Activity of the pure compounds Asperteretal B, Aspulvinone E, Aspulvinone G, Butyrolactone I, Butyrolactone II, Flavipesolide C, Terretonin, and Terretonin A isolated from the fungus Aspergillus terreus ICMP 477 against Mycobacterium abscessus and M. marinum.

  

The fungus Aspergillus terreus ICMP 477 was isolated in September 1961 in Auckland, Aotearoa New Zealand, from sheep’s wool incubated at 30 °C. Forty Potato Dextrose Agar plates were inoculated with ICMP 477 and incubated at room temperature for 3 weeks. Fully grown fungal plates were freeze-dried (26.57 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 an orange oil (2.45 g). The crude product was subjected to C8 reversed-phase column chromatography eluting with a gradient of H₂O/MeOH to give five fractions. The pure compounds were obtained after further fractionation by Sephadex LH-20 and silica gel column chromatography.

Antimicrobial evaluation of the pure compounds was assessed against Mycobacterium abscessus and M. marinum. Because of the slow growth of many mycobacterial species, we routinely use luciferase-tagged strains for our assays. M. abscessus BSG301 and M. marinum 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).

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). M. abscessus was grown at 37 °C and M. marinum at 28 °C. Cultures were grown until they reached stationary phase (approximately 3-5 days for M. abscessus BSG301 and 7-10 days for M. marinum 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 triplicate to the wells of a black 96-well plate (Nunc, Thermo Scientific) at a concentration of 128 μg/mL. Then, 50 μL of diluted bacterial culture was added giving final compound concentrations of 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 M. abscessus and 10 μg/mL for M. marinum. 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).

Data is provided as Area Under Curve (AUC) values of luminescence readings for extracts (column = auc) and controls (column = median_ctrl_auc) and corresponding log reduction in AUC comparing extracts and controls (column = log_reduction_auc). Experiments were performed with three technical replicates of one to two biological replicate of each testing bacterium (column = organism [MA, M. abscessus; MM, M. marinum) depending on the quantity of pure compound available. 

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 Mycobacterium tuberculosis 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 Mycobacterium tuberculosis 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).