title: The use of the microplate alamar blue assay (MABA) to assess the susceptibility of Mycobacterium lepraemurium to anti-leprosy and other drugs.
abstract: Although murine leprosy is no longer a common illness, our understanding of the biology of this disease is incomplete. One particular example of this concerns the etiologic agent Mycobacterium lepraemurium (MLM). MLM is a fastidious microorganism that is difficult to grow in axenic media; in a way, this has hampered attempts to thoroughly study its physiological and metabolic characteristics. MLM is an obligate intracellular bacillus that invades macrophages and replicates profusely with a generation time that oscillates between 0.5 and 11 days. In the present study, we have successfully maintained MLM alive for more than 12 days in vitro, providing us with an opportunity to study its susceptibility to several anti-leprosy agents and other drugs. To achieve this, we used a fluorescence reduction assay of alamar blue (a resazurin) in a microplate format (microplate-alamar-blue-assay; MABA), which is a highly sensitive, practical, and inexpensive method for assaying cell viability. We found that MLM was highly susceptible to clofazimine and rifampicin and was less susceptible to streptomycin, thiacetazone, kanamycin, dapsone, and ethionamide, in that order. MLM was not susceptible to four plant triterpenoids (oleanolic acid, neolignan-c, sitosterol, and ursolic acid) for which bactericidal activity has been reported in M. tuberculosis. Because the MABA has high sensitivity, it can be used to monitor the activity of microorganisms that are difficult to cultivate (such as M. lepraemurium), in response to various drugs, thus offering a method to complement the study of murine leprosy, about which many questions remain unanswered.
authors.affiliation: Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala, Colonia Santo Tomás, 11340, Mexico, D.F., Mexico.
authors.forename: Melby
authors.initials: M
authors.lastname: Mendoza-Aguilar
authors.forename: Laura
authors.initials: L
authors.lastname: Almaguer-Villagrán
authors.forename: Adelina
authors.initials: A
authors.lastname: Jiménez-Arellanes
authors.forename: Patricia
authors.initials: P
authors.lastname: Arce-Paredes
authors.forename: José Luis
authors.initials: JL
authors.lastname: Cid-Gutiérrez
authors.forename: Oscar
authors.initials: O
authors.lastname: Rojas-Espinosa
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D006651:Histocytochemistry; D007202:Indicators and Reagents / Q000737:chemistry; D007917:Leprostatic Agents / Q000494:pharmacology*; D008099:Liver / Q000737:chemistry / Q000382:microbiology / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests / Q000379:methods*; D050296:Microbial Viability / Q000187:drug effects; D009164:Mycobacterium Infections / Q000382:microbiology; D009167:Mycobacterium lepraemurium / Q000187:drug effects* / Q000472:pathogenicity; D010078:Oxazines / Q000737:chemistry*; D010936:Plant Extracts / Q000494:pharmacology; D014966:Xanthenes / Q000737:chemistry*
chemical_list: D007202:Indicators and Reagents; D007917:Leprostatic Agents; D010078:Oxazines; D010936:Plant Extracts; D014966:Xanthenes; C005843:resazurin