High-throughput phenogenotypingof Mycobacteria tuberculosisclinical strains reveals bacterial determinants of treatment outcomes

Abstract

Background: Combatting the tuberculosis (TB) epidemic caused by Mycobacterium tuberculosis (Mtb) necessitates a better understanding of the factors contributing to patient clinical outcomes and transmission. While host and environmental factors have been evaluated, the impact of Mtb genetic background and phenotypic diversity is underexplored. Previous work has made associations between Mtb genetic lineages and some clinical and epidemiological features, but the bacterial traits underlying these connections are largely unknown. Methods: We developed a high-throughput functional genomics platform for defining genotype-phenotype relationships across a panel of Mtb clinical isolates. These phenotypic fitness profiles function as intermediate traits which can be linked to Mtb genetic variants and associated with clinical and epidemiological outcomes. We applied this approach to a collection of 158 Mtb strains from a study of Mtb transmission in Ho Chi Minh City, Vietnam. Mtb strains were genetically tagged in multiplicate, which allowed us to pool the strains and assess in vitro competitive fitness using deep sequencing across a set of 14 host-relevant antibiotic and metabolic conditions. Phylogenetic and monogenic associations with these intermediate traits were identified and then associated with clinical outcomes. Findings: Mtb clinical strains have a broad range of growth and drug response dynamics that can be clustered by their phylogenetic relationships. We identified novel monogenic associations with Mtb fitness in various metabolic and antibiotic conditions. Among these, we find that mutations in Rv1339, a phosphodiesterase, which were identified through their association with slow growth in glycerol, are further associated with treatment failure. We also identify a previously uncharacterized subclade of Lineage 1 strains (L1.1.1.1) that is phenotypically distinguished by slow growth under most antibiotic and metabolic stress conditions in vitro. This clade is associated with cavitary disease, treatment failure, and demonstrates increased transmission potential. Interpretation: High-throughput phenogenotyping of Mtb clinical strains enabled bacterial intermediate trait identification that can provide a mechanistic link between Mtb genetic variation and patient clinical outcomes. Mtb strains associated with cavitary disease, treatment failure, and transmission potential display intermediate phenotypes distinguished by slow growth under various antibiotic and metabolic conditions. These data suggest that Mtb growth regulation is an adaptive advantage for host bacterial success in human populations, in at least some circumstances. These data further suggest markers for the underlying bacterial processes that govern these clinical outcomes.

Ongoing evolution of the Mycobacterium tuberculosis lactate dehydrogenase reveals the pleiotropic effects of bacterial adaption to host pressure

“…[ 30 ]. Our group transformed a subset of these strains with an L5-integrating plasmid carrying a kanamycin resistance cassette and a unique genetic barcode for a separate analysis [ 62 , 63 ]. These barcoded clinical strains were used to conduct the growth curves in the current analysis.…”

“…The clinical strains utilized in this study are part of a larger collection of 1,635 samples originally isolated from patient sputum in Ho Chi Minh City, Vietnam and published by Holt et al [30]. Our group transformed a subset of these strains with an L5-integrating plasmid carrying a kanamycin resistance cassette and a unique genetic barcode for a separate analysis [62,63]. These barcoded clinical strains were used to conduct the growth curves in the current analysis.…”