DOI: 10.4103/ijmy.ijmy_103_26 ISSN: 2212-5531

High-resolution Genotyping of Mycobacterium tuberculosis with 16S Ribosomal Ribonucleic Acid Polymorphisms in Southwest Nigeria

Shola Lawrence Owolabi, Hassan Olalekan Anthony, Adeyemo Samson Adeolu
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Background:

Tuberculosis (TB) remains a leading cause of death worldwide, with Nigeria among the top five high-burden countries. This cross-sectional laboratory-based study performed high-resolution genotyping of Mycobacterium tuberculosis using 16S ribosomal ribonucleic acid (16S rRNA) polymorphisms in Southwest Nigeria.

Methods:

DNA was extracted from 50 archived clinical isolates obtained through convenience sampling from four tertiary hospitals in Oyo, Ogun, and Lagos States (January 2023–March 2024). The 16S rRNA gene was amplified using primers 27F/1525R (58°C annealing, 35 cycles) and sequenced directionally (Phred > 20). After quality filtering, 46 high-quality sequences (1439 bp) were analyzed by BLASTn, aligned with ClustalW, and a neighbor-joining tree (1000 bootstraps, Kimura 2-parameter [K2P + G4 model) was constructed. Genetic diversity, neutrality tests (Tajima’s D, Fu’s Fs), and mismatch distribution (sum of squared deviations [SSD], raggedness) were computed using DnaSP v6.12.03 and Arlequin v3.5.2.2. GC content and nucleotide frequencies were calculated in R v4.5.3.

Results:

All 50 isolates were confirmed as M. tuberculosis (99.86%–100% identity, 99%–100% coverage, E = 0). Four sequences were excluded, leaving 46 with 7 polymorphic sites. Eighteen haplotypes were identified; Haplotype I was most frequent (12 isolates). Mean K2P distance was 0.122% (0%–0.484%). Haplotype diversity (Hd) was 0.876 ± 0.034 (95% confidence interval [CI]: 0.808–0.944), nucleotide diversity 0.00124 ± 0.00019. Tajima’s D was − 1.428 ( P = 0.108), Fu’s Fs − 3.842 ( P = 0.072). Mismatch distribution fit a sudden expansion model (SSD = 0.0032, P = 0.41; raggedness = 0.021, P = 0.68). The phylogenetic tree showed a single cluster with 100% bootstrap support for 45/46 isolates. Mean GC content was 58.1% ( A = 0.219, T = 0.200, G = 0.341, C = 0.240). No nontuberculous mycobacteria or drug resistance-associated mutations were detected.

Conclusions:

16S rRNA sequencing reliably confirms M. tuberculosis in Southwest Nigeria and reveals high Hd despite extremely low nucleotide diversity, consistent with recent population expansion. However, this method cannot detect drug resistance mutations, necessitating integration with whole-genome sequencing or targeted resistance gene analysis for effective TB surveillance.

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