confirmed mycobacterial samples. Species identification used line probe assays and Xpert Mycobacterium tuberculosis /rifampin. Drug susceptibility testing followed World Health Organization standards. Statistical analyses identified associations between resistance patterns, hospital sections, gender, and specimen types. Results: MDR-TB prevalence surged from 0.35% (2017) to 3.27% (2022), peaking during pandemic disruptions. M. simiae dominated nontuberculous mycobacteria (NTM) resistance (55.63% of resistant isolates), with significant increases in 2024 (7.91%). Airborne infection isolation rooms (AIIRs) paradoxically harbored 18.22% of Multidrug-resistant tuberculosis (MDR-TB)isolates ( p &lt; 0.0001), while pulmonary medicine units contained zoonotic M. bovis reservoirs (0.27% MDR prevalence). Male patients carried higher burdens of MDR-TB (26.01% vs. 12.45%, p = 0.005) and M. kansasii (2.05% vs. 0.53%, p = 0.012). Diagnostic challenges included 38.14% smear-negative M. abscessus and gastrointestinal NTMs ( M. genavense 66.66%). Sample type analysis revealed M. fortuitum in 25.0% of abscesses ( p &lt; 0.05) and M. chelonae in 5.55% of synovial fluid (SF) specimens. Conclusion: Iran faces converging epidemics of MDR-TB and climate-adapted NTMs concentrated in hospital hotspots, with significant gender disparities. Precision interventions targeting AIIR protocols, water safety regulations, and gender-specific screening are urgently needed.