A Mycobacterium marinum Zone of Inhibition Assay as a Method for Screening Potential Antimycobacterial Compounds from Marine Extracts

Abstract

A novel screening method for antimycobacterial agents using Mycobacterium marinum was developed. M. marinum was selected as a model organism because it has a close phylogenetic relationship to M. tuberculosis, a relatively rapid doubling time, similar drug susceptibilities to M. tuberculosis, and less stringent safety requirements. More than 1000 crude marine and plant extracts were screened against M. marinum in a Zone of Inhibition (ZOI) assay, and twenty-one target extracts were identified. The crude organic extract of the marine sponge, Haliclona sp.10, was chosen for further investigation as it yielded a ZOI of 20 mm at a concentration of 80 μg/disk. Following bioassay-guided fractionation, (-)-papuamine was isolated, and yielded a 15 mm ZOI at a concentration of 25 μg/disk. In standard assays using M. marinum, (-)-papuamine exhibited both an MIC and an MBC₉₅ of 6.25 μg/mL. This is the first report of antimycobacterial activity for (-)-papuamine. In addition, when (-)-papuamine and other natural product extracts were tested for activity against both M. marinum and M. tuberculosis, activity was comparable between the two species. These data indicate that (-)-papuamine is a promising lead for the development of new antimycobacterial agents and that M. marinum is a useful surrogate for the screening of antimycobacterial compounds.

Antimycobacterial - Haliclona sp. 10 - (-)-papuamine - Mycobacterium marinum

References

• 1 Gbayisomore A, Lardisabal A A, Reichman L B. Update: prevention and treatment of tuberculosis.  Curr Opin Infect Dis. 2000;  13 155-9.
• 2 Dye C, Williams B G, Espinal M A, Raviglione M C. Erasing the world’s slow stain: strategies to beat multidrug-resistant tuberculosis.  Science. 2002;  295 2042-6.
• 3 Aubry A, Jarlier V, Escolano S, Truffot-Pernot C, Cambau E. Antibiotic susceptibility pattern of Mycobacterium marinum .  Antimicrob Agents Chemother. 2000;  44 3133-6.
• 4 Centers for Disease Control and Prevention Emergence of Mycobacterium tuberculosis with extensive resistance to sSecond-line drugs - worldwide, 2000 - 2004. MMWR 2006 55: 301-5.
• 5 Fusetani N, Yasumuro K, Matsunaga S, Hirota H. Haliclamines A and B, cytotoxic macrocyclic alkaloids from a sSponge of the genus Haliclona .  Tetrahedron Lett. 1989;  30 6891-4.
• 6 Falch B S, König G M, Wright A D, Sticher O. Ambigol A and B; new biologically active polychlorinated aromatic compounds from the terrestrial blue-green alga Fischerella ambigua .  J Org Chem. 1993;  58 6570-5.
• 7 Donia M, Hamann M T. Marine natural products and their potential applications as anti-infective agents.  Lancet Infect Dis. 2003;  3 338-48.
• 8 Rogall T, Wolters J, Flohr T, Bottger E C. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium .  Int J Syst Bacteriol. 1990;  40 323-30.
• 9 Tonjum T, Welty D B, Jantzen E, Small P LC. Differentiation of Mycobacterium ulcerans, M. marinum and M. haemophilum: mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA-DNA hybridization and 16S rRNA gene sequence analysis.  J Clin Microbiol. 1998;  36 918-25.
• 10 Stamm L M, Brown E J. Mycobacterium marinum: the generalization and specialization of a pathogenic mycobacterium.  Microbes Infect. 2004;  6 1418-28.
• 11 Kaattari I M, Rhodes M W, Kaattari S L, Shotts E B. The evolving story of Mycobacterium tuberculosis clade members detected in fish.  J Fish Dis. 2006;  29 509-20.
• 12 Barker L P, George K M, Falkow S, Small P LC. Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages.  Infect Immunol. 1997;  65 1497-504.
• 13 Werngren J, Olsson-Liljequist B, Gezelius L, Hoffner S E. Antimicrobial susceptibility of Mycobacterium marinum determined by E-test and agar dilution.  Scand J Infect Dis. 2001;  33 585-8.
• 14 Pagan-Ramos E, Song J, McFalone M, Mudd M H, Deretic V. Oxidative stress response andcharacterization of the oxyR-ahpC and furA-katG loci in Mycobacterium marinum .  J Bacteriol. 1998;  180 4856-64.
• 15 Somoskovi A, Parsons L M, Salfinger M. The molecular basis of resistance to isoniazid, rifampin and pyrazinamide in Mycobacterium tuberculosis .  Respir Res. 2001;  2 164-8.
• 16 Pagan-Ramos E, Master S S, Pritchett C L, Reimschuessel R, Trucksis M, Timmins G S. et al . Molecular and physiological effects of mycobacterial oxyR inactivation.  J Bacteriol. 2006;  188 2674-80.
• 17 Discotere A, Hermans K, Haesebrouck F. Piscine mycobacteriosis: a literature review covering the agent and the disease it causes in fish and humans.  Vet Microbiol. 2004;  99 159-66.
• 18 Sakai R, Kohmoto S, Higa T, Jefford C W, Bernardinelli G. Manzamine B and C, alkaloids from sponge Haliclona sp.  Tetrahedron Lett. 1987;  28 5493-6.
• 19 Kobayashi M, Chen Y J, Aoki S, In Y, Ishida T, Kitagawa I. Four new beta-carboline alkaloids isolated from two Okinawan marine sponges of Xestospongia sp. and Haliclona sp.  Tetrahedron. 1995;  51 3727-36.
• 20 Worthington M, Luo R Q, Pelo J. Copacabana method for spreading E. coli and yeast colonies.  BioTechniques. 2001;  30 738-42.
• 21 Baker B J, Scheuer P J, Schoolery J N. Papuamine and antifungal pentacyclic alkaloid from a marine sponge, Haliclona sp.  J Am Chem Soc. 1988;  110 965-6.
• 22 MARINLIT. A marine literature database maintained by the Marine Chemistry Group, University of Canterbury. Christchurch, New Zealand; 2002.
• 23 Sakai R, Higa T, Jefford C W, Bernardinelli G. Manzamine A, a novel antitumor alkaloid from a sponge.  J Am Chem Soc. 1986;  108 6404-5.
• 24 Yousat M, El Sayed K A, Rao K V, Lim C W, Hu J, Kelly M. et al . 12,34-Oxamanzamines, novel biocatalytic and natural products from manzamine producting indo-paccific sponges.  Tetrahedron. 2002;  58 7397-402.
• 25 Clark R J, Field K L, Charan R D, Garson M J, Brereton I M, Willis A C. The haliclonacyclamines, cytotoxic tertiary alkaloids from the tropical marine sponge Haliclona sp.  Tetrahedron. 1998;  54 8811-26.
• 26 Fahy E, Molinski T F, Harper M K, Sullivan B W, Faulkner D J. Haliclonadiamine, an antimicrobial alkaloid from the sponge Haliclona sp.  Tetrahedron Lett. 1988;  29 3427-8.
• 27 Borzilleri R M, Weinreb S M, Parvez M. Total synthesis of the unusual marine alkaloid (-)-papuamine utilizing a novel imino ene reaction.  J Am Chem Soc. 1995;  117 10 905-13.
• 28 Heifets L, Lindholm-Levy P. Comparison of bactericidal activities of streptomycin, amikacin, kanamycin, and capreomycin against Mycobacterium avium and M. tuberculosis .  Antimicrob Agents Chemother. 1989;  33 1298-301.
• 29 Franzblau S G, Witzig R S, McLaughlin J C, Torres P, Madico G, Hernandez A. et al . Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate alamar blue assay.  J Clin Microbiol. 1998;  36 362-6.
• 30 Xu Z, Barrow W W, Suling W J, Westbrook L, Barrow E, Lin Y. et al . Anti-HIV natural product (+)-calanolide A is active against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis .  Bioorg Med Chem. 2004;  12 1199-207.
• 31 Edrada R A, Proksch P, Wray V, Witte L, Müller W EG, Soest R WM. Four new bioactive manzamine-type alkaloids from the Philippine marine sponge Xestospongia ashmorica. J.  Nat Prod. 1996;  59 1056-60.

1 These authors contributed equally