Heterogeneous Streptomycin Resistance Level Among Mycobacterium tuberculosis Strains From the Same Transmission Cluster

12 páginas, 5 figuras.Widespread and frequent resistance to the second-line tuberculosis (TB) medicine streptomycin, suggests ongoing transmission of low fitness cost streptomycin resistance mutations. To investigate this hypothesis, we studied a cohort of 681 individuals from a TB epidemic in Portugal. Whole-genome sequencing (WGS) analyses were combined with phenotypic growth studies in culture media and in mouse bone marrow derived macrophages. Streptomycin resistance was the most frequent resistance in the cohort accounting for 82.7% (n = 67) of the resistant Mycobacterium tuberculosis isolates. WGS of 149 clinical isolates identified 13 transmission clusters, including three clusters containing only streptomycin resistant isolates. The biggest cluster was formed by eight streptomycin resistant isolates with a maximum of five pairwise single nucleotide polymorphisms of difference. Interestingly, despite their genetic similarity, these isolates displayed different resistance levels to streptomycin, as measured both in culture media and in infected mouse bone marrow derived macrophages. The genetic bases underlying this phenotype are a combination of mutations in gid and other genes. This study suggests that specific streptomycin resistance mutations were transmitted in the cohort, with the resistant isolates evolving at the cluster level to allow low-to-high streptomycin resistance levels without a significative fitness cost. This is relevant not only to better understand transmission of streptomycin resistance in a clinical setting dominated by Lineage 4 M. tuberculosis infections, but mainly because it opens new prospects for the investigation of selection and spread of drug resistance in general.This work has been funded by of the projects NORTE-01-0145- FEDER-000039, NORTE-01-0145-FEDER-000013, and NORTE01-0145-FEDER-00002, supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). It was also funded through the Foundation for Science and Technology (FCT) – project FCT IF/00474/2014, UIDB/50026/2020 and UIDP/50026/2020. DR and CM were funded by FCT Ph.D. scholarships (grant numbers SFRH/BD/135422/2017 and SFRH/BD/132797/2017, respectively). DR was partially funded by the PGCD – Graduate Program Science for Development. MS is supported by FCT through the Estimulo ao Emprego Científico.Peer reviewe