Mueller hinton agar plate

Based on the M7‐A8 guidelines of the Clinical Laboratory Standards Institute (2009), the broth microdilution method was used to evaluate cross‐resistance to Senstitre Antibiotic Plates (TREK Diagnostic Systems, UK). Briefly, S. aureus strains were streaked onto Mueller‐Hinton Agar plates (Sigma Aldrich, USA) and then incubated for 18–24 hr at 37°C. Around four to five S. aureus colonies were transferred to sterile tubes containing 5 ml of 0.9% NaCl solution.
By using the Senstitre Nephelometer (TREK Diagnostic Systems, UK), the turbidity of the growing broth culture was adjusted (ca 1 × 10⁵ KbE/ml). Subsequently, 11 ml of Mueller‐Hinton broth was inoculated with 15 μl of the modified TSB and 50 μl of the mixture was inoculated into each well of the Senstitre plates. The plates were covered with foil and incubated at 37°C for 24 hr. The Senstitre Automatic Reader (TREK Diagnostic Systems, UK) was used to read the plates.

The antibacterial efficacy of the set specimens against Streptococcus mutans (ATCC 25,175) was investigated in vitro using standard disc diffusion method on brain heart infusion (BHI) agar plates. Cement discs (6 mm diameter, 3 mm thickness) were prepared with sterile instruments and sterilized by UV radiation (CAMAG UV Cabinet, CAMAG Germany) at 254 nm for 60 min before proceeding into the next step [29 (link)]. Mueller–Hinton agar plates (Sigma Aldrich, MO, USA) seeded with 1.8 × 10⁸ cfu/mL (0.5 OD⁶⁰⁰) of the test bacteria were checked for the presence of inhibition zones after 24 h of incubation at 37 °C. Inhibition zones surrounding the specimens were measured (mm) using a digital caliper at the outer limit of the inhibition zone generated considering only halos > 6 mm.

The modified agar diffusion well method previously described by Bauer et al. (1966) was used to determine the antibacterial activities of isolated bacteria.
The overnight cultured isolated strains in MRS broth medium at 37 °C were filtered through 0.2 µm filter, and then 50 µL of each filtrate was added to 7 mm diameter wells on Mueller-Hinton agar plates (Sigma-Aldrich, USA), which before were incubated overnight by indicator pathogens at 37 °C. In certain cases, the isolated active supernatants had low pH. Thus, the pH of the isolated active supernatants was adjusted by adding NaOH to the physiological solution (pH 7.2) for use in the antimicrobial assay experiments. After overnight incubation of plates at 37 °C, the clear zones around of each well were measured and considered as positive antibacterial activity.^{25 (link)} According to diameter of inhibition zone; the anti-pathogen activity was divided to strong (≥ 20 mm), moderate (20 mm ≥ diameter ≥ 10 mm), and weak (≤ 10 mm).^{26 (link)} The means data of experiment for twice with three repeats in each time were calculated and considered for each bacterial isolates.

The bioactivity of gentamicin eluted from bone cement was estimated using an agar disk diffusion bioassay. Briefly, basic PMMA bone cement discs containing gentamicin were prepared as described in Section 2.1. (0.1 g gentamicin in 4 g PMMA powder component/2 mL liquid component, Table 1) with a diameter of 5 mm and height of 3 mm. The tested cement discs were placed on Mueller Hinton agar plates (Sigma, St. Louis, MO, USA) inoculated with Pseudomonas aeruginosa (ATCC27853), Staphylococcus aureus (ATCC25923) or Escherichia coli (ATCC25922) in a density of 1 × 10⁶/mL and incubated at 30 °C (n = 4). The tested cements were transferred to new agar plates inoculated with bacteria every 24 h and the diameter of the inhibition zones (zone of inhibition, ZOI; Figure 1) on day 1, 2, 3, 7 and 14 was measured. Bone cement without antibiotic was used as a negative control (ZOI = 5 mm).

A single MRSA colony was planktonically cultured in LB (Invitrogen) medium containing oxacillin (6 μg/ml; Sigma‐Aldrich Co.) for 24 h. The medium containing MRSA was spread onto Mueller‐Hinton agar plates (Sigma‐Aldrich Co.) with oxacillin (6 μg/ml) using a sterile Cotton‐Tipped Applicator (McKesson Medical, San Francisco, CA, USA) and grown in a 35°C incubator for 1 h. Different concentrations of DMF (75, 150, and 300 μg) and vancomycin (1,000 μg) were prepared and 20 μl of each sample was loaded onto sterile blank paper discs (6 mm; Thermo Fisher Scientific, Inc.; catalog no. S70150A) and dried for 1 h. The dried discs were transferred to a plate and then incubated for 24 h. The images were captured with a ChemiDoc™ Touch Imaging System (Bio‐Rad Laboratories) which the diameter of each inhibition zone was measured using ImageJ software (Schneider et al, 2012 (link)).

To test the cross-resistance of Brucella strains resistant to NPs against various antibiotics, broth microdilution was used according to the M7-A8 recommendations of the Clinical Laboratory Standards Institute (2009). The NPs resistant strains were tested against ten different antibiotics (concentration ranges were expressed in μg/ml), including ampicillin (0.016–256), ampicillin-sulbactam (0.016–256), cefuroxime (0.016–256), tetracycline (0.016–256), doxycycline (0.016–256), ciprofloxacin (0.002–32), levofloxacin (0.002–32), trimethoprim- sulfamethoxazole (0.002–32), chloramphenicol (0.016–256), rifampin (0.016–256). In brief, strains of Brucella were plated on Mueller-Hinton agar plates (Sigma Aldrich, USA) and incubated at 37°C for 5–7 consecutive days. Approximately four to five colonies were placed into 5 ml of 0.9% NaCl solution in sterilized test tubes. Using the Sensititre nephelometer, the turbidity of the growing broth culture was adjusted to approximately 1 × 10⁵ KbE/ml. In a subsequent step, 11 ml of Mueller-Hinton broth was inoculated with 15 μl of modified tryptone soya broth (TSB), and 50 μl of this mixture was placed into each well of a microtitre plate. Incubation was carried out at 37°C for 24 hours after foil-wrapping the plates. A Sensititre reader (TREK Diagnostic Systems, UK) was used to read the plates.

10 μl of DPBS (Thermo Fisher Scientific, Inc.) was intraarticularly injected under the patella using a U‐100 Micro‐Fine Insulin Syringe (28‐gauge needle; BD Biosciences), aspirated, and transferred to sterile microcentrifuge tubes. Synovial fluid cell number (1:10 dilution) was measured using a TC20™ Automated Cell Counter (Bio‐Rad Laboratories); MRSA expressive of GFP in synovial fluid was detected using the ZOE™ Fluorescent Cell Imager (Bio‐Rad Laboratories). Synovial fluid cells (1 × 10⁴) were seeded on Mueller–Hinton agar plates (Sigma‐Aldrich Co.) containing oxacillin (6 μg/ml) and incubated for 24–48 h. The images were captured with a ChemiDoc™ Touch Imaging System (Bio‐Rad Laboratories), and MRSA colony forming units (CFU) were quantified using ImageJ software (Schneider et al, 2012 (link)).