Digitally Barcoding Mycobacterium tuberculosis Reveals In Vivo Infection Dynamics in the Macaque Model of Tuberculosis

Abstract

Infection with Mycobacterium tuberculosis causes a spectrum of outcomes; the majority of individuals contain but do not eliminate the infection, while a small subset present with primary active tuberculosis (TB) disease. This variability in infection outcomes is recapitulated at the granuloma level within each host, such that some sites of infection can be fully cleared while others progress. Understanding the spectrum of TB outcomes requires new tools to deconstruct the mechanisms underlying differences in granuloma fate. Here, we use novel genome-encoded barcodes to uniquely tag individual M. tuberculosis bacilli, enabling us to quantitatively track the trajectory of each infecting bacterium in a macaque model of TB. We also introduce a robust bioinformatics pipeline capable of identifying and counting barcode sequences within complex mixtures and at various read depths. By coupling this tagging strategy with serial positron emission tomography coregistered with computed tomography (PET/CT) imaging of lung pathology in macaques, we define a lesional map of M. tuberculosis infection dynamics. We find that there is no significant infection bottleneck, but there are significant constraints on productive bacterial trafficking out of primary granulomas. Our findings validate our barcoding approach and demonstrate its utility in probing lesion-specific biology and dissemination. This novel technology has the potential to greatly enhance our understanding of local dynamics in tuberculosis.

Heterogeneity in tuberculosis

“…There are recent studies 40,51,69 that support this paradigm. In one study, Coleman et al 40 used serial FDG PET–CT imaging to evaluate granuloma dynamics in macaques that subsequently developed active disease or LTBI following a low-dose M. tuberculosis infection 40 .…”

Rhesus Macaques Are More Susceptible to Progressive Tuberculosis than Cynomolgus Macaques: a Quantitative Comparison

“…Dissemination of infection can be defined as the formation of new granulomas after initial establishment of infection, and early dissemination is an indicator of eventual development of active disease (31). Dissemination can be monitored by PET-CT (31,33). Here we assessed the formation of new granulomas that appeared between 4 and 8 weeks postinfection in each macaque ( Fig.…”

“…Serial PET-CT imaging using [ 18 F]fluorodeoxyglucose (FDG) as a PET probe is a relatively noninvasive approach that provides insight into the infection process over time. We have used this imaging technology in macaques to assess early events in M. tuberculosis infection, reactivation kinetics, responses to drug treatment and vaccination, and, where possible, compared with human imaging data (16,26,29,30,32,33). Here, we performed serial PET-CT imaging on each macaque in this study to assess similarities and differences among species during infection.…”

“…We use serial positron emission tomography and computed tomography (PET-CT), which combines a functional map (PET) of the thoracic cavity, depending on the PET probe used, with a spatial map of anatomic structures (CT) (29). Using this imaging strategy in our macaques, we can assess drug efficacy in vivo (29,30) and study critical events during infection (26,(31)(32)(33). Comparative genomic studies have been performed for rhesus and cynomolgus macaques and showed that, while abundant heterogeneity exists in each population, there is an extraordinarily high degree of similarity in both species to human genes (34), and there is great similarity in the T cell responses to M. tuberculosis epitopes between both cynomolgus and rhesus macaques and humans (35).…”

Toward Tuberculosis Vaccine Development: Recommendations for Nonhuman Primate Study Design

“…To avoid variability introduced by different sources and to maximize standardization, a large stock of the Erdman strain has been made for the CTVD NHP Research Community and wider research use, and will be made available through BEI Resources. This strain contains digital barcodes (9), enabling quantification of the number of bacteria that establish infection. This allows for the confirmation of the challenge dose and can be used as a surrogate for prevention of infection; barcodes also allow for assessment of infection dynamics and bottlenecks (as some granulomas are more permissive for mycobacterial growth than others).…”