Antibacterial activity of extracts from Aspergillus terreus ICMP 477 against Escherichia coli and Staphylococcus aureus

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.

菌株ICMP 477于1961年9月在新西兰奥克兰市（奥特亚罗瓦 Aotearoa New Zealand），从30℃培养的绵羊羊毛样本中分离得到。该菌株被鉴定为土曲霉（Aspergillus terreus），这一鉴定结果得到GenBank序列MW862777的支持。将ICMP 477菌株接种于马铃薯葡萄糖琼脂（Potato Dextrose Agar，PDA）平板，于室温下培养。

将40块PDA平板接种ICMP 477菌株，于室温下培养3周。待真菌菌落完全生长后，将平板进行冷冻干燥处理，得到干重26.57 g的样品；随后先后用甲醇（MeOH，2×500 mL）萃取4小时，再用二氯甲烷（CH₂Cl₂，2×500 mL）萃取过夜。合并有机萃取液，经减压浓缩得到橙色油状物2.45 g。将粗产物进行C8反相柱色谱分离，以水/甲醇梯度体系洗脱，最终得到5个组分（F1–F5）。

将萃取所得干燥样品溶于二甲基亚砜（DMSO，Sigma-Aldrich，美国密苏里州圣路易斯市），配制成25 mg/mL的母液；随后用穆勒-辛顿肉汤II（Mueller Hinton broth II，MHB，新西兰奥克兰Fort Richard公司）进行梯度稀释，得到最高浓度为2 mg/mL的待测样品。取200 µL待测样品加入96孔板（Thermo Fisher，货号NUN167008，美国马萨诸塞州沃尔瑟姆市）顶部的相邻两个孔中；向其余孔内加入100 µL MHB培养基，再依次向下对每个孔加入100 µL样品溶液进行两倍梯度稀释，最后弃去多余的100 µL体系。向所有孔内加入光密度600 nm值为0.01（约1×10⁶菌落形成单位/mL）的生物发光菌株悬液：分别为金黄色葡萄球菌（Staphylococcus aureus）Xen 36（PerkinElmer Inc.，美国马萨诸塞州）和大肠杆菌（Escherichia coli）25922 lux。此时体系中待测样品的最高浓度为1 mg/mL，最低浓度为16 µg/mL。所有体系中二甲基亚砜的体积分数最大为4%，因此阴性对照采用相同浓度的二甲基亚砜体系进行平行测试。

采用Victor X-3化学发光微孔板读数仪（PerkinElmer Inc.，美国马萨诸塞州）检测各孔的发光强度，积分时间为1秒，分别在0、2、4和24小时四个时间点读取数值；检测期间将96孔板置于37℃、100 rpm振荡条件下培养。本实验中最低抑菌浓度（minimum inhibitory concentration，MIC）定义为使细菌发光强度降低1个对数级的样品浓度。

本数据集提供了各萃取组分与对照组的发光读数曲线下面积（Area Under Curve，AUC），以及组分与对照组之间AUC的对应对数减少值。所有实验均针对每种受试菌株设置1次生物学重复，并进行2次技术重复。