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Volume 7 Clinical Microbiology: Open Access Clinical Microbiology 2018
July 16-17, 2018
July 16-17, 2018 Melbourne, Australia
2nd International Conference on
Medical and Clinical Microbiology
In vitro starvation model for assessing phenotypic drug tolerance on Mycobacterium tuberculosis
lineages in Ethiopia
Wondimu Ashagre1,2, Endalamaw Gadisa1, Kassu Desta2, Girma Shumie1 and Wakweya Chali 1
1Armauer Hansen Research Institute, Ethiopia
2Addis Ababa University, Ethiopia
Background: Mycobacterium tuberculosis persist in the human host for decades and reactivation can occur at any point.
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 use in vitro starvation model in assessing if nutrient deprivation affects phenotypic drug tolerance in
Mycobacterium tuberculosis lineages circulating in Ethiopia.
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 Jenson medium was 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 we performed culture growth reading, acid fast stain (AFS) by Ziehl Neelson
(ZN), Lipid Bodies (LB) by Sudan black stain and viability by Fluorecin Diacetate (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 sub-cultured on SDW, PBS
and 7H9. Of these, 324 microscopic tests using (108 (ZN) acid fastness, 108 (FDA) viability and 108 (Sudan black stain) 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.
Wondimu Ashagre et al., Clin Microbiol 2018, Volume 7
DOI: 10.4172/2327-5073-C2-036