Spot testing endolysin activity

Spot testing was used to examine the activity of the ORF28 endolysin against the E. faecalis strain isolated from the patient. 0.1 mL of an overnight culture of E. faecalis strain 587A, originally isolated by a clinical laboratory in Bratislava in June 2020, was grown in brain heart infusion (BHI) Broth and was inoculated into 3 mL of molten soft agar (BHI broth containing 0.7% agar). This was poured into plates over a layer of BHI agar (1.5%) and allowed to solidify and dry for approximately 15 min. Drops (3 μL) of dilutions of an ORF28 endolysin suspension were then applied to the surface of the solidified soft agar layer. The drops were allowed to dry into the soft agar layer, and the plates incubated overnight at 37°C. The plates were then examined for clear zones where the drops were originally placed, indicating the lytic activity of the endolysin against the E. faecalis strain.