title: Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence.
abstract: OBJECTIVE
  Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format.
  
  
  METHODS
  We have used strains that express the bacterial lux operon and therefore do not require exogenous substrate to produce light, as well as strains expressing the firefly luciferase that need luciferin substrate. Results were compared with those obtained using the resazurin reduction assay and cfu plating.
  
  
  RESULTS
  Using bioluminescence we were able to reduce the time required to measure the MIC and bactericidal concentrations of antimicrobials to just 3 and 6 days, respectively. Furthermore, antibacterial activity against intracellular mycobacteria was detected within 2 days post-infection. Results were comparable to those obtained by conventional methods.
  
  
  CONCLUSIONS
  We have developed a simple and rapid method for screening antimycobacterial drugs in culture and in macrophages. The use of autoluminescent bacteria also facilitates the determination of growth and inhibition kinetics. The method is cost-effective, can easily be adapted to a larger scale and is amenable to automation. Current efforts are directed towards applying this technology to drug screening in vivo.
authors.affiliation: Microbiology, Department of Medicine,  Imperial College London, South Kensington Campus, London, SW7 2AZ,  UK.
authors.forename: Nuria
authors.initials: N
authors.lastname: Andreu
authors.forename: Taryn
authors.initials: T
authors.lastname: Fletcher
authors.forename: Nitya
authors.initials: N
authors.lastname: Krishnan
authors.forename: Siouxsie
authors.initials: S
authors.lastname: Wiles
authors.forename: Brian D
authors.initials: BD
authors.lastname: Robertson
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D003365:Costs and Cost Analysis; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000191:economics / Q000379:methods; D008264:Macrophages / Q000382:microbiology*; D008826:Microbial Sensitivity Tests / Q000191:economics / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D000995:Antitubercular Agents; D008156:Luciferases
references.citation: Antimicrob Agents Chemother. 1994 Nov;38(11):2557-63
references.pmid: 7872747
references.citation: Antimicrob Agents Chemother. 1996 Sep;40(9):2206-8
references.pmid: 8878609
references.citation: Microbes Infect. 2009 Dec;11(14-15):1114-21
references.pmid: 19682599
references.citation: Curr Microbiol. 1996 Sep;33(3):167-75
references.pmid: 8672093
references.citation: Infect Immun. 2005 Jul;73(7):3878-87
references.pmid: 15972473
references.citation: Antimicrob Agents Chemother. 1993 Jun;37(6):1348-52
references.pmid: 8328785
references.citation: Microbiology (Reading). 2002 Oct;148(Pt 10):2929-2936
references.pmid: 12368426
references.citation: Tuberculosis (Edinb). 2008 Mar;88(2):112-6
references.pmid: 18486045
references.citation: Antimicrob Agents Chemother. 2007 May;51(5):1787-94
references.pmid: 17307984
references.citation: PLoS One. 2010 May 24;5(5):e10777
references.pmid: 20520722
references.citation: Antimicrob Agents Chemother. 2007 Apr;51(4):1380-5
references.pmid: 17210775
references.citation: Antimicrob Agents Chemother. 1996 Jun;40(6):1536-41
references.pmid: 8726034
references.citation: Antimicrob Agents Chemother. 1996 Feb;40(2):400-7
references.pmid: 8834887
references.citation: Antimicrob Agents Chemother. 2002 Aug;46(8):2720-2
references.pmid: 12121966
references.citation: Antimicrob Agents Chemother. 2003 Dec;47(12):3682-7
references.pmid: 14638465
references.citation: Antimicrob Agents Chemother. 2010 Jul;54(7):2806-13
references.pmid: 20421401
references.citation: Antimicrob Agents Chemother. 1996 Jun;40(6):1542-4
references.pmid: 8726035
references.citation: Biochem Biophys Res Commun. 2000 Dec 20;279(2):457-61
references.pmid: 11118308
references.citation: J Clin Microbiol. 2002 May;40(5):1873-4
references.pmid: 11980982
references.citation: Antimicrob Agents Chemother. 2006 Apr;50(4):1245-50
references.pmid: 16569835
references.citation: Antimicrob Agents Chemother. 1997 May;41(5):1004-9
references.pmid: 9145860
references.citation: Clin Vaccine Immunol. 2010 Apr;17(4):513-7
references.pmid: 20107000
references.citation: J Invest Dermatol. 2011 Apr;131(4):907-15
references.pmid: 21191403
references.citation: J Antimicrob Chemother. 2003 Aug;52(2):199-203
references.pmid: 12865388
references.citation: Antimicrob Agents Chemother. 1995 Mar;39(3):754-6
references.pmid: 7793886
references.citation: Tuberculosis (Edinb). 2008 Mar;88(2):162-3
references.pmid: 18486061
references.citation: Biochem Biophys Res Commun. 1998 Dec 18;253(2):431-6
references.pmid: 9878553
references.citation: Antimicrob Agents Chemother. 1997 Mar;41(3):570-4
references.pmid: 9055994
references.citation: Mol Imaging. 2005 Apr-Jun;4(2):137-42
references.pmid: 16105511
references.citation: Antimicrob Agents Chemother. 2001 Jul;45(7):1943-6
references.pmid: 11408205