Drug tolerance in Mycobacterium tuberculosis

Abstract

Although Mycobacterium tuberculosis is eradicated rapidly during therapy in some patients with pulmonary tuberculosis, it can persist for many months in others. This study examined the relationship between mycobacterial drug tolerance (delayed killing in vitro), persistence, and relapse. It was performed with 39 fully drug-susceptible isolates from a prospective trial of standard short-course antituberculous therapy with sputum smear-positive, human immunodeficiency virus-uninfected subjects with pulmonary tuberculosis in Brazil and Uganda. The rate of killing in vitro was determined by monitoring the growth index (GI) in BACTEC 12B medium after addition of drug to established cultures and was measured as the number of days required for 99% sterilization. Drugs differed significantly in bactericidal activity, in the following order from greatest to least, rifampin > isoniazid-ethambutol > ethambutol (P < 0.001). Isolates from subjects who had relapses (n = 2) or in whom persistence was prolonged (n = 1) were significantly more tolerant of isoniazid-ethambutol and rifampin than isolates from other subjects (P < 0.01). More generally, the duration of persistence during therapy was predicted by strain tolerance to isoniazid and rifampin (P = 0.012 and 0.026, respectively). Tolerance to isoniazid-ethambutol and tolerance to rifampin were highly correlated (P < 0.001). Tolerant isolates did not differ from others with respect to the MIC of isoniazid; the rate of killing of a tolerant isolate by isoniazid-ethambutol was not increased at higher drug concentrations. These observations suggest that tolerance may not be due to drug-specific mechanisms. Tolerance was of the phenotypic type, although increased tolerance appeared to emerge after prolonged drug exposure in vivo. This study suggests that drug tolerance may be an important determinant of the outcome of therapy for tuberculosis.

FIG. 1

Killing of M. tuberculosis H₃₇Ra by EMB, INH, and RIF in BACTEC 12B medium. INH resistance emerged in cultures exposed to INH alone. This was prevented by addition of EMB (upright triangles) without otherwise affecting the rate of killing. ○, no drug; ░⃞, EMB; , INH; , INH-EMB; ●, RIF.

FIG. 2

Relationship of CFU of M. tuberculosis H₃₇Ra to DTP in BACTEC 12B medium.

FIG. 3

Relationship of GI to CFU in cultures with INH (A) or RIF (B). CFU values were calculated from DTP values for daily subcultures. The number of viable bacilli in cultures exposed to INH was reduced by 99% (2 log₁₀) after slightly more than 2 days (A). The corresponding GI at that time was 130.

FIG. 4

Killing of clinical M. tuberculosis isolates, grouped according to the duration of persistence in sputum during therapy. Isolates from subjects who had relapses or whose sputum cultures were persistently positive through day 120 of therapy (black circles) showed increased tolerance to INH-EMB and RIF compared to all other isolates.

FIG. 5

Correlation between persistence (sputum culture positivity during therapy) and tolerance to INH (days required for 99% sterilization).

FIG. 6

Lack of effect of INH concentration on the rate of killing of a tolerant clinical M. tuberculosis isolate. All concentrations tested were greater than the MIC for this isolate.

FIG. 7

Correlation between tolerance to RIF and INH in drug-sensitive clinical isolates of M. tuberculosis, measured as DTS.

FIG. 8

Effect of prolonged drug exposure in vitro on expression of tolerance. The surviving bacilli of the isolate from subject 5 were recovered after 11 days of exposure to INH-EMB. The surviving bacilli exhibited tolerance identical to that of the original isolate.

FIG. 9

Effect of prolonged drug exposure in vivo on expression of tolerance to INH. Isolates were collected from the sputum of subject 29 after 7 and 30 days of standard therapy. Tolerance increased in a stepwise manner. The subject ultimately had a relapse.