Determination of ethambutol MICs for Mycobacterium tuberculosis and Mycobacterium avium isolates by resazurin microtitre assay

Department of Microbiology and Molecular Biology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases (Indian Council of Medical Research), PO Box 1101, Dr M. Miyazaki Marg, Tajganj, Agra 282001, India.
Journal of Antimicrobial Chemotherapy (Impact Factor: 5.31). 08/2007; 60(1):152-5. DOI: 10.1093/jac/dkm117
Source: PubMed


To test susceptibilities of Mycobacterium tuberculosis (MTB) isolates to ethambutol by the Löwenstein-Jensen (LJ) proportion method and resazurin microtitre assay (REMA) and to evaluate REMA for the determination of ethambutol MICs for MTB and Mycobacterium avium isolates.
A total of 50 MTB and 20 M. avium isolates were tested to determine the MICs of ethambutol by REMA and agar dilution method. MTB isolates were also tested by the LJ proportion method.
REMA provided ethambutol susceptibility results for all the isolates within 8-9 days. For MTB isolates, REMA showed 96.7% sensitivity, 100.0% specificity and 98.0% accuracy when LJ proportion results were taken as 'gold standard'. For both MTB and M. avium isolates, the MICs determined by REMA were lower than those determined in agar medium, indicating that MIC values determined by REMA are closer to the actual MICs for the isolates.
REMA can be used as a rapid and inexpensive method for mycobacterial drug susceptibility testing against ethambutol. In comparison with the agar method, the MICs determined by REMA can more accurately be correlated with achievable plasma concentrations of antimycobacterial agents.

Download full-text


Available from: Vishnu dutt Sharma,
  • Source
    • "These are not deposited in public strain collection centers. Susceptibility testing for all the drugs was performed by LJ proportion [6] and REMA methods [7] [8]. Cultures were grown in Sauton's liquid medium at 37 °C and harvested in late log phase (4 weeks). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Drug resistance particularly, multi drug resistance tuberculosis (MDR-TB) has emerged as a major problem in the chemotherapy of tuberculosis. Ofloxacin (OFX) has been used as second-line drug against MDR-TB. The principal target of the OFX is DNA gyrase encoded by gyrA and gyrB genes. Many explanations have been proposed for drug resistance to OFX but still some mechanisms are unknown. As proteins manifest most of the biological processes, these are attractive targets for developing drugs and diagnostics/therapeutics. We examined the OFX resistant Mycobacterium tuberculosis isolates by proteomic approach (2DE-MALDI-TOF-MS) and bioinformatic tools under OFX induced conditions. Our study showed fourteen proteins (Rv0685, Rv0363c, Rv2744c, Rv3803c, Rv2534c, Rv2140c, Rv1475c, Rv0440, Rv2245, Rv1436, Rv3551, Rv0148, Rv2882c and Rv0733) with increased intensities in OFX resistant and OFX induced as compared to susceptible isolates. Bioinformatic analysis of hypothetical proteins (Rv2744c, Rv2140c, Rv3551 and Rv0148) revealed the presence of conserved motifs and domains. Molecular docking showed proper interaction of OFX with residues of conserved motifs. These proteins might be involved in the OFX modulation/neutralization and act as novel resistance mechanisms as well as potential for diagnostics and drug targets against OFX resistance.
    Journal of proteomics 07/2015; 127. DOI:10.1016/j.jprot.2015.07.031 · 3.89 Impact Factor
  • Source
    • "Five total sensitive (rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin, kanamycin and amikacin) and six amikacin as well as kanamycin resistant (sensitive to other first line and second line drugs) M. tuberculosis isolates were obtained from the Mycobacterial Repository Centre of National JALMA Institute for Leprosy & Other Mycobacterial Diseases, Agra, India. Susceptibility testing for all the drugs was performed by LJ proportion [16] and REMA methods [17] [18] "
    [Show abstract] [Hide abstract]
    ABSTRACT: Unlabelled: Kanamycin (KM) and amikacin (AK) are the key aminoglycoside drugs against tuberculosis (TB) and resistance to them severely affects the options for treatment. Many explanations have been proposed for drug resistance to these drugs but still some mechanisms are unknown. Proteins are the functional moiety of the cell and manifest in most of the biological processes; so, these are potential foci for the development of new therapeutics, diagnostics and vaccine. We examined the KM and AK resistant isolates of Mycobacterium tuberculosis using proteomic analysis comprising of two dimensional gel electrophoresis (2DGE), matrix assisted laser desorption ionization time-of-flight/time-of flight (MALDI-TOF/TOF) and bioinformatic tools like BLASTP, InterProScan, KEGG motif scan and molecular docking. Proteins intensities of twelve spots were found to be consistently increased in KM and AK resistant isolates and these were identified as Rv3867, Rv1932, Rv3418c, Rv1876, Rv2031c, Rv0155, Rv0643c, Rv3224, Rv0952, and Rv0440. Among these, Rv3867 and Rv3224 were identified as proteins with unknown function. All the proteins identified were cellular proteins. Molecular docking shows the proper interaction of both drugs with these molecules. Also, Rv1876 and Rv3224 were found to be probably involved in iron regulation/metabolism indicating the role of iron in imparting resistance to second line drugs. Biological significance: The study that was carried out shows that two dimensional electrophoresis along with mass spectrometry is still the best approach for proteomic analysis. To the best of our knowledge it is the first ever report on proteomic analysis of M. tuberculosis isolates resistant to second line drugs (kanamycin and amikacin). The major finding implicates that the genes/proteins involved in iron metabolism and the two hypothetical proteins (Rv3867 and Rv3224) might be playing some crucial role in contributing resistance to second line drugs. Further exploitation in this direction may lead to the development of newer therapeutics against tuberculosis.
    Journal of proteomics 09/2013; 94. DOI:10.1016/j.jprot.2013.08.025 · 3.89 Impact Factor
  • Source
    • "scordant results ( 3 false resistant and 23 false sensi - tive ) ( Table 2 ) , resulting in a lower value of sensitivity , low value of specificity ( Montoro et al . , 2005 ) and reasonable correlation ; previous sensitivity results ranged between 92 and 98% ( 6 , 8 , 13 ) , specificity results between 98 and 100% ( Luna - Herrera et al . , 2003 , Jadaun et al . , 2007 ) and correla - tion was reasonable ( Madison et al . , 2002 ) . According to the literature , the INH high - resistance isolates showed sim - ilar results to those of Palomino et al . ( Palomino et al . , 2002 ) , RMP and STR high - resistance isolates showed worse results than Palomino et al . ( 2002 ) and Tudó et al . ( 2010 ) , resp"
    [Show abstract] [Hide abstract]
    ABSTRACT: We assessed the performance of REMA in comparison with BACTEC MGIT 960 in the susceptibility testing of 80 Mycobacterium tuberculosis clinical isolates from Clemente Ferreira Institute against four drugs. REMA proved to be a rapid and accurate method, providing excellent correlation with BACTEC MGIT 960, with the exception of results for the ethambutol drug.
    Brazilian Journal of Microbiology 05/2013; 44(1):281-5. DOI:10.1590/S1517-83822013005000028 · 0.59 Impact Factor
Show more