title: A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability.
abstract: 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 5days 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: >4mg/L, 8mg/L and 2mg/L, respectively) compared to extracellularly (MIC90: 0.5mg/L for AMI and EMB; 0.25mg/L for LEV). The reverse was the case for INH (IC: 0.064mg/L vs EC: 0.25mg/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.
authors.affiliation: Department of Clinical Microbiology, Linköping University Hospital, Sweden.
authors.forename: Marie C
authors.initials: MC
authors.lastname: Larsson
authors.affiliation: Department of Medical Microbiology, Linköping University, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Lerm
authors.affiliation: Department of Clinical Microbiology, Karolinska University Hospital Department of Clinical Microbiology, MTC-Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden; Department of Microbiology, The University of the West Indies, Kingston, Jamaica.
authors.forename: Kristian
authors.initials: K
authors.lastname: Ängeby
authors.affiliation: Department of Clinical Microbiology, Linköping University Hospital, Sweden.
authors.forename: Michaela
authors.initials: M
authors.lastname: Nordvall
authors.affiliation: Public Health Agency of Sweden, Stockholm, Sweden.
authors.forename: Pontus
authors.initials: P
authors.lastname: Juréen
authors.affiliation: Department of Medical Microbiology, Linköping University, Sweden; Department of Clinical Microbiology and Infectious Diseases, Kalmar County Hospital, Sweden; Department of Medicine and Optometry, Linnaeus University, Kalmar, Sweden. Electronic address: tschon@hotmail.com.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schön
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival; D015169:Colony Count, Microbial; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000032:analysis*; D008163:Luminescent Measurements; D008264:Macrophages / Q000382:microbiology* / Q000502:physiology; D008826:Microbial Sensitivity Tests / Q000379:methods*; D050296:Microbial Viability / Q000187:drug effects*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000502:physiology
chemical_list: D000995:Antitubercular Agents; D008156:Luciferases
keywords: Drug susceptibility testing; Intracellular; Levofloxacin; Luminescence; Minimal inhibitory concentration; Tuberculosis