Effectiveness of tuberculosis chemotherapy correlates with resistance to Mycobacterium tuberculosis infection in animal models

Abstract

Objectives: It is widely believed that persistent Mycobacterium tuberculosis inhabits necrotic lung granulomas in humans and that the microenvironmental conditions encountered therein render the bacilli phenotypically tolerant to antibiotics, accounting for the long duration required for successful treatment of tuberculosis (TB). To validate this belief, we directly compared the activity of rifampicin/isoniazid/pyrazinamide (RHZ) against chronic TB infection in guinea pigs, which exhibit caseous granulomas histologically resembling human caseous foci, and in mice, which lack necrotic granulomas.

Methods: Guinea pigs and mice were aerosol-infected with M. tuberculosis CDC1551 and twice weekly treatment with RHZ was started 4 weeks later. Culture-positive relapse was assessed in subgroups of guinea pigs after 3 months and 4 months of treatment.

Results: All guinea pig lungs exhibited histological evidence of granulomas with central caseation, while mouse lungs exhibited cellular lesions at the initiation of antibiotic treatment. Guinea pig lungs became culture-negative after 2 months of RHZ given twice weekly at human-equivalent doses. Relapse rates in guinea pigs were 0% (0/10) both after 3 months and 4 months of treatment. In contrast, all mouse lungs remained culture-positive after 4 months of equivalent RHZ exposures.

Conclusions: Caseous necrosis does not reduce the sterilizing activity of the standard antituberculosis regimen of RHZ. Our findings have important implications for the use of alternative animal models in testing novel TB drug regimens and for modelling M. tuberculosis persistence.

Figure 1.

Antibiotic treatment reduces lung and spleen weights in M. tuberculosis-infected guinea pigs. Normalized lung (a) and spleen (b) weights are shown in untreated guinea pigs (continuous lines) and those treated with 2 months of human-equivalent doses of rifampicin, isoniazid and pyrazinamide, followed by 2 months of rifampicin and isoniazid (2RHZ/2RH) (broken lines). n = 5 mice and 4 guinea pigs per timepoint.

Figure 2.

Mouse and guinea pig histopathology following antibiotic treatment. (a) Mouse lungs at treatment initiation; lesions are loose aggregates of primarily lymphocytes with few histiocytes and plasma cells, and AFB were detected primarily within foamy macrophages (inset). Low power (b) and high power (c) magnification of mouse lungs after 4 months of treatment; lesions are smaller and cell composition is maintained, and rare intracellular AFB were detected (c, inset). (d) Guinea pig lungs at treatment initiation; large, well-circumscribed granulomas comprising mainly histiocytes and a few lymphocytes are seen, with central necrosis. Acid-fast staining revealed the presence of organisms within histiocytes and in the necrotic debris of granulomas (inset). Low-power (e) and high-power (f) magnification of guinea pig lungs at month 4 of treatment; necrotic granulomas are smaller and confined mostly to the periphery of the lungs, and bacilli can be seen within the necrotic centre of one such granuloma (f, inset). Sections were stained with haematoxylin-eosin and images were obtained at low power (a, b, d and e; 2× magnification) and high power (c and f; 20× magnification). Insets: images of acid-fast-stained sections were obtained at 60× magnification. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 3.

Equivalent exposures of the combination regimen comprising rifampicin, isoniazid and pyrazinamide (RHZ) show greater bactericidal activity against M. tuberculosis in guinea pig lungs relative to mouse lungs. Twice weekly administration of RHZ was initiated 28 days after aerosol infection (M0) in each species for a total of 2 months (2RHZ). Animals received human-equivalent doses of RH during the remaining 2 months of treatment (2RH). In guinea pigs, the following doses were used: 50 mg/kg rifampicin; 60 mg/kg isoniazid; and 300 mg/kg pyrazinamide. In mice, the following doses were used: 10 mg/kg rifampicin; 25 mg/kg isoniazid; and 150 mg/kg pyrazinamide. Data represent mean cfu counts per lung ± SD. n = 5 mice and 4 guinea pigs per timepoint. GP, guinea pigs.