Description of bacterial RNA transcripts detected in Mycobacterium tuberculosis - infected cells from peripheral human granulomas.

Mycobacterium tuberculosis (Mtb) remains a global human health threat. However, understanding effects of the microbe on cellular interactions in infected tissue has been hindered by inability to discriminate between infected versus un-infected cells. We included the H37Rv Mtb reference genome when assembling scRNA seq libraries from fine needle aspirate samples of peripheral nodal TB patients. Using the 10X Genomics Cell Ranger tool to align sequencing reads, we consistently detected bacterial small and large ribosomal subunit RNA sequences. We interpret Mtb reads associated with a cell's UMI and transcriptome to indicate infection of that individual host cell. This provides a new window into the status of infected cells in the context of the bystander cells in the infected tissue. We investigated these Mtb transcripts to explore their clinical utility. The Mtb transcripts showed frequent sequence variation from the reference genome, with greater than 90% of the rrs or rrl reads from many clinical samples having at least one sequence difference. The highly conserved nature of the rrs and rrl gene sequences limited the ability to assign bacterial lineage based solely transcriptome analysis. However, rapid improvements in sequencing depth may soon allow transcriptome analysis of infecting microbes and improved certainty regarding their lineage, drug resistance, and virulence factors.