ICMP_477_Extract_Testing_Ecoli_Saureus_Figshare.csv (10.63 kB)
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Antibacterial activity of extracts from Aspergillus terreus ICMP 477 against Escherichia coli and Staphylococcus aureus

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posted on 22.06.2022, 04:58 authored by Siouxsie WilesSiouxsie Wiles

ICMP 477 was isolated in September 1961 in Auckland, Aotearoa New Zealand, from sheep’s wool incubated at 30 °C. The identification of this culture as Aspergillus terreus is supported by GenBank sequence MW862777. Cultures of ICMP 477 were grown at room temperature on Potato Dextrose Agar (PDA). 

40 PDA 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 (F1–F5).    

Dry samples of extracts were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) to make a 25 mg/mL solution and then further diluted into Mueller Hinton broth II (MHB) (Fort Richard, Auckland, New Zealand) to achieve a maximum concentration of 2 mg/mL. Each extract (200 µL) was added to two adjacent wells along the top of the 96-well plate (Thermo Fisher, NUN167008, Waltham, MA, USA). MHB (100 µL) was then added to the remaining wells and extract solution (100 µL) serially diluted two-fold down the plate and discarded. Aliquots of bioluminescent derivatives of S. aureus (Xen 36 (PerkinElmer Inc., MA, USA)) and E. coli (25922 lux), at an optical density at 600 nm of 0.01 (approximately 1 × 106 colony forming units (CFU)/mL) were then added to all the wells. This gave a maximum concentration of 1 mg/mL and a minimum concentration of 16 µg/mL. The maximum volume/volume concentration of DMSO in all extracts was 4%; therefore, the negative control was tested at an identical concentration.

Luminescence was measured using a Victor X-3 luminescence plate reader (PerkinElmer Inc., MA, USA) with an integration time of 1s at 0, 2, 4 and 24 h to determine the minimum inhibitory concentration (MIC), between which times the plates were incubated at 37 °C with shaking at 100 rpm.  MIC was defined as causing a 1 log reduction in bacterial light production. 

Data is provided as Area Under Curve (AUC) of luminescence readings for extracts and controls and corresponding log reduction in AUC comparing extracts and controls. Experiments were performed with two technical replicates of one biological replicate of each testing bacterium.


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