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

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.

SUBMITTER: den Hertog AL

PROVIDER: S-EPMC4227065 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

den Hertog Alice L AL Menting Sandra S Smienk Ernst T ET Werngren Jim J Hoffner Sven S Anthony Richard M RM

BMC infectious diseases 20140710

<h4>Background</h4>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 ...[more]

PMID: 25011623

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Project description:A novel method for the detection of any alteration within a defined sequence has recently been demonstrated (A. Lishanski, N. Kurn, and E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin. Chem. 46:9, 2000). Essential to this method are the generation of partial duplexes that are capable of forming four-stranded structures and the ability to detect inhibition of branch migration in these structures (I. G. Panyutin and P. Hsieh, J. Mol. Biol. 230:413-424, 1993). Inhibition of branch migration indicates the presence of sequence alteration. This mutation detection method, termed branch migration inhibition (BMI), is suitable for the detection of drug resistance in M. tuberculosis, which is frequently associated with multiple mutations within known genes. We describe the genotypic determination of the rifampin (RMP) and pyrazinamide (PZA) susceptibilities of M. tuberculosis isolates, using BMI coupled with the luminescence oxygen channeling immunoassay (LOCI) (E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426-5430, 1994). RMP and PZA resistances are associated with multiple mutations within the rpoB and pncA genes, respectively. M. tuberculosis genomic DNA samples prepared from 46 clinical isolates were used for genotypic determination of RMP resistance in a "blind study." Similarly, PZA resistance was determined using genomic DNA samples prepared from 37 clinical isolates. Full agreement of the genotypic and phenotypic determinations of drug susceptibility was demonstrated. RMP susceptibility determination directly from cells of 10 clinical isolates grown in culture was also demonstrated. The genotypic result of only 1 out of 10 isolates did not agree with the phenotypic susceptibility testing result. Sequence analysis of the rpoB gene of this clinical isolate revealed a single base substitution, most likely a silent point mutation. The new BMI-LOCI mutation detection method is a rapid and accurate procedure for the genotypic determination of the RMP and PZA susceptibilities of M. tuberculosis clinical isolates. BMI can also be detected by using commercially available automated enzyme-linked immunosorbent assay plate formats (Lishanski et al., Nucleic Acids Res. 28:E42, 2000).

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

Publications

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

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