M 1 c evaluator

M.I.C.Evaluator (Oxoid), ETEST^® (bioMérieux, Marcy-l’Étoile, France), or MIC (Liofilchem, Roseto degli Abruzzi, Italy) gradient strips were used to evaluate susceptibility. The following panel of 18 antibiotics spanning 13 classes were used: ampicillin and amoxicillin/clavulanic acid (penicillins); cefotaxime and cefepime (cephalosporins); ciprofloxacin (fluoroquinolones); amikacin, gentamicin, and streptomycin (aminoglycosides); trimethoprim (trimethoprims); trimethoprim/sulfamethoxazole (trimethoprim/sulfonamides); bacitracin (bacitracins); erythromycin (macrolides); tetracycline (tetracyclines); nitrofurantoin (nitrofurans); fosfomycin (fosfomycins); and imipenem and meropenem (carbapenems). Susceptibility profiles to colistin (polymyxins) was determined through the broth micro-dilution method using ComASP^TM Colistin (Liofilchem). All testing was performed in accordance to the manufacturer’s instructions. MIC clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to determine susceptibility (European Committee on Antimicrobial Susceptibility Testing, 2019 ). Multidrug resistance was defined as phenotypic resistance to three or more classes of antibiotics.

The antimicrobial susceptibility was determined by the E-test according to the protocol of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for fastidious organisms. C. jejuni ATCC 33560 was used for quality control. All Campylobacter isolates were suspended into Brain Heart Infusion (BHI) broth to a turbidity equivalent to a 0.5 McFarland standard. Mueller–Hinton agar plates supplemented with 5% of defibrinated horse blood (Oxoid) and 20 mg/L β-NAD (Sigma Aldrich) were inoculated with the suspension prepared. The following M.I.C.Evaluator (Oxoid) strips were placed on the surface of dry plates: erythromycin (E, 0.015–256 μg/ml), gentamicin (CN, 0.015–256 μg/ml), ciprofloxacin (CIP, 0.002–32 μg/ml), amoxicillin (AML, 0.015–256 μg/ml), tetracycline (TET, 0.015–256 μg/ml), amoxicillin/clavulanic acid (AMC, 0.015–256 μg/ml), ceftriaxone (CRO, 0.002–32 μg/ml), and trimethoprim/sulfamethoxazole (SXT, 0.002–32 μg/ml). The plates were incubated at 41 ± 1°C for 24–48 h at microaerophilic atmosphere. Zones of inhibited growth for erythromycin, ciprofloxacin, and tetracycline were determined according to EUCAST breakpoints for Campylobacter³. Since the MIC breakpoints for the remaining tested antimicrobials were not specified for Campylobacter by EUCAST, we used the breakpoints for Enterobacteriaceae.

The samples obtained were cultured on bloody agar and EMB agar, and conventional methods plus a VITEK-2 Compact Automated System (bioMerieux, Marcy-l’Etoile, France) were used for bacterial identification and antibiograms. The antibiotic susceptibility of the isolated strains was determined by the Kirby Bauer disk diffusion method (CLSI 2010) [18 ]. Ampicillin-sulbactam (10/10 μg), piperaciline-tazobactam 100/10 μg, cefepime (30 μg), ceftazidime (30 μg), imipenem (10 μg), meropenem (10 μg), gentamicin (10 μg), amikacin (30 μg), tobramicin (10 μg), netilmicin (30 μg), trimethoprim/sulfamethoxazole (1.25 μg/23 μg), ciprofloxacin (5 μg), colistin (10 μg), and tigecycline (15 μg) antibiotic disks (Oxoid, UK) were used. P.aeruginosa ATCC 27853 was studied as the quality control strain. For colistin, the interpretive criteria of Galani et al. [19 (link)], and for tigecycline, the interpretive criteria of Jones et al. [20 (link)], were applied. The imipenem-resistant strains were detected by the disk diffusion method and confirmed by the imipenem E-test (Oxoid M.I.C Evaluator, UK).