Ceftazidime

The combined disc method was used for the confirmatory tests for ESBL-producing E. coli (CLSI, 2020 ). Therefore, the following discs were used: cefotaxime, 30 μg, cefotaxime + clavulanic acid, 30 + 10 μg; ceftazidime, 30 μg; ceftazidime + clavulanic acid, 30 + 10 μg (Oxoid, Basingstoke, United Kingdom). For the identification of pAmpC-producing E. coli, the cefoxitin disc (30 μg) was used. Additionally, the cefepime disc (30 μg) was also used. Moreover, all isolates were subjected to susceptibility testing by broth microdilution (VetMIC GN-mo, National Veterinary Institute, Uppsala, Sweden) to the following antimicrobial agents: ampicillin (1–128 mg/L), cefotaxime (0.016–2 mg/L), ceftazidime (0.25–16 mg/L), nalidixic acid (1–128 mg/L), ciprofloxacin (0.008–1 mg/L), gentamicin (0.12–16 mg/L), streptomycin (2–256 mg/L), kanamycin (8–16 mg/L), chloramphenicol (2–64 mg/L), florfenicol (4–32 mg/L), trimethoprim (1–128 mg/L), sulfamethoxazole (8–1,024 mg/L), tetracycline (1–128 mg/L), and colistin (0.5–4 mg/L). The E. coli ATCC 25922 was used as a control strain. Isolates were considered to be wild-type or non-wild-type based on epidemiological cut-off (ECOFF) values defined by EUCAST¹. ECOFF separates wild-type populations from isolates that have developed reduced susceptibility for the antimicrobial and may differ from clinical breakpoints used for clinical settings (EFSA, 2017 ).

The detection of ESBL production involves two steps, which are the screening test and confirmation. For the screening test, the disk diffusion method was used [30 ]. Three individual cephalosporin disks, including cefotaxime (30 μg) (Oxiod, Hampshire, UK), cefpodoxime (10 μg) (Oxiod), and ceftazidime (30 μg) (Oxoid) were placed on Muller Hilton (MHA) (Difco) agar plates, which were fully spread with a bacterial suspension prepared in 0.9% NaCl solution at 0.5 McFarland standard. The MHA plates were incubated at 37°C for 24 hr. The diameter of the inhibition zone was measured and analyzed according to the CLSI standard. Subsequently, the bacterial isolates, which were resistant to at least one single cephalosporin, were confirmed using the combination disk diffusion method. Two single cephalosporin disks containing cefotaxime (30 μg) (Oxiod) and ceftazidime (30 μg) (Oxoid) were applied in combination with clavulanic acid (10 μg) (Oxiod) to confirm the presence of ESBL production. The isolates were identified as producing ESBL if the difference in diameter of the inhibition zones between the individual cephalosporin disks and the disks with added clavulanic acid was greater than 5 mm.

ESBL production was initially screened and confirmed using disk diffusion method in all the Salmonella and E. coli isolates [46 ]. Initial screening was performed using ceftazidime (30 ug), cefotaxime (30 ug) and cefpodoxime (10 ug) (Oxoid, Hampshire, England). All the isolates resistant to at least one of the indicator cephalosporins were subjected to confirm ESBLs production by using combination disk diffusion method with ceftazidime (30 µg) and cefotaxime (30 µg) alone and in combination with clavulanic acid (10 µg) (Oxoid). A difference of ≥5 mm between the inhibition zone of the cephalosporin/clavulanic acid combination and corresponding cephalosporin disks alone was interpreted as positive ESBL phenotype E. coli ATCC 25922 was used as a quality control strain.

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.