Columbia agar

Milk samples (10 µL) were plated on BD Columbia agar with 5% sheep blood (Becton-Dickinson, Franklin Lakes, NJ, USA) and MacConkey-3 agar (Oxoid, Basingstoke, Hampshire, UK) and incubated at 37°C for 24–48 hours. Strain identification was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS).

The hemolysis testing was conducted on BD™ Columbia agar with 5% sheep blood (Becton Dickinson GmbH, Germany) to determine the type of hemolysis. VN2, VN3, VN5, and VN7 were cultured in BD™ Columbia agar with 5% sheep blood agar and incubated at 37°C for 24 hr for the determination of the hemolysis pattern. Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus epidermidis were used as the positive control of α-hemolysis, β-hemolysis, and γ-hemolysis, respectively.

The Kirschner wires were explanted, rolled over nutrient agar (BD™ Columbia Agar, Becton Dickinson, Heidelberg, Germany), and placed in 2.5 ml sterile TSB. Agar plates and TSB were incubated at 37.8 °C. After 24 h, CFU on agar plates were counted and bacterial growth in the tryptic soy solution was evaluated (cloudy: positive growth; clear: no growth) [12 (link)].

Microbiological samples were taken during the second surgery and after the animals were sacrificed. 10 μl of swabs and clinical samples were cultured on BD Columbia Agar with 5% Sheep Blood (Becton Dickinson, Heidelberg, Germany) at 37 °C with 5% CO₂ for 24 h. Colonies with morphological consistency with S. aureus were confirmed by a slide agglutination test (Pastorex Staph Plus, Bio-rad, Germany), as described elsewhere [18 (link)]. Strain typing was performed by Sanger sequencing and comparison of the polymorphic protein A gene (spa typing) as previously described [18 (link)].

Bacterial strains. The in vitro effect of the HA nanocrystals loaded with the monocyclic azetidinones was evaluated against Gram-positive and Gram-negative reference bacterial strains: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922). In addition, clinical isolates obtained from surgical bone biopsies were included in the study and they were categorized based on their antimicrobial susceptibility to methicillin. The tested strains were isolated on BD Columbia Agar with 5% sheep blood (Becton Dickinson, Germany) and confirmed by MALDI-TOF MS (Bruker Daltonik, Germany)^{44 (link)}. Their susceptibility was analyzed by the Vitek2 semi-automated system (bioMerieux, France) and interpreted following EUCAST guidelines. MRSA strains were confirmed by growth on BD oxacillin screen agar (Becton Dickinson, Germany), as in the clinical microbiology laboratory resistance to oxacillin is the marker for detecting methicillin resistance^{45 (link)}. SCV phenotypic characterization was carried out by identification of very small pinpoint colonies on blood agar plate following 48 h of growth.

Throat and rectal swabs were plated on BD Columbia Agar with 5 % sheep blood (Becton-Dickinson) with a 10 µg colistin disk (Oxoid) placed in the first streak and plates were incubated at 37°C under a 5% CO₂ atmosphere for 48 hours. Strain identification was performed by MALDI-TOF from all phenotypically different colonies in the colistin disk’s inhibition zone.

The string test was performed to identify the hypermucoviscous (HMV) phenotype. The strains were grown in BD Columbia agar with 5% sheep blood (Beckton Dickinson) at 37°C overnight. Afterwards, a bacteriological inoculation loop was used to stretch a mucous bacterial colony. The HMV phenotype was positive when a string > 5 mm in length was observed (Fang et al., 2004 (link)).