Mannitol salt agar

Semi-quantitative inoculation of the catheter according to Maki was performed using the following culture media: Columbia agar with 5% sheep blood (bioMérieux, France), MacConkey agar (bioMérieux, France), Mannitol salt agar (bioMérieux, France), and Sabouraud medium with chloramphenicol (bioMérieux, France). The plates were incubated at 35–37°C under aerobic conditions.

Wound swabs were taken and placed in Amies transport media (bioMérieux, France), and biopsy tissue samples and peritoneal fluid were stored in thioglycollate broth (bioMérieux, France). A semi-quantitative technique was used to assess bacterial growth from the swabs. The samples were inoculated on Columbia agar (bioMérieux, France), chocolate agar (bioMérieux, France), MacConkey agar (bioMérieux, France), Mannitol salt agar (bioMérieux, France), and Sabouraud agar (bioMérieux, France) for overnight incubation at 37°C + 5% CO₂.

The clinical and laboratory standard guidelines were followed to assess the tested agents minimum inhibitory concentration (MIC) [8 ]. In brief, some isolated colonies of S. aureus were recovered from mannitol salt agar (bioMérieux, Marcy I'Etoile, France) and subsequently resuspended in tryptic soy broth (NutriSelect® Plus, Sigma-Aldrich, Schnelldorf, Germany) to an optical density of 0.5 McFarland (ca 1.5 × 10⁸ colony forming [CFU] units per mL). This suspension was then dispersed in a 96-well microplate containing serial twofold dilutions of the testing molecules to a final concentration of 10⁵ CFU/mL. MIC values were visually assessed after 48 h. The following antibacterial agents were assessed against the planktonic form of S. aureus: (1) vancomycin (Vianex, Greece); (3) Al₂O₃ nanowires (diameter × L 2–6 nm × 200–400 nm; Sigma); (3) TiO₂ nanoparticles (Nanografi, Turkey); daptomycin (Accord Healthcare SLU); Fucidic Acid (LEO Pharmaceutical); linezolid (Pfizer, Hellas A.E.). The above compounds were diluted in a twofold fashion with NaCL 0.9%.

Specimen collection and identification of the microorganism was performed in accordance to the routine microbiological diagnostics procedures. To determine the presence of S. aureus, a swab from affected skin and from nose of every patient were obtained. Specimen was collected from each patient from the actually most inflamed skin site. All swabs were cultured onto Columbia Agar with 5% of sheep blood (bioMerieux, France) and Mannitol - Salt Agar (bioMerieux, France) and incubated for 18 h in 37 °C. Determination of S. aureus species was performed on the basis of ability to ferment mannitol, to coagulate rabbit plasma and on biochemical properties (VITEK System, bioMerieux). The isolated strains were also subsequently used in the preparation of the autovaccines.

The study included oral S. aureus isolated from all 2327 oral microbiological samples analysed consecutively at the Laboratory of Department of Oral Microbiology of the Medical University of Gdansk during routine clinical laboratory procedures, over a period of three years. The samples were obtained with sterile cotton swabs from the oral mucosa, the dorsal surface of the tongue, denture surface and angular cheilitis lesions. The analysed S. aureus were not specifically isolated for this research, they were part of the diagnostic laboratory procedure and no humans were involved in the experiments.
All samples were plated onto Columbia blood agar (GrasoBiotech, Starogard Gd., Poland) and mannitol salt agar (bioMérieux, Marcy l'Etoile, France) and were incubated 18–24 h at 37 °C. Suspected staphylococcal colonies were identified by standard methods, on the basis of colony characteristics, pigment production, Gram-staining, haemolysis and Pastorex StaphPlus latex agglutination kit (Bio-Rad, Marnes la Coquette, France). Further, all isolates eventually identified as S. aureus based on PCR amplification of species-specific thermostable nuclease gene (nuc)^{82 (link)}.
After final identification, the isolates were stored at − 80 °C in Trypticase Soy Broth (Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 20% glycerol.

Bacteria were isolated from clinical and environmental samples using standard microbiological procedures as described in the Manual of Clinical Microbiology (editions 8–10, issued by the American Society for Microbiology). In short, blood agar, McConkey and mannitol salt agar plates (all purchased from bioMérieux, Brussels, Belgium) were inoculated and incubated overnight at 37°C. Colonies were identified using the gram-negative (GN) and gram-positive (GP) identification cards for the VITEK 2 microbial ID system (bioMérieux, Brussels, Belgium), according to the manufacturer’s instructions.

S. aureus strain ATCC 25923 was used in this study as described in our recent study [24] . Briefly, bacteria were cultured at 37°C overnight onto Mannitol Salt Agar (BioMerieux, France) and incubated into Brain Heart Infusion Broth (BioMerieux) at 37°C for 16 hours. The bacterial suspension was suspended in PBS to obtain a 0.5 McFarland turbidity (equal to about 1×10⁸ CFU/mL), then serially diluted with sterile saline solution and counts were performed to check for bacterial inoculum used for the experiments.