The effect of growth rate on pyrazinamide activity in Mycobacterium tuberculosis - insights for early bactericidal activity?

Abstract

BackgroundPyrazinamide (PZA) plays an essential part in the shortened six-month tuberculosis (TB) treatment course due to its activity against slow-growing and non-replicating organisms. We tested whether PZA preferentially targets slow growing cells of Mycobacterium tuberculosis that could be representative of bacteria that remain after the initial kill with isoniazid (INH), by observing the response of either slow growing or fast growing bacilli to differing concentrations of PZA.MethodsM. tuberculosis H37Rv was grown in continuous culture at either a constant fast growth rate (Mean Generation Time (MGT) of 23.1 h) or slow growth rate (69.3 h MGT) at a controlled dissolved oxygen tension of 10 % and a controlled acidity at pH 6.3 ± 0.1. Cultures were exposed to step-wise increases in the concentration of PZA (25 to 500 μgml−1) every two MGTs, and bacterial survival was measured. PZA-induced global gene expression was explored for each increase in PZA-concentration, using DNA microarray.ResultsAt a constant pH 6.3, actively dividing mycobacteria were susceptible to PZA, with similar responses to increasing concentrations of PZA at both growth rates. Three distinct phases of drug response could be distingished for both slow growing (69.3 h MGT) and fast growing (23.1 h MGT) bacilli. A bacteriostatic phase at a low concentration of PZA was followed by a recovery period in which the culture adapted to the presence of PZA and bacteria were actively dividing in steady-state. In contrast, there was a rapid loss of viability at bactericidal concentrations. There was a notable delay in the onset of the recovery period in quickly dividing cells compared with those dividing more slowly. Fast growers and slow growers adapted to PZA-exposure via very similar mechanisms; through reduced gene expression of tRNA, 50S, and 30S ribosomal proteins.ConclusionsPZA had an equivalent level of activity against fast growing and slow growing M. tuberculosis. At both growth rates drug-tolerance to sub-lethal concentrations may have been due to reduced expression of tRNA, 50S, and 30S ribosomal proteins. The findings from this study show that PZA has utility against more than one phenotypic sub-population of bacilli and could be re-assessed for its early bactericidal activity, in combination with other drugs, during TB treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1533-z) contains supplementary material, which is available to authorized users.

Linezolid Kills Acid-Phase and Nonreplicative-Persister-Phase Mycobacterium tuberculosis in a Hollow-Fiber Infection Model

“…In either event, linezolid demonstrated acid-phase bacterial killing activity at least as great as that seen with pyrazinamide. As pyrazinamide is credited with playing a major role in the shortening of M. tuberculosis therapy to its current 6-month standard (15)(16)(17), the finding of substantial killing activity for linezolid against acid-phase M. tuberculosis is important, particularly so as the prevalence of pyrazinamide resistance has increased over time (18).…”

Direct Determination of Pyrazinamide (PZA) Susceptibility by Sputum Microscopic Observation Drug Susceptibility (MODS) Culture at Neutral pH: the MODS-PZA Assay

“…In vitro, a similar condition can be obtained when the culture is performed at pH 5.5 (using an MIC of 50 g/ml). However, an acidic pH has been reported to inhibit M. tuberculosis growth (30,31). Theoretically, higher concentrations of PZA can show antituberculosis activity at a higher pH.…”

“…Performing direct phenotypic DST for PZA has been hampered by the difficulty in demonstrating anti-TB activity of the drug when analyzed under conventional culture conditions. The widely accepted explanation for this is the requirement of an acidic environment for PZA to exert anti-TB activity versus the pH close to neutral of the culture medium (9,10,18,(31)(32)(33). At present, the Bactec MGIT 960 PZA (MGIT-PZA) assay is the accepted standardized phenotypic DST (34).…”

MODS-Wayne, a Colorimetric Adaptation of the Microscopic-Observation Drug Susceptibility (MODS) Assay for Detection of Mycobacterium tuberculosis Pyrazinamide Resistance from Sputum Samples

“…The advantage of using a neutral pH is that acidity inhibits the growth of M. tuberculosis and therefore reduces the test’s specificity (44). MODS-Wayne overcomes other limitations of the traditional DSTs for PZA, in that there is no requirement for an isolate to be grown first (indirect test), or for a large inoculum size (3 to 5 mg) in order to avoid false-negative results (30).…”