Mutation Analysis of <i>rrs</i> Gene and Its Association with Second-Line Drug Resistance in <i>Mycobacterium tuberculosis</i> Isolates from Khyber Pakhtunkhwa, Pakistan

Background: The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) poses a significant threat to global TB control efforts. Resistance to second-line injectable drugs (SLIDs) in Mycobacterium tuberculosis is primarily associated with mutations in the rrs gene. Understanding the molecular basis of resistance is crucial for improving diagnostic strategies and treatment outcomes in high-burden settings. Methods: A total of 147 sputum samples were collected from confirmed TB patients at Hayatabad Medical Complex, Peshawar, Pakistan. Samples were processed using standardized protocols including fluorescent microscopy, culture on BACTEC MGIT 960 system, and drug susceptibility testing against first-line (isoniazid, rifampicin, ethambutol, streptomycin) and second-line (amikacin, kanamycin, capreomycin) drugs. DNA extraction was performed using the GenoLyse Kit, followed by line probe assays (MTBDRplus and MTBDRsl) to detect mutations in the rrs gene. Statistical analysis was performed to correlate mutation patterns with phenotypic resistance. Results: Among 147 M. tuberculosis isolates, 141 (95.9%) were from pulmonary and 6 (4.1%) from extrapulmonary specimens. The highest infection rate (31.97%) was observed in patients aged >45 years. Phenotypic drug susceptibility testing revealed that 126 (85.7%) isolates were MDR, 15 (10.2%) were resistant (non-MDR), 3 (2.0%) were XDR, and 3 (2.0%) were fully sensitive. Mutations in the rrs gene were detected in 18 (12.2%) isolates, comprising two distinct mutation types: A1401G substitution in 7 (38.9%) isolates and G1484T substitution in 11 (61.1%) isolates. Strong correlation was observed between rrs mutations and resistance to second-line injectable drugs. Isolates harboring rrs mutations exhibited 55-100% resistance to amikacin, kanamycin, and capreomycin, whereas non-mutated strains showed 99% susceptibility to these agents. G1484T mutations demonstrated stronger positive correlation with multidrug resistance compared to A1401G. Conclusions: This study confirms that rrs gene mutations, particularly A1401G and G1484T, are significantly associated with resistance to second-line injectable drugs in clinical M. tuberculosis isolates from Pakistan. The strong correlation between genotypic markers and phenotypic resistance supports the utility of molecular assays for rapid detection of SLID resistance. These findings have important implications for TB control programs in high-burden regions and emphasize the need for routine molecular surveillance to guide appropriate treatment regimens.