Conditions that may affect the results of susceptibility testing of Mycobacterium tuberculosis to pyrazinamide

Abstract

Pyrazinamide (PZA) is an important front-line anti-tuberculosis drug that is active only at acid pH. However, acid pH causes signi®cant dif®culty for PZA susceptibility testing. A common problem in PZA testing is false resistance caused by large bacterial inocula. This study investigated the relationship of false resistance to numbers of bacilli, pH and other factors that potentially affect susceptibility to PZA. Large inocula (10 7±8 bacilli=ml) of M. tuberculosis H37Ra caused signi®cant increase in medium pH from 5.5 towards neutrality, and thus produced false resistance results. The increase in medium pH was determined to be a function of live bacilli; heat-killed bacilli had little or no effect. Susceptibility to PZA and its active derivative pyrazinoic acid (POA) was comparable on 7H11 agar medium, but POA was less active than PZA in liquid medium containing bovine serum albumin (BSA), suggesting that susceptibility to PZA or POA was reduced in the presence of BSA, because of its neutralising effect on medium pH and signi®cant POA binding. A 3-month-old H37Ra culture was shown to be more susceptible to PZA exposure than a 4-day log-phase culture, suggesting that PZA is more active for non-growing bacilli. Finally, reserpine, an inhibitor of POA ef¯ux pump, increased susceptibility to PZA even near neutral pH 6.8, with an MIC of 400 mg=L compared with 1000 mg=L without reserpine. These ®ndings should have implications for understanding the mode of action of PZA and for PZA susceptibility testing.

Anaerobic incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis

“…This was expected, as PZA has less activity on young, growing cultures at acid pH (5 . 5) than on old cultures (Zhang et al, 2002). These results suggest that the level of oxygen has an effect on PZA activity such that the lower the oxygen level, the greater the PZA activity.…”

“…20 %) where tubercle bacilli grow well. PZA is a peculiar and unconventional drug that does not kill growing cultures very well, but kills old, non-growing cultures with less energy reserves more effectively (Zhang et al, 2002). In this study we have shown that anaerobic or hypoxic conditions, which do not allow tubercle bacilli to grow, enhanced the activity of PZA (Fig.…”

“…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).…”

Novel targeting of the lepB gene using PCR with confronting two-pair primers for simultaneous detection of Mycobacterium tuberculosis complex and Mycobacterium bovis

“…Additionally, due to the need for acidic conditions in pyrazinamide drug susceptibility testing, this is not routinely performed in the laboratory (Zhang et al, 2002). Hence, the treatment of M. bovisinfected patients with the front-line drug regimen is not only presumed to be totally inadequate but is also believed to lead to the emergence of drug-resistant mycobacterial strains.…”

Wayne’s Assay: A Screening Method for Indirect Detection of Pyrazinamide Resistance in Mycobacterium Tuberculosis Clinical Isolates

“…Despite its remarkable sterilizing activity in vivo, [13,14] it is not active against MTB under 'normal' culture conditions near neutral pH, [15]. It is only active at acid pH [16] an environment that is produced during active inflammation, and its activity is closely related to the acidity of the medium [7,17]. Even at acid pH 5.6, PZA kills MTB slowly and incompletely with no more than 76% of the bacterial population being killed by 1000 mg/L PZA, [18] which is 10-12 times higher than the already high minimum inhibitory concentration (MIC) of 50-100 mg/L [12].…”