2.6.5. Antibacterial Activity against *Cutibacterium Acnes*

Susceptibility of the *Cutibacterium acnes* bacteria (ATCC 6919) was tested using the microdilution broth assay as described by Lall et al. [21]. Briefly, 0.1 mL of extract (2 mg/mL) and tetracycline (0.2 mg/mL) were serially diluted in 100 μL of Brain Heart Infusion (BHI) broth in a 96-well plate. Concentrations of plant extract ranged from 3.91 to 500 μg/mL, while the positive control, tetracycline was tested from 0.39 to 50 μg/mL. Cultures of *C. acnes* grown for 72 h at 37 ◦C on BHI agar supplemented with 1% glucose were inoculated in BHI broth at a concentration of 1.5 <sup>×</sup> 108 CFU/mL (OD600 <sup>=</sup> 0.132). One hundred microlitres of the bacterial suspension was then added to the relevant test wells. The 96-well plates were then incubated at 37 ◦C for 72 h in an Anaerocult® jar with Anaerocult® A (Merck, Darmstadt, Germany) for the generation of CO2. Control plates were included and consisted of *C. acnes* without treatment, an extract vehicle DMSO treatment (2.5% v/v) and a BHI media control. After 72 h, 0.02 mL of PrestoBlue® (LTC Tech (Pty) Ltd., Johannesburg, South Africa) reagent was added. The MIC was then determined after 1 h of incubation.
