Antibacterial efficacy via agar diffusion test

Agar diffusion test was used to test loaded suture tapes and cerclage wires for their antibacterial activity.

Staphylococcus aureus (S. aureus; clinical isolate) was grown over night in 10 ml LB-broth (2 g yeast extract, 4 g tryptone (both Applichem GmbH, Darmstadt, Germany), 2 g NaCl (Sigma-Aldrich, Germany) ad 400 ml H₂O double dest) at 37 °C under aerobic conditions. 100 µl of this bacterial culture were spread onto LB-agar plates (LB-medium containing 1.5% (w/v) agar (Applichem GmbH, Darmstadt, Germany). The vancomycin- or NaCl-loaded test bodies were placed on one agar plate inoculated with S. aureus each and incubated at 37 °C for 24 h. After 24 h the test bodies were transferred onto a new agar plate inoculated with S. aureus to detect the remaining antibacterial activity. This procedure was repeated after 48 and 72 h. Pictures with a reference scale were taken of the samples before and after removal of the suture tape or the cerclage wire, respectively, using a digital camera. Photographic documentation was performed 1, 2, 3, and 6 days after incubation in vancomycin or NaCl solution.

The digital photographs were processed for planimetric analysis, using the open source image processing program ImageJ (ImageJ, NIH, Bethesda, Maryland, USA). The planimetric analysis was performed by two independent observers (AEi and IS). For further analysis the mean value of the collected data was used. After initial scaling, the visible zone of bacterial growth inhibition (quantified in mm²) and the surface of the test body (quantified in mm²) were measured. The relative zone of inhibition was calculated by dividing the zone of bacterial growth inhibition by the surface of the test body and multiplied by 100%.