Anaerobic incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) is an unconventional front line tuberculosis drug characterized by high in vivo sterilizing activity, but poor in vitro activity. This disparity in PZA activity may reflect differences between the in vivo tissue environment and in vitro culture conditions. This study examined the effect of anaerobic conditions, which exist in granulomatous lesions in vivo, on PZA activity in vitro. Low oxygen enhanced the activity of PZA against Mycobacterium tuberculosis, with anaerobic conditions resulting in greater enhancement than microaerobic conditions. ATPase and respiratory chain enzyme inhibitors enhanced PZA activity under normal atmospheric conditions, but not under anaerobic conditions. Furthermore, the inhibitors did not enhance isoniazid or rifampicin activity. Nitrate as an alternative electron acceptor antagonized PZA activity under anaerobic conditions. These findings provide further support for a proposed mechanism of action of PZA in which the active form of PZA (pyrazinoic acid) depletes the membrane energy reserve. They also provide another explanation for the higher sterilizing activity of PZA within in vivo lesions with low oxygen than under in vitro drug susceptibility testing conditions with ambient oxygen. INTRODUCTIONPyrazinamide (PZA) is an important front line tuberculosis (TB) drug responsible for the shortening of TB therapy from 9-12 months to the current 6 months, presumably due to its ability to kill 'semi-dormant' bacilli that are not killed by other drugs (Mitchison, 1985;Heifets & Lindholm-Levy, 1992). Interestingly, PZA is not active in vitro under normal culture conditions near neutral pH (Tarshis & Weed, 1953) but is only active under acidic conditions (e.g. pH 5 . 5) (McDermott & Tompsett, 1954), with an MIC of 50-100 ìg ml À1 at pH 5 . 5-6 . 0 (Zhang & Mitchison, 2003). This may well be a reflection of the in vivo conditions present during active inflammation. However, acid pH alone cannot be the only reason for the difference in activity of PZA observed in vivo versus in vitro, as the serum concentration of PZA is somewhat lower (30-60 ìg ml À1 ) than the MIC in vitro (Zhang & Mitchison, 2003). Although various factors could affect PZA activity (Zhang et al., 2002), exactly what accounts for the overall high sterilizing activity in vivo remains unknown. Recently our laboratory has shown that iron, the level of which could be elevated in local inflammatory lesions (Aisen, 1980), can enhance the activity of PZA in vitro (Somoskovi et al., 2004). However, other potential mechanisms that contribute to PZA activity need to be investigated. One such factor could be the difference in the level of oxygen present in vivo versus in vitro. Tubercle bacilli in granulomatous lesions in vivo are believed to reside in a low oxygen environment (Canetti, 1955). We wondered whether a hypoxic environment in vivo could enhance the activity of PZA, thereby contributing to the differences observed in the activity of PZA in vivo and in vitro. In the current study, we have examined the effe...

Regulation of nitrate reductase activity in Mycobacterium tuberculosis by oxygen and nitric oxide

“…In immunocompetent mice, the mutant shows decreased ability to persist in the lungs, liver and kidneys, but no decrease in the spleen (Fritz et al, 2002). A role for nitrate reductase in maintaining ATP levels in M. tuberculosis during hypoxia has been demonstrated from work with pyrazinamide (Wade & Zhang, 2004;Zhang et al, 2003).…”

Nitrate Enhances the Survival of Mycobacterium tuberculosis during Inhibition of Respiration

“…Pyrazinamide, one of the frontline antibiotics in the treatment of tuberculosis, is proposed to act by collapsing the proton gradient (32). Survival was increased in the presence of nitrate as an alternate electron acceptor under anaerobic conditions (26). Since nitrate present in the medium can change drug susceptibility, it should be included when testing compounds that affect respiration.…”

Tuberculosis Chemotherapy: the Influence of Bacillary Stress and Damage Response Pathways on Drug Efficacy

“…That is, a functional immune system might be required to potentiate drug activity, thus preventing the evolution of resistance. Although direct evidence is scant, the enhanced sterilizing activity of PZA in vivo, in contrast with its poor activity in vitro (257), is suggestive of a synergistic interplay between drug and host (236). The large bacterial populations associated with M. tuberculosis-infected immunocompromised individuals might provide an expanded subset for selection and transmission of rare mutation events.…”

“…However, the influence of the in vivo environment on drug efficacy should not be viewed as inevitably negative: pyrazinamide (PZA), for example, is inactive in vitro under standard culture conditions but displays strong sterilizing activity in vivo (257,259). Moreover, the activity of PZA in vivo correlates with enhanced in vitro activity under low pH (257) and limiting oxygen (236), suggesting the relevance of these factors to the in vivo environment (discussed below). The absolute dependence on environmental factors for PZA activity has profound implications for the discovery of new anti-TB drugs, since it would almost certainly render this drug unidentifiable according to standard drug screening criteria, thereby eliminating a mainstay of current TB chemotherapy.…”