Pyrazinamide is one of the most important drugs in the treatment of latent
Mycobacterium tuberculosis infection. The emergence of strains resistant to
pyrazinamide represents an important public health problem, as both first- and second-line treatment regimens include
pyrazinamide. The accepted mechanism of action states that after the conversion of
pyrazinamide into
pyrazinoic acid by the bacterial
pyrazinamidase enzyme, the
drug is expelled from the bacteria by an efflux pump. The
pyrazinoic acid is protonated in the extracellular environment and then re-enters the mycobacterium, releasing the
proton and causing a lethal disruption of the membrane. Although it has been shown that mutations causing significant loss of
pyrazinamidase activity significantly contribute to
pyrazinamide resistance, the mechanism of resistance is not completely understood. The
pyrazinoic acid efflux rate may depend on multiple factors, including
pyrazinamidase activity, intracellular
pyrazinamidase concentration, and the efficiency of the efflux pump. Whilst the importance of the
pyrazinoic acid efflux rate to the susceptibility to
pyrazinamide is recognized, its quantitative effect remains unknown. Thirty-four M. tuberculosis clinical isolates and a Mycobacterium smegmatis strain (naturally resistant to PZA) were selected based on their susceptibility to
pyrazinamide, as measured by Bactec 460TB and the Wayne method. For each isolate, the initial velocity at which
pyrazinoic acid is released from the bacteria and the initial velocity at which
pyrazinamide enters the bacteria were estimated. The data indicated that
pyrazinoic acid efflux rates for
pyrazinamide-susceptible M. tuberculosis strains fell within a specific range, and M. tuberculosis strains with a
pyrazinoic acid efflux rate below this range appeared to be resistant. This finding contrasts with the high
pyrazinoic acid efflux rate for M. smegmatis, which is innately resistant to
pyrazinamide: its
pyrazinoic acid efflux rate was found to be 900 fold higher than the average efflux rate for M. tuberculosis strains. No significant variability was observed in the
pyrazinamide flux rate. The
pyrazinoic acid efflux rate explained 61% of the variability in Bactec
pyrazinamide susceptibility, 24% of Wayne activity, and 51% of the Bactec 460TB growth index. In contrast,
pyrazinamidase activity accounted for only 27% of the Bactec
pyrazinamide susceptibility. This finding suggests that mechanisms other than pncA mutations (reduction of
pyrazinamidase activity) are also implicated in
pyrazinamide resistance, and that
pyrazinoic acid efflux rate acts as a better proxy for
pyrazinamide resistance than the presence of pncA mutations. This is relevant to the design of molecular diagnostics for
pyrazinamide susceptibility, which currently rely on pncA gene mutation detection.