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.

Keywords: Mycobacterium tuberculosis; caseum; drug tolerance; in vitro potency model; persistence; pharmacokinetics-pharmacodynamics.

FIG 1

Bacterial burden of 10 different ex vivo samples of rabbit caseum incubated at 37°C for 7 days, measured on day 1 (D1) and day 8 (D8) in the absence of drug. Data are expressed as log₁₀ of CFU per gram of caseum (undiluted) and plotted on a log scale. Statistically significant differences in CFU counts are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.005.

FIG 2

Bactericidal activity of eight standard TB treatment drugs in caseum. Data are expressed as log₁₀ of average CFU per milliliter of homogenized caseum from three replicates. A red dot highlights data points below the limit of detection (LOD [approximately 20 CFU]); i.e., no CFU was recovered from the lowest dilution of caseum homogenate plated. Standard deviations are indicated by error bars.

FIG 3

Bactericidal activity of four rifamycins in caseum. Data are expressed as log₁₀ of average CFU per milliliter of homogenized caseum obtained from three replicates. Red dots highlight data points below the limit of detection (LOD [approximately 20 CFU]); i.e., no CFU was recovered from the lowest dilution of caseum homogenate plated. Standard deviations are indicated by error bars.

FIG 4

Comparative levels of accumulation of intracellular lipid inclusions and acid-fastness of M. tuberculosis bacilli from caseum and from a replicating culture spiked in synthetic caseum (31). Caseum smears (A to C) and actively replicating M. tuberculosis (D to F) were stained with Auramine O (green; acid-fast stain) and Nile red (red; neutral lipid stain) and examined by confocal laser scanning microscopy (Nikon Eclipse Ti) at the same laser intensity for all samples with Z-stacking to get the depth of the scan field. Scanned samples were analyzed with NIS Elements software for image projection. (A and D) Overlaid image of dual-stained M. tuberculosis. (B and E) Nile red signal only. (C and F) Auramine O signal only. The diffuse red signal in panels E and F is due to staining of lipids present in synthetic caseum. (G) Venn diagrams showing the distribution of auramine-positive and Nile red-positive M. tuberculosis cells in caseum and in an exponentially growing culture spiked in synthetic caseum (control).