Extreme Drug Tolerance of Mycobacterium tuberculosis in Caseum

Abstract

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 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 ex vivo assay developed to measure the cidal activity of anti-TB drugs against M. tuberculosis bacilli present in cavity caseum obtained from rabbits with active TB. We show that caseum M. tuberculosis 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 M. tuberculosis exhibits higher drug tolerance than nonreplicating M. tuberculosis in the Wayne and Loebel models, and (ii) pyrazinamide is cidal in caseum but has no detectable activity in these classic nonreplicating assays. Thus, ex vivo 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.

PerSort facilitates characterization and elimination of persister subpopulation in mycobacteria

“…These physiological changes (either stochastically- or environmentally-induced) result in antibiotic tolerance, in which MTB is genetically susceptible to antibiotics but exists in a physiological state rendering it refractory to drug killing. These persistent states are a major reason why long courses of antibiotic therapy are required to treat human tuberculosis (TB) (2); standard chemotherapy of TB requires 6 months of treatment and 5% are not cured even then (3, 4). There is an urgent need for new strategies that shorten the duration of treatment and target drug tolerant MTB.…”

Treatment-shortening effect of a novel regimen combining high-dose rifapentine and clofazimine in pathologically distinct mouse models of tuberculosis

“…The authors linked the diminished activity of clofazimine with the development of hypoxic, caseous necrotic lesions containing extracellular bacteria, suggesting that clofazimine was less active within these lesions. This hypothesis was further supported by studies demonstrating that clofazimine diffuses relatively poorly into caseous necrotic lesions in humans TB (27), does not have bactericidal activity in ex vivo caseum from M. tuberculosis -infected rabbits (41), and the long-standing observation that clofazimine accumulates in macrophages (26, 27, 42, 43). Rifapentine has been shown to diffuse into necrotic caseous lesions less rapidly than rifampin in a rabbit model of cavitary TB (28), a finding that correlates with clinical data indicating that replacing rifampin with rifapentine added proportionally less activity in patients with cavitary versus non-cavitary TB disease (44).…”

Drug permeation and metabolism in Mycobacterium tuberculosis: Prioritising local exposure as essential criterion in new TB drug development

“…For intracellular bacilli, the drug must also overcome the barrier of the host cell membrane and, in many cases, an encapsulating vesicular membrane, as well as potential sequestration by the different organelles and intracellular bodies within different cell types (11,12). The occupation of discrete host loci (13–15) presents an additional confounder (Figure 1), in that individual bacteria from a clonal infecting population are characterized by different physiological states, and this can impact drug susceptibility (11,16,17) and the ability of the drug to penetrate the complex mycobacterial cell wall (18), as well as subverting the active compound’s activity against a metabolic target that might be essential only under specific conditions (19,20). As if that weren’t already sufficiently complex, host-mediated (21,22) and/or mycobacterium-mediated (23,24) biotransformation might further complicate the passage of drug from ingestion by the patient to its intrabacillary target (7).…”

“…Using a combination of New Zealand White rabbits, imaging mass spectrometry, three first-line anti-TB drugs (INH, RIF, and PZA), and the fluoroquinolone MXF, the group demonstrated that drug plasma concentration was indeed a poor proxy for drug concentration in TB lesions (8). The lack of data on how this might translate into humans combined with the absence of cellular protein binding data were acknowledged as weaknesses by the authors; nevertheless, this work prompted a growing appreciation of the need to include permeation studies in their drug development pipeline (16,129,130). …”

“…Using the TB-infected rabbit model, along with microdissection and LC-MS/MS, the group determined that EMB partitions into the caseous lesions with great efficiency, potentially explaining its efficacy and again motivating for permeation studies to be conducted in early pre-clinical drug development (30). This work was followed up by a further study using the infected rabbit model, where ex vivo MBC measurements were taken in caseum (MBC caseum ) for several first-line TB drugs (16). The Wayne (133) and Loebel (134) models for non-replicating persistence failed to predict the extent to which resistance was being generated in the caseous environment, whilst also indicating that PZA has activity in the caseum, a fact which in the current non-replicating models would have missed.…”