Activity of Dpo42 against Biofilms

A spot test was performed to observe Dpo42 activity as described previously (Verma et al., 2009). In brief, LB agar was overlaid with 0.7% top agar inoculated with 200 μL of fresh log-phase bacterial culture. After drying, the purified Dpo42 (1 μg) was spotted onto the double-layer agar plate. Serial dilutions of Dpo42 (50, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, and 0.078 ng) were also dropped onto host strain double-layer agar plates. The elution buffer diluted in PBS was used as a control. After overnight incubation at 37°C, plates were observed for the formation of semi-clear spots.

The lytic activity of Dpo42 (0.25 μg/μL) toward E. coli isolates was tested at 37°C (Gu et al., 2014). Cell counts were performed at 0 min, 30 min, 1, 2, and 4 h. The elution buffer diluted in PBS was used as a negative control.

Escherichia coli isolates E9 and HXM were used to test the preventative effects of Dpo42 on biofilm formation as described previously (O’Toole et al., 1999), with some modifications. Briefly, E. coli E9 and HXM were cultured to logarithmic phase, centrifuged, resuspended in equal amounts of TSB medium, mixed and (100 μL) added to each well of a 96-well micro-titre plate. The wells were then divided into four groups. The elution buffer diluted in PBS, 10 μg/well Dpo42, 25 μg/well Dpo42 or 50 μg/well Dpo42 at the volume of 100 μL was added to different groups; 200 μL of sterile TSB medium was used as a negative control. Each group contained triplicate samples. The plate was covered, and bacteria were permitted to adhere and grow at 37°C for 24, 72, and 96 h without agitation. After incubation, the residual biofilms were stained, and the OD₅₉₀ was measured.

The effects of Dpo42 on 24 and 96 h old biofilms were also detected. E. coli E9 and HXM were cultured in the 96-well micro-titre plate to form 24 h old biofilm and 96 h old biofilm. The biofilms were treated with 10 μg/well Dpo42, 25 μg/well Dpo42, 50 μg/well Dpo42 or elution buffer diluted in PBS for 12 h. As a control, 200 μL of sterile TSB medium was used. Each group contained triplicate samples. After incubation, the residual biofilms were stained, and the OD₅₉₀ was measured.