Open Access Research article

Evaluation of a microcolony growth monitoring method for the rapid determination of ethambutol resistance in Mycobacterium tuberculosis

Alice L den Hertog1*, Sandra Menting1, Ernst T Smienk1, Jim Werngren2, Sven Hoffner2 and Richard M Anthony1

Author Affiliations

1 Royal Tropical Institute, KIT Biomedical Research, Meibergdreef 39, 1105, AZ, Amsterdam, The Netherlands

2 Department of Diagnostics and Vaccines, Unit of Highly Pathogenic Microorganisms, Swedish Institute for Communicable Disease Control, S-171 82, Solna, Sweden

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BMC Infectious Diseases 2014, 14:380  doi:10.1186/1471-2334-14-380

Published: 10 July 2014

Abstract

Background

Due to the increasing prevalence of Mycobacterium tuberculosis strains resistant to one or more antibiotics, there is a need for new quantitative culture methods both for drug susceptibility testing and for validation of mutations putatively associated with drug resistance. We previously developed a (myco) bacterial culture method, in which multiple growing microcolonies are monitored individually. Transfer of the growing microcolonies to selective medium allows the effect on the growth rate of each individual colony to be determined. As entire growing colonies are exposed to antibiotics rather than re-subbed, a second lag phase is avoided and results are obtained more rapidly. Here we investigate the performance of the microcolony method to differentiate between ethambutol (EMB) resistant, intermediate and susceptible strains.

Methods

One week old microcolonies from a reference panel of four strains with known EMB susceptibility were transferred to different concentrations of EMB. Growth rates during the 1st 2 days of exposure were used to set up classification criteria to test and classify a blinded panel of 20 tuberculosis strains with different susceptibilities.

Results

For 18 strains (90%) reference culture results corresponded to our classifications based on data collected within 9 days of inoculation. A single strain was classified as Intermediate instead of Susceptible, and 1 strain could not be classified due to a contamination.

Conclusions

Using a microcolony growth monitoring method we were able to classify, within 9 days after inoculation, a panel of strains as EMB susceptible, intermediate or resistant with 90% correlation to the reference methods.

Keywords:
Ethambutol; Drug susceptibility testing; Mycobacterium tuberculosis; Culture