Ultrasound bath

Specimens were placed in separate sterile containers and sterile normal saline was added to cover the sample completely. After vortexing for 30s, the sample was sonicated by use of an ultrasound bath (VWR, Milan, Italy) for 5 min at a frequency of 30 kHz and vortexed for another 30s [78 (link)]. The outside of the container was treated with 70% ethanol. The resulting sonication fluid was centrifuged at 4000 rpm for 15 min, and the sediment was used for subsequent microbiological cultures onto chocolate agar, Columbia blood agar, Mannitol salt agar, MacConkey agar, Sabouraud agar and Schaedler agar plates (Biomerieux, Marcy-l’Etoile, France) and inoculated into Brain Heart Infusion and Thyoglycollate broths (TermoFhisher, Cornaredo, Italy). Anaerobic and aerobic agar plates were incubated at 37 °C for 5 days while broths were incubated for 15 days in the same conditions. Microorganisms were identified using conventional methods.

Freeze-dried and ground leaf samples were extracted with 100% acetone and subjected to an ultra-sound bath (VWR International, Germany) for 1 min to ensure complete disaggregation of the leaf material. Extraction occurred in the dark for 24 h at −20 °C, after which the samples were centrifuged at 4000× g at 4 °C for 15 min. Supernatants were scanned from 350 nm to 750 nm in 1 nm steps, using a dual-beam spectrophotometer (Shimadzu UV/VIS UV1601 Spectrophotometer, Country) and the absorbance data were analysed employing the Gauss-Peak Spectra (GPS) method [22 (link),24 (link)]. The De-Epoxidation State (DES) was calculated as

To quantify bacteria within the explanted samples (n = 4 per group), serial dilutions from sonicated fluids were plated onto Mueller-Hinton agar plates (Biomerieux, Marcy l'Etoile, France) and incubated for 16 h at 37°C. Briefly, sterile container containing explanted samples was filled with 1 ml of sterile saline and sonicated in an ultrasound bath (VWR, Milan, Italy) for 5 min with a frequency of 30 kHz and a power output of 300 W at room temperature. All samples were assayed by serial 10-fold dilution in sterile saline solution and then plated on solid growth medium. After incubation of the plates at 37°C for 16 h, colonies of Gram-positive catalase positive cocci, resembling those of S. aureus, were tested for coagulase activity with Coagulase Plasma (Remel Europe Ltd. Dartford, UK) and identified by means of API Staph assay (BioMerieux, Mercy L'Etoile, France). Positivity to catalase test consists in the development of gaseous oxygen when the colony was put in contact with oxygenate water. Coagulase test results positive when S.aureus colonies in contact with coagulase plasma, form a visible clot after incubation for 4–6 h at 37°C. Colonies identified as S. aureus grown on Mueller-Hinton agar plates were then counted. The detection limit (L. o. D) was ≤1.300 (Log CFU)/g of bone.