Pyrazinamide Is Active against Mycobacterium tuberculosis Cultures at Neutral pH and Low Temperature

Abstract

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, ...

Pyrazinamide resistance-conferring mutations in pncA and the transmission of multidrug resistant TB in Georgia

“…This finding was corroborated by a recent study where the pncA -I6L, mutation was also associated with susceptibility to PZA in a panel of clustered isolates [26] and was not categorised as a high confidence resistance mutation [10]. Interestingly, strain 12-17889 was tested phenotypically resistant by an alternative method for PZA DST not using low pH [40]. …”

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

“…PZA is a prodrug in which the antimycobacterial activity requires hydrolysis by MTB pyrazinamidase (PZase)/nicotinamidase encoded by pncA to convert into its active form pyrazinoic acid (POA) causing cytoplasmic acidification and depletion of membrane potential . The activity of PZA is generally thought to be dependent on acidic pH; however, it has recently been reported that the PZA activity can be independent of acidic pH and intrabacterial acidification . The latter finding can be confusing as it does not mean that PZA works at neutral pH in general, irrespective of the metabolic status of the TB bacteria.…”

“…21,48 The activity of PZA is generally thought to be dependent on acidic pH 49,50 ; however, it has recently been reported that the PZA activity can be independent of acidic pH and intrabacterial acidification. 51,52 The latter finding can be confusing as it does not mean that PZA works at neutral pH in general, irrespective of the metabolic status of the TB bacteria. Rather, it indicates that the bacterial metabolic status is important in determining the activity of PZA where in dormant persister bacteria with low metabolisms PZA could even show activity at near neutral pH, even then PZA would be expected to show more activity under acidic pH.…”

Impact of the host environment on the antitubercular action of pyrazinamide

“…Future experimentation by McDermott and Thompsett would show that the culture medium needed to be mildly acidic (pH of 5.8) for PZA to inhibit growth of M. tuberculosis [7] . Subsequent studies revealed enhanced PZA activity in the presence of alkaline pH [7] , low temperature [8] , nutrient limitation [9] , and hypoxia [10] . Further testing of experimentally infected mice and guinea pigs by Malone [11] , Dessau [12] and colleagues showed that PZA treatment resulted in decreased lung pathology and showed superior antitubercular activity compared to para -aminosalicylic acid and nicotinamide.…”