DOI: 10.17608/k6.auckland.14503584.v1
title: Activity of the pure compounds (2E)–cillifuranone, taiwapyrone, and pachybasin isolated from the fungus Neodidymelliopsis sp. against Mycobacterium abscessus and M. marinum.

description: The ascomycete fungus <i>Neodidymelliopsis</i> sp. (ICMP 11463) was isolated in October 1991 from a leaf spot on New Zealand native Pittosporum. Fifty-five Potato Dextrose Agar plates were inoculated with ICMP 11463 and incubated at room temperature for 4 weeks. Fully grown fungal plates were freeze-dried (108.6 g, dry weight) and extracted with MeOH (2 × 500 mL) for 4 h followed CH2Cl2 (2 × 500 mL) overnight. Combined organic extracts were concentrated under reduced pressure to afford a red/brown gum (0.51 g). The crude product was subjected to C8 reversed-phase column chromatography eluting with a gradient of H2O/MeOH to afford five fractions (F1–F5). The pure compounds taiwapyrone, pachybasin, and (2E)–cillifuranone were obtained after further fractionation by Sephadex LH-20 and silica gel column chromatography. <br>Antimicrobial evaluation of the pure compounds was assessed against <i>Mycobacterium abscessus </i>and <i>M. marinum</i>. Because of the slow growth of many mycobacterial species, we routinely use luciferase-tagged strains for our assays. <i>M. abscessus</i> BSG301 and <i>M. marinum</i> BSG101 (1) are stable bioluminescent derivatives transformed with the integrating plasmid pMV306G13ABCDE (2). As bacteria only produce light when alive, bioluminescence is an excellent non-destructive real-time reporter to assay for anti-mycobacterial activity in microtitre plate formats using a luminometer (1,3,4) or in vivo using sensitive imaging equipment (5). <br>Mycobacterial cultures were grown with shaking (200 rpm) in Middlebrook 7H9 broth (Fort Richard, Auckland) supplemented with 10% Middlebrook ADC enrichment media (Fort Richard), 0.4% glycerol (Sigma-Aldrich) and 0.05% tyloxapol (Sigma-Aldrich). <i>M. abscessus </i>was grown at 37 °C and <i>M. marinum</i> at 28 °C. Cultures were grown until they reached stationary phase (approximately 3-5 days for <i>M. abscessus </i>BSG301 and 7-10 days for <i>M. marinum</i> BSG101) and then diluted in Mueller Hinton broth II (MHB) (Fort Richard) supplemented with 10% Middlebrook ADC enrichment media and 0.05% tyloxapol to give an optical density at 600 nm (OD600) of 0.001 which is the equivalent of ~106 bacteria per mL. Pure compounds were dissolved in DMSO and added in duplicate to the wells of a black 96-well plate (Nunc, Thermo Scientific) at doubling dilutions with a maximum concentration of 128 μg/mL. Then, 50 μL of diluted bacterial culture was added to each well of the compound containing plates giving final compound concentrations of 0-64 μg/mL and a cell density of ~5 × 105 CFU/mL. Rifampicin (Sigma-Aldrich) was used as positive control at 1000 μg/mL for <i>M. abscessus</i> and 10 μg/mL for <i>M. marinum</i>. Between measurements, plates were covered, placed in a plastic box lined with damp paper towels and incubated with shaking at 100 rpm at 37 °C for M. abscessus and 28 °C for M. marinum. Bacterial luminescence (as relative light units (RLU) was measured at regular intervals using a Victor X-3 luminescence plate reader (PerkinElmer) with an integration time of 1 s. More detailed protocols are available at protocols.io (6, 7).<br>References:1. Dalton JP, Uy B, Okuda K, Hall CJ, Denny WA, Crosier PS, Swift S, Wiles S (2017). Screening of anti-mycobacterial compounds in a naturally infected zebrafish embryo model. Journal of Antimicrobial Chemotherapy 72(2):421-427 (doi: 10.1093/jac/dkw421).2. Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible UE, Robertson BD, Wiles S (2010). Optimisation of bioluminescent reporters for use with Mycobacteria. PLOS One. 5(5): e10777 (doi:10.1371/journal.pone.0010777).3. Andreu N, Fletcher T, Krishnan N, Wiles S, Robertson BD (2012). Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence. Journal of Antimicrobial Chemotherapy. 67(2): 404-14 (doi: 10.1093/jac/dkr472). 4. Dalton JP, Uy B, Phummarin N, Copp BR, Denny WA, Crosier PS, Swift S, Wiles S (2016). Effect of common and experimental anti-tuberculosis treatments on <i>Mycobacterium tuberculosis </i>growing as biofilms. PeerJ. 4:e2717 (doi: 10.7717/peerj.2717).5. Andreu N, Zelmer A, Sampson SL, Ikeh M, Bancroft GJ, Schaible UE, Wiles S, Robertson BD (2013). Rapid in vivo assessment of drug efficacy against <i>Mycobacterium tuberculosis</i> using an improved firefly luciferase. Journal of Antimicrobial Chemotherapy. 68(9):2118-27 (doi: 10.1093/jac/dkt155).6. Grey A & Wiles S (2021). Bioluminescence-based Minimum Inhibitory Concentration (MIC) testing of pure compounds isolated from fungi against Mycobacterium marinum. Protocols.io. (doi: dx.doi.org/10.17504/protocols.io.3x7gprn). 7. Grey A & Wiles S (2021). Bioluminescence-based Minimum Inhibitory Concentration (MIC) testing of pure compounds isolated from fungi against Mycobacterium abscessus. Protocols.io. (doi: dx.doi.org/10.17504/protocols.io.bumcnu2w).<br>