For the past decades, an acidic pH has been used to render Mycobacterium tuberculosis susceptible to pyrazinamide for in vitro testing. Here, we show that at the standard breakpoint concentration and reduced culture temperatures, pyrazinamide (PZA) is active against tuberculosis (TB) at neutral pH. This finding should help unravel the mechanism of action of PZA and allow drug susceptibility testing (DST) methods to be optimized. P yrazinamide (PZA) is an important drug for TB treatment. PZA is used in standard first-and second-line therapies and is also included in many new regimens due to its unique ability to shorten therapy (1, 2).The mechanism of action of PZA is unresolved (3), but it is commonly assumed that a low pH is required for PZA activity against Mycobacterium tuberculosis. In a widely accepted model proposed by Zhang and Mitchison (4), low pH causes the protonation of extracellular pyrazinoic acid (POA; the enzymatically activated form of PZA) required for POA to reenter mycobacteria and exert its antimicrobial effect. In addition, the reduced membrane potential at low pH was proposed to facilitate energy depletion by PZA (5). However, the activity of PZA in vivo and in vitro is directed against nonmetabolizing, or slowly metabolizing, mycobacteria (1, 6), and the role of low pH on the transcriptional remodeling of M. tuberculosis known to occur under those conditions (7-9) might also be related to the antimicrobial effects of PZA at low pH. We believe the relative contribution of the protonation and metabolic effects deserves investigation and might help elucidate PZA's mechanism of action in vivo.Due to the incompletely resolved mechanism, developments in drug susceptibility testing (DST) have been limited to testing at reduced pH. Partly due to the suboptimal growth of the bacteria at low pH, the conditions are difficult to control, and PZA DST results in more failures and a lower test accuracy and reproducibility than those of other first-line drugs (10-12).It was previously demonstrated that under acidic conditions, PZA activity is enhanced by lowering the temperature (13), but the effect of low temperature alone was not assessed. To investigate how dependent the action of PZA is on low pH, we determined the susceptibility of TB to PZA at reduced temperature at neutral pH.
MATERIALS AND METHODS
Strains.The tested strains are presented in Table 1. M. tuberculosis strains 12-17995 and 12-17889 are clinical isolates from Georgia (14) from the Beijing lineage. Strain 12-17889 is closely related to the previously described clade A strains sharing a pncA I6L mutation (15). Apart from the pncA I6L mutation, no additional mutation in pncA is present in this strain.Microcolony-based growth rate determination. Measurement of the effect of antimicrobials on TB microcolonies on solid medium was performed essentially as previously described (16). In short, aliquots of liquid cultures, sieved through a 5-m-pore filter, were inoculated on 8 by 8-mm squares of porous supports on nonselective MB7H11 agar (BD, ...
