New Agar Medium for Testing Susceptibility of Mycobacterium tuberculosis to Pyrazinamide

Abstract

A new agar medium to perform pyrazinamide (PZA) susceptibility testing with Mycobacterium tuberculosis has been developed. This medium has an acidic pH of 6.0 instead of the usual for agar media, pH 6.8, to provide optimal conditions for PZA activity, and it also differs from conventional Middlebrook 7H10/7H11 agar in that animal serum (fetal or calf bovine or fetal equine serum) is used instead of oleic acid-albumin-dextrose-catalase to support good growth of M. tuberculosis at the low pH of 6.0. A critical concentration of 900 or 1,200 μg of PZA/ml in this medium made it possible to differentiate between PZA-susceptible and PZA-resistant clinical isolates. This agar medium has the following advantages compared to a liquid medium: it allows determination of the actual proportion of PZA-resistant bacteria in the isolate and it is simple and inexpensive. In addition, it has the potential of being used for a direct susceptibility test with PZA, but this approach will require further confirmation. Further studies to develop critical concentrations of other drugs for this low-pH medium, as well as to investigate the possibility of cultivation in regular (non-CO2) incubators, are in progress.

Susceptibility testing of Mycobacterium tuberculosis to pyrazinamide

“…Information provided by Zhang and his colleagues [18,19] supports the previous suggestions to use PZA at 300 ìg=ml instead of 100 ìg=ml in a single concentration test by the Bactec method in pH 6.0 medium [2,7,13]. For the same reason, PZA concentrations of 900 or 1200 ìg=ml have been suggested for the new agar medium with pH 6.2 that contains 10% of FBS [14].…”

“…More reliable distinction between PZA-susceptible and PZA-resistant strains was observed when 300 ìg=ml of PZA was used as a critical concentration [12], or when three concentrations (100, 300, 900 ìg=ml) were employed to determine the MIC of the drug with corresponding reports as`susceptible',`intermediate', or`resistant' [13]. Another recently developed and less expensive option is a special agar medium with pH 6.2 with a PZA concentration of 900 or 1200 ìg=ml [14].…”

Unravelling the Secrets of Mycobacterial Cidality through the Lens of Antisense

“…We further investigated the vulnerability of selected genes by checking cidality under all the in-vivo simulated multiple physiological conditions (the equivalent of macrophages and the lung granuloma) by performing survival kinetics in-vitro. All the strains were tested under six different physiological in-vitro assay conditions for investigations in parallel, in triplicate: replicating, hypoxia [ 7 ], nutrient starvation [ 5 ], low pH [ 2 ], nitric oxide stress [ 3 ], and nitrogen starvation [ 6 ]. The survivors were monitored by enumerating up to 35 days in a kinetic manner from all the screens in parallel using SPOT-MBC assay [ 22 ].…”

“…Mtb encounters complex physiological situations due to inflammatory immune pressures in the human host, starting with phagocytosis by the macrophages and ending in the same niche- the macrophages. The phagosome-lysosome fusion causes an acidic environment [ 2 ], and a strong chemistry of nitroxidative free radicals [ 3 , 4 ] produced by the macrophages in the granuloma; accompanied by gradual deficiency of nutrients: Carbon [ 5 ], Nitrogen [ 6 ], Oxygen [ 7 ], etc. However, some Mtb populations may be replicating logarithmically [ 8 ].…”

Evaluation of Etest for Susceptibility Testing of Mycobacterium tuberculosis

“…Pyrazinoic acid is active at an acidic pH (5.5), while M. tuberculosis grows poorly at this pH. However, efforts are being made to develop new agar media for susceptibility testing of M. tuberculosis to pyrazinamide (7).…”