Mueller hinton broth (mhb)

Catechin, chlorogenic acid, quercetin, cyanidin chloride, and gallic acid used as standards for the HPLC-DAD-ESI-MS analysis were purchased from Sigma-Aldrich (Steinheim, Germany). Folin–Ciocalteu’s phenol reagent, sodium carbonate (Na2CO3), sodium nitrate (NaNO2), hydrochloric acid (HCl), aluminum chloride (AlCl3), sodium hydroxide (NaOH), acetic acid, acetonitrile, methanol, ethanol, DPPH (2,2-diphenyl-1-picrylhydrazyl), and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Sigma-Aldrich (Steinheim, Germany). For antimicrobial assays, Mueller–Hinton agar, thioglycollate broth with resazurin, and Mueller–Hinton broth were purchased from BioMerieux (France), and Tween 80 and Broth Malt medium were purchased from Sigma-Aldrich (Steinheim, Germany).

The white wheat bran (WB) and oat bran (OB) (Solaris, Romania), commercially available on the local market, were used for present experiments. Firstly, they were defatted thrice with hexane (1:5 w/v), for 5 min at room temperature while stirring, followed by filtration, and were then allowed to dry for 24 h at room temperature. The bran material obtained was immediately used to extract the bioactive compounds (detailed in Section 2.2.), and was considered further as fresh (F) samples, precisely fresh wheat bran (WBF) and fresh oat bran (OBF) samples. The same bran materials were further used to make the thermal samples (TP), precisely the thermally processed wheat bran (WBTP) and thermally processed oat bran (OBTP), by mixing each type of bran with water (5:1 v/w for wheat bran and 2:1 v/w for oat bran) and were thermally processed (10 min at 80 °C), followed by their extraction (detailed in Section 2.2.).
Folin–Ciocalteu’s phenol reagent, DPPH (1,1-diphenyl-2-picrylhydrazyl), sodium carbonate, acetonitrile, acetic acid, and methanol were obtained from Sigma-Aldrich (Steinheim, Germany). The Mueller–Hinton agar, thioglycollate broth with resazurin, and Mueller–Hinton broth were obtained from BioMerieux (Craponne, France).

After the collection, all samples were processed within 2h. Each swab was inoculated in 3 mL of Mueller-Hinton broth (bioMérieux, France) supplemented with 6.5% NaCl, and incubated 24h at 35°C, in atmospheric air. A 50 μl amount of the culture was inoculated onto chromogenic media MRSA-ID (bioMérieux, France), oxacillin resistance screening agar (ORSA; HiMedia, India), and mannitol salt agar (MSA; bioMérieux, France) supplemented with 2 mg/L of oxacillin, which represent the available MRSA-screening media on the Serbian market. All inoculated plates were incubated at 35°C, in air, and read after 24h and 48h of incubation.

We modified routine methods to determine the biofilm formation capability of the isolates [15 (link)]. Briefly, the isolate to be tested was cultured in Mueller-Hinton broth (bioMérieux, Marcy-l’Étoile, France) at 37°C for 20 h; then the concentration was adjusted to 0.5 McFarland, the suspension was diluted 100 times, and 200 μL of the resulting suspension was inoculated into a sterile flat-bottomed 96-well polystyrene microplate. After a 20-h incubation at 37°C, the non-adherent cells were removed, and the plate was washed three times with 300 μl of 1% sterile phosphate-buffered saline (PBS) per well. Biofilms were fixed with methanol for 15 min, the remaining methanol was removed, and the well was air-dried. Subsequently, 220 μl 1% crystal violet was added per well and incubated for 10 min. The well was then rinsed with PBS, air dried, and 200 μl 75% ethanol was used to dissolve the dyed biofilm. Finally, the optical density (OD) of the dissolved biofilm was measured with a microplate reader at a wavelength of 460 nm. Three repeat tests were conducted for each strain. Sterile Mueller-Hinton broth medium (Solarbio, Beijing, China) was used as a negative control. The cut-off for biofilm formation was defined as three standard deviations above the mean of the negative control OD (ODc). The criteria were as follows: OD

The antibacterial properties of the tested compounds were evaluated by dilution method adapted to the requirements of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), Clinical Laboratory and Standard Institute (CLSI), and other studies [20 ,21 (link),22 (link),23 (link),24 (link)].
From each strain, standardized suspensions were prepared to a concentration of 0.5 McFarland and this inoculum was adjusted by dilution at 10⁵ CFU/mL. Then, 100 µL of each test compound dilution, 400 µL Mueller-Hinton broth (bioMérieux, Marcy-l’Etoile, France), supplemented with horse blood and β-NAD for streptococci, and 500 µL bacterial suspension were added to the test tubes and incubated for 24 h at 37 °C. Thus, the final dilutions of the test compounds were considered ten times lower than the initial serial dilutions. The higher dilution with no observable growth was considered the minimum inhibitory concentration (MIC). To determine the minimum bactericidal concentration (MBC), which is the higher dilution that killed 99.9% of the bacteria, 1 µL from the obtained suspension with no visible growth was inoculated on Columbia agar with sheep blood (bioMérieux, Marcy-l’Etoile, France) and incubated for 24 h at 37 °C.
The negative control consisted of 100 µL of DMSO, 400 µL broth, and 500 µL bacterial suspension.

Catheter pieces, which were rinsed three times with 0.9% NACl for 30 min by shaking, were placed in control broth (Mueller–Hinton Broth, bioMérieux, Marcy l’Etoile, France) in different tubes which contain DAPT (2 mg/ml) (50 mg/lt Ca^+ 2 added at physiological concentration), VAN (10 mg/ml), LIN (2 mg/ml), and combination with NAC (6 mg/ml), respectively for 24 h at 37 °C [18 (link)]. All antibiotic solutions, daptomycin (Novartis Health, Food and Agriculture Products Industry and Trade Inc., Istanbul, Turkey), vancomycin (Meditera Group, Izmir, Turkey), linezolid (Pfizer Ltd. Şti., Istanbul, Turkey), and NAC (Merc, Millipore, Germany), were prepared fresh from commercially available forms in accordance with the manufacturer’s recommendations. Catheter parts were washed with 0.9% NACl, placed in 5 ml of 0.9% NACl prepared in a different tube, and vortexed for 30 s, and then 10 μl of liquid was taken and inoculated on 5% sheep blood agar. Single dropped colonies were counted following 24 h of incubation at 37 °C [18 (link)]. All microbial steps were performed according to our previous study, and the method described in Fig. 1 is adapted from our previous work (Fig. 1) [19 (link), 20 (link)].Fig. 1

Schematic illustration of bacterial enumeration. S. aureus strains were cultured (A). Dilution series were schematized (B). The bacterial colonies in biofilm layers were observed (C)