A Simple In Vitro Bioassay Protocol Against Latent Phase Mycobacterial Cells
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Date
2016-06
Authors
Lai, Hung Wei
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Abstract
Development of new drugs against latent tuberculosis (TB) is partly hindered by the complexity of laboratory protocol for dormant in vitro assay. Therefore, the main aim of this study was to improvise a simple protocol for latent TB by manipulating normal growth parameters using four surrogate Mycobacterium species: M. smegmatis, M. fortuitum, M. kansasii, and M. tuberculosis H37Ra. Initial growth optimizations by manipulating nutrient availability, incubation condition, O2 content, and aeration of the culture system were carried out in a batch-culture system over 10 - 12 days. The results showed that an addition of 10 % enrichment supplement in combination with a static culture system in sub-anaerobic environment of 4 % CO2, and an O2 content of 0.25 headspace ratio induced the mycobacterial cells to enter latent phase of growth. These growth manipulations also extended the duration of latent phase to 5 - 6 days compared to the control cells undergoing normal growth. Replication of the optimized parameters in a 96-well microplate system for use in the assay of multiple samples produced longer duration of latent phase. Assessment of the metabolic rate of the latent phase cells by measuring the adenosine 5’-triphosphate levels using luciferase bioluminescence assay showed a decline in luminescence values of 1.6 – 2.3 folds compared to the log phase of the Mycobacterium species. Observation of the external structural morphology of these dormant cells under a scanning electron microscope showed that these cells were healthy and intact but lacked septate formation, indicating that they were alive but
not undergoing active replication. Finally, the protocol for activity against latent phase mycobacteria was improvised by adapting the procedure of colorimetric microplate assay using tetrazolium bromide as a redox reagent. The protocols were tested using known TB drugs. The results based on minimum inhibitory concentration (MIC) values of the drugs were compared to their activity against log phase cells. The newly established protocol for measuring drug susceptibility against dormant Mycobacterium species were successful to a certain extent, which can be seen by the difference in MIC value between log and latent phase culture of M. smegmatis and M. fortuitum. However, the protocol did not correspond well against dormant M. tuberculosis H37Ra, suggesting further improvement of the assay protocol is still required. In conclusion, the findings in this study showed that normal growth parameters at the right proportion could indeed induce mycobacterial cells to enter a longer latent phase of growth and using such simulation, it is possible to develop a simple laboratory protocol to test the activity of potential agents against latent TB.
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To improvise a simple protocol for latent TB , by manipulating normal growth parameters