Mueller hinton agar medium

Susceptibility testing of the GNB was examined by the Kirby–Bauer disk diffusion assay on Mueller-Hinton agar medium (Oxoid, England) against 18 antibiotic disks following the Clinical and Laboratory Standard Institute guidelines [17 ]. The following antibiotics were examined: amikacin (30 μg), amoxicillin/clavulanate (20/10 μg), aztreonam (30 μg), cefepime (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), cefuroxime (30 μg), ciprofloxacin (5 μg), colistin (10 μg), gentamicin (10 μg), imipenem (10 μg), meropenem (10 μg), nitrofurantoin (50 μg), piperacillin(100 μg), piperacillin/tazobactam (100/10 μg), tobramycin (10 μg), and trimethoprim/sulfamethoxazole (23.75 μg/1.25 μg) (Oxoid, England). In brief, standardized suspension of each isolate conforming 0.5 McFarland turbidity was inoculated onto two Mueller-Hinton agar plates. Then, nine antibiotic disks were placed onto each plate with recommended distance, followed by overnight incubation at 37°C. The strain of E. coli ATCC 25922 was used as control and was tested each time when susceptibility testing was performed.

Antimicrobial susceptibility testing of E. coli isolates were performed by the Kirby-Bauer disk diffusion assay on Mueller-Hinton agar medium (Oxoid, Basingstoke, England) as recommended by Clinical Laboratory Standard Institute (CLSI, 2011), against 15 antimicrobial agents from different categories including: amikacin (30 μg), amoxicillin (10 μg), amoxicillin-clavulanic acid (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg) cefuroxime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), nalidixic acid (30 μg), nitrofurantoin (50 μg), ofloxacin (5 μg), tetracycline (30 μg), tobramicin (10 μg) and trimethoprim- sulfamethoxazole (25 μg) (Oxoid, Basingstoke, England). E. coli isolate was considered non-susceptible to an antimicrobial agent when it tested resistant, intermediate or non-susceptible when using clinical breakpoints as interpretive criteria, provided by the CLSI, (2011). MDR patterns of E. coli isolates were defined as non-susceptibility to at least one agent in three or more antimicrobial categories (Magiorakos et al., 2012 ).

The disk diffusion (Kirby–Bauer) method was used to evaluate the antimicrobial activity of each nanoparticle compound. Bacterial cultures were diluted in the Mueller–Hinton broth, and achieved an optical density corresponding to 0.5 MacFarland standards, in which the concentration of bacteria was 1.5 × 10⁸ CFU ml⁻¹. The Mueller–Hinton agar medium (Oxoid) was prepared and sterilized at 121 °C in an autoclave, then about 15 ml of the melted agar media was poured aseptically into each of the sterilized Petri plates and kept at room temperature for solidification. 100 μl of the prepared bacterial cell suspension was pipetted and spread across the dried surface of the Muller Hinton agar. Sterile filter paper disks loaded with each nanoparticle (at a concentration of 0.005 g/10 ml) were placed on the surface of the Mueller–Hilton agar plate using sterile forceps. The plates were incubated at 37 °C for 24 h. Following incubation, the zones of inhibition were measured in mm from four sides of each well to determine an average mean value. The presence of inhibition zones was measured by vernier caliper, and was recorded and considered as an indication for antibacterial activity.^{51,52 (link)}