Studying the metabolic activity of Mycobacterium tuberculosis through a new resazurin reduction based assay

Abstract

Lately several approaches have been performed to study the biological aspects of the mycobacteria metabolism and elucidate some bacterial strategies to survive to host responses, but little is known about mycobacteria adaptation to adverse environmental conditions. In this study we developed a simple and rapid method to evaluate the metabolic rate Mycobacterium tuberculosis (Mtb) strains in liquid culture media based on resazurin reduction kinetic. We tested 4 mycobacterial growth conditions: physiological (pH 6.7), acidic (pH 5.5 and 4.5) and anaerobic; the avirulent strain Mtb H37Ra was cultured until log phase, and 3 different bacterial concentrations (10(3), 10(4) and 10(6) CFU/ml) were inoculated in triplicate, and incubated at 37ºC with 5% Co2 atmosphere. After inoculation, 20 microL of re sazurin 0.01% were added, and the indicated optical density was meassured at several time-points (0h, 6h and 24h). This procedure was repeatedthree days after the inoculation, concluding a 5 day periodof incubation. CFU counting was performed at the time of inoculation, and at the end of experience for each condition.Globaly, our results show an increasing resazurin reduction curve dependent on the normal growth curve of viable bacteria. However, after a 3 day incubation period the percentage of resazurin reduction kinetics was shown to be linear only for cultures innoculated with 10(6) CFU/mL. This pattern was also observed under acidic and anaerobic conditions. The method was found to be reproducible with low standard deviations. Thus we selected the 10(6) CFU/mL as the best bacterial inoculum concentration for this assay.Afterwards, we analysed the growth curve of 10(4)CFU/ml culture for each conditions, and CFU counting as well. Although the H37Ra strain has the capability to grow in all conditions tested, which was confirmed by CFU counting, it was possible to distinguish the rate of resazurin reduction across the different environments. As expected, a smaller number of CFU/mL was obtained in acidic conditions when compared to anaerobic and aerobic conditions. Taking into account these results, we believe that the methodology here presented holds potential for mycobacterial metabolic studies concerning different strain physiology.