Transmission dynamics of a Mycobacterium tuberculosis complex outbreak in an indigenous population in the Colombian Amazon Region

Whole genome sequencing (WGS) has become the main tool for studying the transmission of Mycobacterium tuberculosis complex (Mtbc) strains, however, the clonal expansion of one strain often limits its application for local Mtbc outbreaks. The use of an alternative reference genome and the inclusion of repetitive regions in the analysis have been suggested as measures to increase the resolution for outbreak investigations, but the added value has not yet been defined. Here, we leveraged short and long WGS read data of a previously reported Mtbc outbreak in the Colombian Amazon Region to analyze possible transmission chains among 74 patients in the indigenous setting of Puerto Nariño (March-October 2016). In total, 90.5% (67/74) of the patients were infected with one distinct Mtbc strain belonging to lineage 4.3.3. Employing a newly established reference genome from the outbreak strain and the inclusion of highly confident SNPs in repetitive genomic regions, e.g., PE/PPE gene family, increased the phylogenetic resolution as compared to a classical H37Rv reference mapping approach. Precisely, the number of differentiating SNPs increased from 890 to 1094 which resulted in a more granular transmission network as judged by an increasing number of individual nodes in a maximum parsimony tree, i.e., 9 versus 14 nodes. We also found in 29.9% (20/67) of the outbreak isolates, heterogenous alleles at phylogenetically informative sites suggest that these patients are infected with more than one clone. In conclusion, customized SNP calling thresholds and using a local reference genome for a mapping approach can improve the phylogenetic resolution in highly clonal Mtbc populations and help elucidate within host Mtbc diversity.