There are currently renewed efforts to develop drugs that could shorten the duration of antituberculosis therapy. This is best achieved by optimizing the sterilizing effect. However, the current pathway for the development of new molecules with the potential to have a sterilizing effect is inefficient. We designed an in vitro pharmacokinetic-pharmacodynamic model in which Mycobacterium tuberculosis replicating slowly at pH 5.8 was exposed to pyrazinamide by use of the concentration-time profiles encountered in patients. The sterilizing effect rates and the time to the emergence of drug resistance were examined. Daily pyrazinamide dosing for 28 days accurately achieved (i) the pyrazinamide pharmacokinetic parameters, (ii) the lack of early bactericidal activity, (iii) a sterilizing effect rate of 0.10 log 10 CFU/ml per day starting on day 6 of therapy, and (iv) a time to the emergence of resistance of the from 2 to 3 weeks of monotherapy encountered in patients with tuberculosis. Next, dose-scheduling studies were performed. Current therapy for tuberculosis (TB) consists of rifampin (rifampicin), isoniazid, and pyrazinamide (5). In order to improve therapy, several studies have examined the pharmacokinetic (PK)-pharmacodynamic (PD) relationship between isoniazid and rifampin treatment and the response of Mycobacterium tuberculosis (22,25,29,30). However, those studies were of short duration and mainly reflected the bactericidal activities of these agents, defined as the killing of bacilli in log-phase growth. No similar studies have examined the sterilizing activities of anti-TB drugs. An anti-TB drug is considered to have a sterilizing effect when it is able to kill one of two subpopulations of M. tuberculosis: slowly growing bacilli in an acidic environment (slowly replicating bacilli [SRB]) and nonreplicating persistent bacilli (5, 26). The sterilizing activity of a drug also reflects its ability to shorten anti-TB therapy duration (41). Pyrazinamide has unique sterilizing activity against SRB, which are estimated to constitute the majority of bacilli in the pulmonary cavities of patients with TB (31,35). A measure of the size of the SRB population is that the addition of pyrazinamide to isoniazid-and rifampin-containing regimens led to a one-third reduction in the duration of therapy and a two-thirds reduction in the rate of TB relapse (2, 48). Given the size of this population and the central role of pyrazinamide in eradicating it, pyrazinamide PK-PD studies could help with the further optimization of therapy.After oral administration, pyrazinamide is almost completely systematically absorbed and eventually enters the pulmonary cavities containing M. tuberculosis. Pyrazinamide works in these cavities under acidic conditions (38). There is conflicting evidence as to whether pyrazinamide enters the bacilli by simple diffusion or by active uptake (44, 56). However, once the bacilli are in the cell, bacillary nicotinamidase deaminates the pyrazinamide to pyrazinoic acid (POA), which is transported to the extrace...

These data, based on a preclinical model, demonstrate that nonadherence alone is not a sufficient condition for MDR-tuberculosis emergence.

ABSTRACTWe hypothesize that low-level efflux pump expression is the first step in the development of high-level drug resistance in mycobacteria. We performed 28-day azithromycin dose-effect and dose-scheduling studies in our hollow-fiber model of disseminatedMycobacterium avium-M. intracellularecomplex. Both microbial kill and resistance emergence were most closely linked to the within-macrophage area under the concentration-time curve (AUC)/MIC ratio. Quantitative PCR revealed that subtherapeutic azithromycin exposures over 3 days led to a 56-fold increase in expression of MAV_3306, which encodes a putative ABC transporter, and MAV_1406, which encodes a putative major facilitator superfamily pump, inM. avium. By day 7, a subpopulation ofM. aviumwith low-level resistance was encountered and exhibited the classic inverted U curve versus AUC/MIC ratios. The resistance was abolished by an efflux pump inhibitor. While the maximal microbial kill started to decrease after day 7, a population with high-level azithromycin resistance appeared at day 28. This resistance could not be reversed by efflux pump inhibitors. Orthologs of pumps encoded by MAV_3306 and MAV_1406 were identified inMycobacterium tuberculosis,Mycobacterium leprae,Mycobacterium marinum,Mycobacterium abscessus, andMycobacterium ulcerans. All had highly conserved protein secondary structures. We propose that induction of several efflux pumps is the first step in a general pathway to drug resistance that eventually leads to high-level chromosomal-mutation-related resistance in mycobacteria as ordered events in an “antibiotic resistance arrow of time.”