In vitro starvation model for Assessing Phenotypic Drug Tolerance in Mycobacterium tuberculosis Lineages of Ethiopia
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Date
2018-04
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Addis Ababa University
Abstract
Background: Mycobacterium tuberculosis persist in the human host for decades and reactivation can occur at any point. It becomes dormant and phenotypically drug tolerant when exposed adverse conditions. Understanding of the signals and processes which allow the bacteria to achieve this feat could potentially be used as a baseline to design new types of drugs or modify old drug regimens for improved cure and avert development of drug resistance.
Objective: To assess the level of phenotypic drug tolerance of Mycobacterium tuberculosis using in vitro nutrition starvation model.
Methods: Three MTB lineages and one standard susceptible reference strain (H37Rv) were tested by different test methods at different time point from March to September 2017. All lineages tested to be sensitive to first-line anti-TB drugs. Log phase (highest colony count on week 3-4) culture from Lowenstein Jensen medium sub cultured to Middle-brook7H9 with 10% Oleic Acid Albumin Dextrose Catalase as a normal, Phosphate Buffer Solution (PBS) (PH 7.2) and Sterile Distilled water (SDW) as starvation media were used. Each week culture growth reading was performed, Acid Fast Stain (AFS) by Ziehl Neelson(ZN), Lipid Bodies (LB)by Sudan black stain and viability by Fluorescein DiAcitate (FDA) staining. On week 0, 3 and 6 drug susceptibility test was done by colorimetric MTT assay. Graph pad prism 6 and SPSS V20 used for data analysis.
Results: A total of 576 experiments were performed using4 strains of Mycobacterium tuberculosis subcultured on SDW, PBS and 7H9 and. Of these, 324 microscopic tests using ZN(108)acid fastness, FDA(108) viability, and Sudan black stain(108) for lipid bodies, 108 culture growth reading done. After week 6 acid fastness, viability and culture growth decreased. 144 phenotypic DST done using MTT assay. A higher inhibitory drug concentration was required at the 6th week compared to the baseline and C50 (RMP=0.5;INH=0.1; STM=2.0 and for EMB=4.0), yet the proportion of lipid body containing bacilli increased continuously in all lineages.
Conclusion: Our study showed that the mycobacteria lineages behaved similarly in all media systems and reached stationary phase at similar time. The increased drug concentration observed at the 6th week coincided with the decline in viable bacilli in all media systems, thus attributing this phenomena to lipid body accumulation alone was difficult.
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Mycobacterium tuberculosis, LB%, Drug Tolerance, and MTT Assay.