Extreme Drug Tolerance of Mycobacterium tuberculosis in Caseum

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 drug susceptibility of 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 toward this goal and describe an assay developed to measure the cidal activity of anti-TB drugs against bacilli present in cavity caseum obtained from rabbits with active TB. We show that caseum bacilli are largely nonreplicating, maintain viability over the course of the assay, and exhibit extreme tolerance to many first- and second-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 nonreplicating models, but with notable qualitative and quantitative differences: (i) caseum exhibits higher drug tolerance than nonreplicating in the Wayne and Loebel models, and (ii) pyrazinamide is cidal in caseum but has no detectable activity in these classic nonreplicating assays. Thus, caseum constitutes a unique tool to evaluate drug potency against slowly replicating or nonreplicating bacilli in their native caseous environment. Intracaseum cidal concentrations can now be related to the concentrations achieved in the necrotic foci of granulomas and cavities to establish correlations between clinical outcome and lesion-centered pharmacokinetics-pharmacodynamics (PK-PD) parameters.