Evaluation of crystal violet decolorization assay and resazurin microplate assay for antimycobacterial screening

Abstract

The main obstacle in antimycobacterial discovery is the extremely slow growth rates of pathogenic mycobacteria that lead to the long incubation times needed in antimycobacterial screening. Some in vitro testings has been developed and are currently available for antimycobacterial screening. The aim of the study was to compare Resazurin Microplate Assay (REMA) and Crystal Violet Decolorization Assay (CVDA) for testing mycobacteria susceptibility to isoniazid and rifampicin as well as for antimycobacterial screening of natural products (NP). Mycobacterium tuberculosis strain H37Rv and Mycobacterium smegmatis strain mc2 155 were used as tested mycobacteria. Serial two-fold dilutions from 0.0625 to 1.0 μg/mL for the isoniazid and rifampicin and from 6.25 to 100.0 μg/mL for the NP A and B were prepared. Tested mycobacteria were then incubated with tested drugs or NPs in each growth medium at 37 °C for 7 days for M. tuberculosis and 3 days for M. smegmatis . MIC values against M. tuberculosis were interpreted 24–48 h after adding resazurin or at least 72 h after adding crystal violet, whereas MIC values against M. smegmatis were interpreted 1 h after adding resazurin or 24 h after adding crystal violet. The MIC values against M. tuberculosis interpreted by REMA were 0.0625, 0.0625, 6.25, and >100 μg/mL for rifampicin, isoniazid, NP A, and NP B, respectively, and those interpreted by CVDA were 0.0625, 0.0625, 6.25, and >100 μg/mL for rifampicin, isoniazid, NP A, and NP B, respectively. Moreover, the MIC values against M. smegmatis interpreted by REMA were 0.0625, >1, 6.25, and 100 μg/mL for rifampicin, isoniazid, NP A, and NP B, respectively, and those interpreted by CVDA were 0.125, >1, 6.25, and >100 μg/mL for rifampicin, isoniazid, NP A, NP B respectively. In conclusion, REMA is faster and easier than CVDA to interpret MIC values, however CVDA produces higher MIC values than REMA for rifampicin and NP B in M. smegmatis susceptibility testing. Therefore, REMA and CVDA can be used for antimycobacterial screening.

Antibiotic susceptibility of mycobacteria isolated from ornamental fish

“…Nontuberculous mycobacteria are known to be ubiquitous in the environment. Mycobacterium marinum , M. chelonae , M. peregrinum , M. gordonae , M. fortuitum , M. abscessus , M. mucogenicum , M. neoaurum , M. septicum , M. senegalense and M. szulgai have been isolated from diseased fish ( 1 , 11 , 16 , 44 , 45 , 63 , 64 ) and have been shown to be the causative agents of pulmonary, skin, soft tissue and disseminated diseases in humans ( 1 , 10 , 11 , 16 , 36 , 46 , 54 , 55 ). As in other countries, in Poland also Kwiatkowska et al .…”

High-throughput, multiplex microfluidic test strip for the determination of antibiotic susceptibility in uropathogenic E. coli with smartphone detection

“…Resazurin has been used in many different plate assays studying bacterial and cellular growth, mitochondrial function, and MIC of antimicrobial agents. However, with many cell viability test methods the resazurin is added towards the end of the experiment and incubated for several hours to measure metabolic activity after a primary incubation period with antimicrobial agent but no dye, to reduce the risk of the dye interacting with the antimicrobial (Kim and Jang, 2018, Teh et al, 2017, Rakhmawatie et al, 2019, Elshikh et al, 2016, Sarker et al, 2007). In contrast, for the novel microcapillary BMD method, the resazurin is present from the start.…”

Cinnamomum sp. and Pelargonium odoratissimum as the Main Contributors to the Antibacterial Activity of the Medicinal Drink Horchata: A Study Based on the Antibacterial and Chemical Analysis of 21 Plants

“…Ethanol (95% v / v ) was then used to dissolve the biofilm, and its eradication was measured by spectrophotometry at an optical density of 570 nm, by comparing it with positive controls (bacteria growth in media only). Finally, the percentage of biofilm eradication was calculated [ 55 , 56 , 58 ]. The assays were performed in triplicate in three independent experiments, using the same negative, sterile, and positive controls previously described.…”