Mycobacterium tuberculosis Sub-Lineage 4.2.2/SIT149 as Dominant Drug-Resistant Clade in Northwest Ethiopia 2020-2022: In-silico Whole-Genome Sequence Analysis

Introduction: Drug resistance (DR) in Mycobacterium tuberculosis complex (MTBC) is mainly associated with certain lineages and varies across regions and countries. The Beijing genotype is the leading resistant lineage in Asia and western countries. M. tuberculosis (Mtb) (sub) lineages responsible for most drug resistance in Ethiopia are not well described. Hence, this study aimed to identify the leading drug resistance sub-lineages and characterize first-line anti-tuberculosis drug resistance-associated single nucleotide polymorphisms (SNPs). Methods: A facility-based cross-sectional study was conducted in 2020-2022 among new and presumptive multidrug resistant-TB (MDR-TB) cases in Northwest Ethiopia. Whole-genome sequencing (WGS) was performed on 161 isolates using Illumina NovaSeq 6000 technology. The SNP mutations associated with drug resistance were identified using MtbSeq and TB profiler Bioinformatics softwares. Results: Of the 146 Mtb isolates that were successfully genotyped, 20 (13.7%) harbored one or more resistance-associated SNPs. L4.2.2.ETH was the leading drug-resistant sub-lineage, accounting for 10/20 (50%) of the resistant Mtb. MDR-TB isolates showed extensive mutations against first-line anti-TB drugs. Ser450Leu/(tcg/tTg) for Rifampicin (RIF), Ser315Thr/(agc/aCc) for Isoniazid (INH), Met306Ile/(atg/atA(C)) for Ethambutol (EMB), and Gly69Asp for Streptomycin (STR) were the leading resistance associated mutations which accounted for 56.5%, 89.5%, 47%, and 29.4%, respectively. The presence of both clustered and non-clustered drug resistance (DR) isolates indicated that the epidemics is driven by both new DR development and acquired resistance. Conclusion: The high prevalence of drug-resistant TB due to geographically restricted sub-lineages (L4.2.2.ETH) indicates the ongoing local micro epidemics. The Mtb drug resistance surveillance system must be improved. Further evolutionary analysis of L4.2.2.ETH strain is highly desirable to understand evolutionary forces that leads L4.2.2.ETH in to high level DR and transmissible sub-lineage.Sample collection was funded by the Institute of Biotechnology, Bahir Dar University, through the Endalkachew Nibret Mega Project. The Mtb culture and identification-related laboratory supply was supported by the Amhara Public Health Institute, Bahir Dar, Ethiopia. Whole-genome sequencing was performed with the great support of National Center of Microbiology, Institute of Carlos III, Madrid, Spain. The International Federation for Clinical Chemistry (IFCC) provided financial support to Daniel Mekonnen through the IFCC Professional Exchange Program (PEP) for three months stay in Madrid Spain for conducting the WGS analysis.S