Imipenem

We determined antimicrobial drug susceptibility by the disk-diffusion method on Mueller-Hinton agar plates as recommended by the Clinical Laboratory Standard Institute [19 ]. We tested the following antimicrobial agents: ampicillin 10 μg, tetracycline 30 μg, oxy-tetracycline 30 μg, which were used in the farms and the following agents used in the management of E.coli infections; amoxicillin/clavulanic acid 10 μg, cephalothin 30 μg, cefotaxime 30 μg, ceftazidime 30 μg, imipenem 10 μg, norfloxacin 10 μg, ciprofloxacin 5 ug, enrofloxacin 5 μg, amikacin 30 ug, chloramphenicol 10 μg, kanamycin 30 μg, streptomycin 10 μg, gentamicin 10 μg and sulfamethoxazole/trimethoprim (cotrimoxazole) 25 μg (Mast Diagnostics). Results obtained were used to classify isolates as being resistant or susceptible to a particular antibiotic using standard reference values [19 ].

Antimicrobial susceptibility testing was performed on Mueller-Hinton agar (MHA) (Merck, Co., Germany) by agar disk diffusion (DD) method as recommended by the Clinical and Laboratory Standards Institute (CLSI document M100-S14) [17 ]. The tested antibiotics were as follows: amikacin (AK; 30 μg), ciprofloxacin (CP; 5 μg), ceftazidime (CAZ; 30 μg), gentamicin (GM; 10 μg), imipenem (IMP; 10 μg), meropenem (MER; 10 μg) and piperacillin/tazobactam (PTZ; 100/10 μg) (MAST diagnostics, Merseyside, UK). A. baumannii ATCC 17978 was used as a positive quality control (PQC) and P. aeruginosa ATCC 25853 and E. coli ATCC 25922 were used as a negative quality control (NQC) in this study.

Standard discs of 17 antibiotics were obtained from Mast Diagnostics (Merseyside, Liverpool, UK) to be used in this assay; amikacin (30 µg), ampicillin (10 µg), augmentin (30 µg), aztreonam (30 µg), cefoxitin (30 µg), cefepime (30 µg), ceftazidime (30 µg), cephalothin (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), cotrimoxazole (25 µg), fucidic acid (10 µg), gentamicin (10 µg), imipenem (10 µg), oxacillin (1 µg), piperacillin (100 µg), and vancomycin (30 µg).
Antimicrobial activity of four cLfs isolated from milk of four different breeds of Saudi camels (cLf1, cLf2, cLf3, and cLf4), bLf, hLf, and the standard antibiotics against S. typhimurium and S. sonnei was tested by agar disc-diffusion technique. Plates of Mueller-Hinton (MH) agar were overlaid with 2 × 10⁶ CFU/ml of Salmonella typhimurium LT2 and Shigella sonnei. Wells of about 5 mm diameter were made on inoculated plates then different concentrations of lactoferrins (0.00, 0.125, 0.250, 0.50, 0.750, 1, 1.5, 2, 2.5, and 3 mg/ml) were added, and left to diffuse in agar at 4 °C for 2 h before plates finally incubated overnight at 37 °C. The diameters of clear inhibition zones were measured in millimeters. Antibacterial effects of different lactoferrins on growth of S. typhimurium and S. sonnei after 1, 3, 6, 12, and 24 h of incubation at 37 °C were monitored spectrophotometrically (measuring OD at 620 nm).

According to the clinical and laboratory standards Institute protocol (CLSI; M100-S14) [13 ], the antibiotic susceptibility was tested by disk agar diffusion method on the Mueller-Hinton agar plates (MHA) (Merck, Darmstadt, Germany) for ceftazidime (CAZ: 30 µg), cefotaxime (CTX: 30 µg), imipenem (IPM: 10 µg), meropenem (MEM: 10 µg), ciprofloxacin (CIP: 5 µg), cefepime (FEP: 30 µg), ceftriaxone (CRO: 30 µg), amikacin (AN: 30 µg), gentamicin (GM; 10 µg), and trimethoprim-sulfamethoxazole (SXT; 5 µg) (MAST Diagnostics, Merseyside, UK).
Strains non-susceptible to at least three or more antimicrobial classes were defined as MDR, and those non-susceptible to at least one agent in all but two or more antimicrobial categories were considered as possible XDR, and the strains that non-susceptibility to all agents in all antimicrobial categories were defined as possible pan drug-resistant (PDR) [14 (link)]. Escherichia coli ATCC 25922 was used as control organism in susceptibility testing.