Redox dyes in the detection of mycobacterial viability
Niemi, Jenni (2021)
2021
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2021-01-20
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202012048480
https://urn.fi/URN:NBN:fi:tuni-202012048480
Tiivistelmä
In this master’s thesis, I optimize the conditions of use of redox dyes to detect the viability of mycobacteria. Mycobacterium tuberculosis causes tuberculosis that infects millions of people every year. In 2019, 1.4 million people died of tuberculosis infection. M. tuberculosis is tolerant to several antibiotics, and therefore its investigation is important.
M. tuberculosis research can utilize Mycobacterium marinum, which is genetically very close to M. tuberculosis. In in vitro cultures, both M. tuberculosis and M. marinum, form biofilms containing antibiotic-tolerant persisting bacteria. In addition to bacteria, biofilms consist of extracellular polymeric material.
Redox staining of mycobacteria was studied with two different redox dyes, resazurin and 2,3,5-triphenyltetrazolium chloride (TTC). Metabolism of living cells reduces blue resazurin to pink, fluorescent resorufin. The yellowish colour of TTC, on the other hand, turns red by the cell metabolism as it is reduced to insoluble triphenylformazane crystals. Different concentrations of dyes and different measurement time points were optimized. Initial optimizations were performed using wild-type M. marinum. In optimized conditions, resazurin staining was used in the minimum duration of killing assay (MDK) of M. marinum and the sensitivity of resazurin staining was compared to bioluminescence from luciferase over-expressing M. marinum strain (MM-lux). Finally, resazurin staining was used to study the development of antibiotic tolerance in biofilms of avirulent M. tuberculosis.
A suitable protocol was found for resazurin staining of mycobacteria. Resazurin staining was found to act reliably with M. tuberculosis as well as with longer antibiotic exposures with M. marinum. In contrast, TTC staining of mycobacteria did not give reliable results under the conditions studied.
Based on resazurin staining, biofilm cultures of M. tuberculosis were found to be more tolerant to the antibiotic exposure than planktonic cultures. In biofilm cultures, the level of tolerance remained similar between five- and 12-day old biofilms.
Based on the results, resazurin staining is a rapid and promising way to study the viability of mycobacteria in samples, but it is less sensitive than for example luminescence-based detection methods. If it is not possible to measure the fluorescence or luminescence from the samples, the viability of the bacteria can also be detected visually by colour change.
M. tuberculosis research can utilize Mycobacterium marinum, which is genetically very close to M. tuberculosis. In in vitro cultures, both M. tuberculosis and M. marinum, form biofilms containing antibiotic-tolerant persisting bacteria. In addition to bacteria, biofilms consist of extracellular polymeric material.
Redox staining of mycobacteria was studied with two different redox dyes, resazurin and 2,3,5-triphenyltetrazolium chloride (TTC). Metabolism of living cells reduces blue resazurin to pink, fluorescent resorufin. The yellowish colour of TTC, on the other hand, turns red by the cell metabolism as it is reduced to insoluble triphenylformazane crystals. Different concentrations of dyes and different measurement time points were optimized. Initial optimizations were performed using wild-type M. marinum. In optimized conditions, resazurin staining was used in the minimum duration of killing assay (MDK) of M. marinum and the sensitivity of resazurin staining was compared to bioluminescence from luciferase over-expressing M. marinum strain (MM-lux). Finally, resazurin staining was used to study the development of antibiotic tolerance in biofilms of avirulent M. tuberculosis.
A suitable protocol was found for resazurin staining of mycobacteria. Resazurin staining was found to act reliably with M. tuberculosis as well as with longer antibiotic exposures with M. marinum. In contrast, TTC staining of mycobacteria did not give reliable results under the conditions studied.
Based on resazurin staining, biofilm cultures of M. tuberculosis were found to be more tolerant to the antibiotic exposure than planktonic cultures. In biofilm cultures, the level of tolerance remained similar between five- and 12-day old biofilms.
Based on the results, resazurin staining is a rapid and promising way to study the viability of mycobacteria in samples, but it is less sensitive than for example luminescence-based detection methods. If it is not possible to measure the fluorescence or luminescence from the samples, the viability of the bacteria can also be detected visually by colour change.