Direct Transmission of Within-Host Mycobacterium Tuberculosis Diversity to Secondary Cases Can Lead to Variable Between-Host Heterogeneity Without De Novo Mutation: A Genomic Investigation

Abstract

Background: Whole genome sequencing (WGS) has enabled the development of new approaches to track Mycobacterium tuberculosis (Mtb) transmission between tuberculosis (TB) cases but the utility may be challenged by the discovery that Mtb diversifies within hosts. Nevertheless, there is limited data on the mechanisms of within-host evolution. Methods: We profiled a well-documented Mtb transmission cluster with three pulmonary TB cases to investigate within-host evolution and describe its impact on recent transmission estimates. We used deep sequencing to track minority allele frequencies (<0·5% abundance) during transmission and standard treatment. Findings: Pre-treatment (n=3) and serial samples collected over two-months of chemotherapy (n=16) from all three cases were analyzed. Consistent with the epidemiological data, zero high confidence fixed SNPs separated all genomes. However, we identified five subclones in the index case. We recovered all subclones in one secondary case, while only the dominant clone was observed in the other secondary case. By tracking the frequency of these heterogeneous alleles over the two-month therapy, we observed distinct signatures of drift and negative selection, but limited evidence for de novo mutations, even under drug pressure. Interpretation: Within-host diversity is composed of minority alleles selected against during transmission and disease latency. Incorporating data on heterogeneous alleles may refine our understanding of Mtb transmission dynamics. However, more evidence is needed on the role of transmission bottleneck on observed heterogeneity between cases. Funding: This research was supported in part by a grant from the National Institute of Health, National Center for Advancement of Translational Science sponsored University of Florida Career Development Grant (5KL2TROO1429-04) to MNS. Declaration of Interest: We declare no competing interests. Ethical Approval: Data were collected as part of public health practice and shared anonymously after cases completed therapy, thus informed consent was not required. The use of the data for this study was approved by the Institutional Review Boards (IRB) of the University of Florida and the Florida Department of Health, respectively.