Population structure and transmission dynamics of multidrug resistant Mycobacterium tuberculosis strains in Sierra Leone Harriet NA Blankson1, 2, Christian Utpatel1, Rashidatu Kamara3, Ivan Barilar1, Sönke Andres4, Tobias Dallenga5, Katharina Kranzer6, Florian Maurer4, Lynda Foray3, Stefan Niemann1, 2 1Molecular and Experimental Mycobacteriology, Research Center Borstel Leibniz Lung Center, Borstel, Germany 2German Center for Infection Research, Partner Site Hamburg-Lübeck-Borstel-Reims, Borstel, Germany 3National Leprosy and Tuberculosis Control Programme Sierra Leone, Freetown, Sierra Leone 4National Reference Center for Tuberculosis, Research Center Borstel Leibniz Lung Center, Borstel, Germany 5Cellular Microbiology, Research Center Borstel Leibniz Lung Center, Borstel, Germany 6Clinical Research Department, London School of Hygiene and Tropical Medicine, London, United Kingdom Introduction: Drug resistant Mycobacterium tuberculosis complex (Mtbc) strains represent an increasing problem for global tuberculosis control. Particular important are rifampicin/multidrug resistant (RR/MDR) strains, that represent approx. 2.5% of new TB cases in Sierra Leone. As treatment of MDR TB cases is less effective, much longer and more expensive compared to susceptible TB cases, the control of the MDR TB epidemic is of outmost importance. Therefore, it is vital to understand the determinants of the MDR TB epidemic on a regional level to develop effective control measures. Here, we performed whole genome sequencing (WGS) to determine the population structure, resistance levels and transmission patterns of MDR Mtbc strains from Sierra Leone. Methods: A total of 326 culture positive samples from 2003 to 2004 (85 samples, retreatment cases) and from 2016 to 2020 (241samples) were subjected to WGS. RR/MDR strains were selected based on the genotypic drug susceptibility test (gDST). WGS RR/MDR strains were then used for phylogenetic strain classification, and a cluster analysis based on a maximum of ≤12 single nucleotide polymorphisms (SNPs) distances between two strains. Results: There were 247 RR/MDR strains and they were classified into seven major lineages. Most prevalent were lineage 4 strains 61% (151), followed by lineage 6 strains 21% (51) and lineage 2 strains 10% (25). Overall, 19% (47) of the strains were RR, 81% (199) were MDR, and 0.004% (1) was preXDR. 78% (118) and 86% (44) of the lineage 4 and 6 strains were MDR, while 100% (n=10 & 25) of the Lineage 1and 2 strains were MDR. The most prevalent resistance conferring mutations were rpoB S450L and katG S315T for rifampicin and isoniazid respectively. The overall clustering rate was 30%, a total of 106 strains were grouped in 32 clusters, ranging in size from 2 to 16 strains. The largest cluster among MDR strains was formed by 2.2.1 Beijing Ancestral 3 strains, and the second largest cluster by strains of the 4.8 mainly T sublineage. Conclusions: MDR was mostly found in strains of endemic lineages while larger transmission was found in the Beijing ancestral 3 strains. Although MDR tuberculosis is developed, resistance to second line injectables and fluoroquinolone was scarce, an indication that most of the MDR cases in the country could be treated based on the WHO treatment guidelines.