CRISPR-Based Typing and Population Structure Analysis of Multidrug-Resistant Mycobacterium tuberculosis Lineages in Northwest Nigeria: Toward a National Strain Database

Understanding the population structure and transmission dynamics of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is essential for designing effective TB control strategies, yet a CRISPR-based molecular typing database for Nigerian MDR-TB strains has not been established. This study applied spoligotyping, whole-genome sequencing, and CRISPR-spacer array analysis to characterize the population structure of MDR-TB strains from across the North West zone of Nigeria, encompassing Kano, Kaduna, Sokoto, Zamfara, Kebbi, Katsina, and Jigawa States. One hundred and sixty MDR-TB isolates from National Reference Laboratory Abuja-confirmed resistant cases over a 24-month period were analyzed. Spoligotyping was performed using standard membrane hybridization. Whole-genome sequencing was conducted on an Illumina platform, with phylogenetic analysis using RAxML and BEAST2 for molecular clock dating. CRISPR-spacer arrays were manually curated and compared. Population structure analysis used STRUCTURE software and principal component analysis. Seven major spoligotype clusters were identified, with the Euro-American lineage 4 dominant at 58.1 percent, followed by lineage 2 Beijing at 26.9 percent. Molecular clock analysis estimated the lineage 2 introduction into the North West at approximately 30 years before present. Drug resistance mutations in rpoB, katG, and rrs loci were linked to specific spoligotype families. The CRISPR spacer database established from this study represents Nigeria's first regional MDR-TB molecular typing resource and enables real-time strain discrimination for outbreak investigation and transmission tracing. Keywords: MDR-TB, CRISPR typing, spoligotyping, population structure, Northwest Nigeria.