Extreme drug tolerance of Mycobacterium tuberculosis in caseum [PublishAheadOfPrint]

Tuberculosis (TB) recently became the leading infectious cause of death in adults, while attempts to shorten therapy have largely failed. Dormancy, persistence and drug tolerance are among the factors driving the long therapy duration. Assays to measure in situ drug susceptibility of Mycobacterium tuberculosis (Mtb) bacteria in pulmonary lesions are needed if we are to discover new fast acting regimens and address the global TB threat. Here we take a first step towards this goal and describe an ex vivo assay developed to measure the cidal activity of anti-TB drugs against Mtb bacilli present in cavity caseum, obtained from rabbits with active TB. We show that caseum Mtb bacilli are largely non-replicating while maintaining viability over the course of the assay, and exhibit extreme tolerance to many 1^st and 2^nd line TB drugs. Among the drugs tested, only the rifamycins fully sterilized caseum. A similar trend of phenotypic drug resistance was observed in the hypoxia and starvation induced non-replicating models, but with notable qualitative and quantitative differences: (i) caseum Mtb exhibits higher drug tolerance than non-replicating Mtb in the Wayne and Loebel models, and (ii) pyrazinamide is cidal in caseum but has no detectable activity in these classic non-replicating assays. Thus, ex vivo caseum constitutes a unique tool to evaluate drug potency against slow- or non-replicating bacilli in their native caseous environment. Intra-caseum cidal concentrations can now be related to concentrations achieved in the necrotic foci of granulomas and cavities, to establish correlations between clinical outcome and lesion-centric PK-PD parameters.

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