The development and application of a high throughput methodology to determine MICs of Mycobacterium tuberculosis isolates against antimicrobial agents.

Abstract

Chapter 1 of this dissertation entails the introduction, aims, objectives and the literature review. Drug susceptibility testing of Mycobacterium tuberculosis is time consuming and expensive. Multi-point inoculation offers the advantage of testing multiple isolates on a series of solid media with a single breakpoint concentration of a drug in each plate or a series of different drug concentrations of one drug. We aimed to determine the reproducibility of MIC determination for anti-TB drugs of M. tuberculosis isolates using agar dilution with multi-point inoculation and thereafter validating the results by comparing it to classic agar dilution on quadrant plates and the MTT assay. Chapter 2 contains the manuscript that has been submitted for publication. This manuscript contains a brief introduction with the aim and objectives, and a detailed description of the methodology, results and discussion. Thirty M. tuberculosis isolates were grown in Middlebrook 7H9 broth with 20% Tween until mid-log phase was reached. Agar dilution MICs were determined on Middlebrook 7H10 agar for 11 anti-TB drugs at concentrations ranging from 128 to 0.125 mg/L. The agar plates were inoculated using a multi-point inoculation device with 36 points each delivering 1μL of a suspension of 1×104 cfu/ml. For the quadrant plate method and the MTT assay 100 μl of the same suspension was used. All tests were done 3 times in triplicate. Agar dilution with multi-point inoculation was found to be reproducible within the 11 anti-TB drugs tested and correlated well with agar dilution on quadrant plates and the MTT assay for the three anti-TB drugs tested. Chapter 3 entails a brief summary (synthesis) of the discussion found in the above-mentioned manuscript. The multi-point inoculation method has potential for wide scale application in breakpoint drug susceptibility testing as well as MIC testing of M. tuberculosis isolates. Lastly this dissertation contains the required references and appendices.