A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability
2014 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 106, 146-150 p.Article in journal (Refereed) Published
The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5 days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: greater than4 mg/L, 8 mg/L and 2 mg/L, respectively) compared to extracellularly (MIC90: 0.5 mg/L for AMI and EMB; 0.25 mg/L for LEV). The reverse was the case for INH (IC: 0.064 mg/L vs EC: 0.25 mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs.
Place, publisher, year, edition, pages
Elsevier, 2014. Vol. 106, 146-150 p.
Tuberculosis; Levofloxacin; Drug susceptibility testing; Intracellular; Luminescence; Minimal inhibitory concentration
Clinical Medicine Basic Medicine
IdentifiersURN: urn:nbn:se:liu:diva-112299DOI: 10.1016/j.mimet.2014.08.015ISI: 000343358200024PubMedID: 25194234OAI: oai:DiVA.org:liu-112299DiVA: diva2:765678
Funding Agencies|Swedish Heart and Lung Foundation (Oscar II Jubilee Foundation); Marianne and Marcus Wallenberg Foundation; Research Council of Southeast Sweden (FORSS)2014-11-242014-11-242015-03-12Bibliographically approved