**5. Conclusions**

Our study shows that there is second-line drug resistance and the majority of cases had *gyrA* A90V, *rrs* A1401G mutation. Our results show that monitoring and further investigations with more discriminatory methods are required to determine whether these strains are transmitted or if second-line drug resistance is acquired during treatment.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-0817/8/4/208/s1, Table S1. Clinical characteristics and treatment outcomes of the drug resistant isolates in the study.

**Author Contributions:** Conceptualization: S.G., methodology: E.M.S., R.M.W., T.M., S.G., S.D.L., B.D.; formal analysis: T.M., P.M., S.M., E.M.S., R.M.W., S.D.L.; software: S.G.; validation: E.M.S., S.D.L., B.D., P.M.; visualization: E.M.S., S.D.L., B.D., T.M.; investigation: E.M.S., S.D.L., B.D., T.M.; resources: S.G., R.M.W.; data curation: S.G., B.K., G.R.-P., T.T.T., M.M.; writing original draft: T.M., S.D.L., writing review & editing: T.M., E.M.S., B.D., R.M.W., S.D.L., S.G., S.M., I.K., T.I., P.M., L.M., M.M., G.R.-P., T.T., B.K.; supervision: I.K., S.G.; project administration: S.G., T.M., I.K., R.M.W.; funding acquisition: S.G. All authors read and approved the final manuscript.

**Funding:** This work was supported through the Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE), a DELTAS Africa Initiative [grant # DEL-15-006]. The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)'s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa's Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust [grant # 107752/Z/15/Z] and the UK government. The views expressed in this publication are those of the author(s) and not necessarily those AAS, NEPAD Agency, Wellcome Trust or the UK government.

**Acknowledgments:** We would like to thank the National Tuberculosis Reference Laboratory and Ministry of Health and Wellness Botswana for providing clinical *M.tb* isolates for this study.

**Conflicts of Interest:** The authors declare no conflicts of interest.
