Novel rrs mutations in second-line injectable drug-resistant clinical isolates of Mycobacterium tuberculosis from the Punjab province of Pakistan

Phenotypic drug susceptibility testing is the most common approach to assess drug-resistant isolates; however, molecular methods of drug susceptibility testing are fast, accurate hence, offer less time for transmission during the diagnosis period. As data on the molecular methods regarding injectable drug resistance in the Punjab province of Pakistan is limited, therefore in this study, we aimed to analyze the mutations in the rrs gene behind second-line injectable drug resistance. Mycobacterium tuberculosis isolates were collected from the sputum of 5362 TB suspects. The strains confirmed for resistant to injectable drugs through drug susceptibility testing were further proceeded. The 1537bp rrs gene was amplified with the help of three sets of primers with overlapping regions and DNA sequencing was performed. Obtained sequences were aligned with reference sequence to find mutations. RFLP-PCR method was also optimized for rapid detection of a common (143bp and 205bp) rrs gene mutation. Among 172 rifampicin resistance isolates, 163(95%) were resistant to both rifampicin and isoniazid, and 9 (5%) were resistant to only rifampicin. Among the resistant samples, 12 (6.9%) samples were resistant to all three injectable drugs. Sixty out of 172 (34.9%) samples showed resistance to at least one drug and 10 (5.8%) samples were resistant to two drugs among the 3 s-line drugs. Sequencing analysis showed novel mutations in different samples at positions 443InsC, 19DelT, 29G>A, 48C>T, 50G>C, 265InsT, 423T>G, 476InsA, 446A>G, 563DelA, 695G>A, 805DelA, 900G>A, and 1510A>G, while some already reported mutations at position 1401A>G, 1402A>G, and 1484G>T were also observed. MIC of novel rrs gene mutations in KAN, CAP, and AMK resistant isolates were found between 2.5 mg/L-3.05 mg/L, 2.08 mg/L-3.0 mg/L, and 2.1 mg/L-2.7 mg/L respectively. Novel mutations in the rrs gene reported in this study may confer second-line injectable drugs resistance in Mtb. This molecular insight into second-line injectable drug resistance is useful for better management of resistance Mtb in high burden countries.