Antimicrobial susceptibility of rapidly growing mycobacteria using the rapid colorimetric method

Abstract

Drug susceptibility testing (DST) of rapidly growing mycobacteria (RGM) are recommended for guiding the antimicrobial therapy. We have evaluated the use of resazurin in Mueller-Hinton medium (MHR) for MIC determination of RGM and compared the results with those obtained with the reference standard broth microdilution in Mueller-Hinton (MH) and with the resazurin microtiter assay (REMA) in 7H9 broth. The MIC of eight drugs: amikacin (AMI), cefoxitin (FOX), ciprofloxacin (CIP), clarithromycin (CLA), doxycycline (DOX), linezolid (LZD), moxifloxacin (MXF) and trimethoprim-sulfamethoxazole (TMP-SMX) were evaluated against 76 RGM (18 species) using three methods (MH, MHR, and REMA) in a 96-well plate format incubated at 37 °C over 3-5 days. Results obtained in the MH plates were interpreted by the appearance of turbidity at the bottom of the well before adding the resazurin. MHR and 7H9-REMA plates were read by visual observation for a change in color from blue to pink. The majority of results were obtained at day 5 for MH and 1 day after for MHR and 7H9-REMA. However, the preliminary experiment on time to positivity results using the reference strain showed that the resazurin can be added to the MH at day 2 to produce the results at day 3, but future studies with large sets of strains are required to confirm this suggestion. A high level of agreement (kappa 1.000-0.884) was obtained between the MH and the MHR. Comparison of results obtained with 7H9-REMA, on the other hand, revealed several discrepancies and a lower level of agreement (kappa 1.000-0.111). The majority of the strains were resistant to DOX and TMP-SMX, and the most active antimicrobials for RGM were AMI and FOX. In the present study, MHR represented an excellent alternative for MIC determination of RGM. The results could be read reliably, more easily, and more quickly than with the classical MH method.

Resazurin Microtiter Assay for Clarithromycin Susceptibility Testing of Clinical Isolates of Mycobacterium abscessus Group

“…The visual reading may not be accurate, especially when bacterial growth is very thin, making it difficult to read visually. In addition, previous validation of resazurin in MIC determination alerts for the importance of clarithromycin induced resistance in RGM , which is the aim of the present study. Based on this test, we verified the accuracy of resazurin reading in clarithromycin susceptibility testing, which minimizes possible reading errors enabling an accurate reading of the susceptibility testing.…”

“…Ramis et al. for the first time described the use of resazurin as a good alternative method for MIC determination in Mycobacterium species, but the detection of inducible resistance by REMA was not verified. In order to fulfill this issue, we aimed to evaluate the performance of resazurin in a determining routine MIC of clarithromycin on M. abscessus group strains from a reference laboratory in Brazil.…”

Rifamycin O, An Alternative Anti-Mycobacterium abscessus Agent

“…The plates were covered and incubated at 37 • C for 3 days. Then, 20 µL of 0.025% (w/v) resazurin was added to each well and the plates were re-incubated overnight [23]. Fluorescence was measured (ex.…”

Down-regulation of N -acetylglucosamine-1-phosphate transferase (WecA) enhanced the sensitivity of Mycobacterium smegmatis against rifampin

“…; Ramis et al . ) as well as a traditional agar dilution method. The drug against which the sensitivity of WecA down‐regulation strain enhanced was selected and co‐treated with an existing WecA inhibitor tunicamycin on the growth of wild type Myco.…”

“…The morphological alterations of WecA down-regulation strain were observed by scanning electron microscopy (SEM). The susceptibility of WecA low-expression strain to anti-tuberculosis drugs was detected through a rapid resazurin microtitre assay (REMA) (Gold et al 2015;Ramis et al 2015) as well as a traditional agar dilution method. The drug against which the sensitivity of WecA down-regulation strain enhanced was selected and cotreated with an existing WecA inhibitor tunicamycin on the growth of wild type Myco.…”