Antibiotic tolerance as a stepping stone to tuberculosis drug-resistance

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PROJECT SUMMARY Antibiotic tolerance describes differential susceptibility of an isogenic bacterial population to bactericidal antibiotics. Antibiotic tolerance contributes to two clinically relevant phenotypes: populations with increased antibiotic tolerance are harder to eradicate and need prolonged antibiotic therapy – a hallmark of tuberculosis; and antibiotic tolerance has been shown in some organisms to act as a stepping stone to bona fide drug- resistance. However, there are many forms of antibiotic tolerance – ranging from non-replicating persisters to growing phenotypic resister cells – and these most likely represent diverse physiological states, mediated by distinct molecular mechanisms. The relative prevalence of these forms of tolerance in clinical isolates of Mycobacterium tuberculosis – the cause of tuberculosis – is completely unknown, as is the relative contribution of distinct types of tolerance to in vivo antibiotic susceptibility and development of antibiotic resistance. We have developed assays that can measure the relative tolerant subpopulation caused by non-replicating persistence and growing phenotypic resistance to the first-line anti-TB antibiotic rifampicin. Using these assays, we will measure the relative prevalence of these distinct forms of tolerance in clinical isolates of M. tuberculosis representing all major phylogenetic groups. By transposon insertion mutagenesis and deep-sequencing (Tnseq), we will dissect the genetic requirements for tolerance both in vitro and in a novel murine model of tuberculosis infection, using B6.Sp140 -/- mice that recapitulate the hallmarks of infection such as necrotic granulomata of C3H/FeJ ‘Kramnik’ mice. We will also determine the contribution of these two forms of rifampicin tolerance to the development of rifampicin-resistance in select clinical isolates of M. tuberculosis, and perform Tnseq to identify genetic contributors to resistance. Together, these studies will further our understanding of the molecular mechanisms of distinct forms of rifampicin tolerance in clinical strains of tuberculosis, and their relevance to the development of genetic resistance.