In Vitro Disc Diffusion Assay

Modified Kirby-Bauer disc diffusion assays were performed using lysogeny broth agar plates. Six millimeter diameter Number 1 Whatman® paper discs (Sigma-Aldrich, St Louis, MO, USA) were prepared in a sterile environment with 10 μg to 500 μg CSA-90 and compared with 10-μg gentamicin discs as a positive control (Oxoid Limited, Hampshire, UK). Plates were spread with 1.5 x 10⁸ CFU (equivalent to 0.5 McFarland turbidity standard), the discs applied, and incubated at 37^o C for 24 hours before two independent observers (TLC, RM) measured the maximal zone of bacterial growth inhibition (radius from the center of the disc). Assays were run in triplicate with S aureus, MRSA, and MRSE.

Potential delivery methods for CSA-90 were tested in the same assay (S aureus only) using a 10-μL carrier agent to deliver 500 μg CSA-90 per disc. The delivery agents tested were: saline; 1% poly(vinylpyrrolidone) (PVP); 0.25% dihydrous calcium sulphate; and sucrose acetate isobutyrate mixed 50:50 with absolute ethanol to obtain a suitable viscosity for coating the discs. These reagents were obtained from Sigma-Aldrich. Similar 6-mm diameter x 2.5-mm bone cement discs were prepared in a silicone mold created from a three-dimensional-printed reverse template. Twenty-five milligrams of CSA-90 were added to 10 g CMW^TM 1 bone cement mix (DePuy, Blackpool, UK) compared with 80 mg gentamicin prepared in the same volume as a positive control.