High genetic diversity of rifampicin-resistant Mycobacterium tuberculosis strains in Botswana

Abstract Background The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) strains remains a threat to tuberculosis (TB) prevention and care. Understanding the circulating strains' drug resistance profiles is crucial for effective TB control. This study aimed to describe the genetic diversity of rifampicin-resistant M.tb strains circulating in Botswana using whole genome sequencing (WGS). Methods This study included 202 stored M.tb isolates from people diagnosed with rifampicin-resistant TB (RR-TB) between January 2016 and June 2023. Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. Library preparation was performed using the Illumina DNA prep kit following the manufacturer's instructions. Sequencing was done on Illumina NextSeq2000. TBProfiler software was used to identify known M.tb lineages and drug resistance profiles. Statistical analyses were performed on STATA version 18. Results WGS analysis revealed multidrug resistance (57.9%: 95% CI; 50.7-64.8), Pre-XDR (16.8%, 95%CI:11.9-22.7), RR-TB (20.2%: 95%CI: 14.98-26.5), and HR-TB (0.5%, 95%CI; 0.01-2.7). We identified a high genetic diversity with three predominant lineages: lineage 4 (60.9%, 95% CI;53.8-67.7), lineage 1 (22.8%: 95%CI;17.2-29.2), and lineage 2 (13.9%, 95% CI:9.4-19.4). The most frequently observed drug resistance mutations for rifampicin, isoniazid, ethambutol, streptomycin, pyrazinamide, and fluoroquinolones were rpoB S450L (28.6%), katG S315T (60.5%), embA_c.-29_-28delCT, embB Q497R (31.7%), rrs_n.517C>T (47.1%), pncA_c.375_389delCGATGAGGTCGATGT (36.0%) and gyrA A90V (79.4%), respectively. No bedaquiline and delamanid resistance-associated mutations were detected.