2.6. Antimicrobial susceptibility test
The susceptibility of E. ictaluri isolates to antibiotics was examined using the disk diffusion method according to the guidelines of the Clinical Laboratory Standards Institute (CLSI, 2018). Sixteen antibiotics (Oxoid, Hampshire, United Kingdom) comprising 11 antibiotic classes/subclasses were tested, including two penicillins: oxacillin (Ox, 1 µg) and amoxicillin (Ax, 10 µg); one β-lactam/β-lactamase inhibitor combination ( BL/BLIs): amoxicillin-clavulanic acid (Ac, 20/10 µg); three cephalosporins: cefotaxime (Ct, 30 µg), cefuroxime (Cu, 30 µg), and ceftriaxone (Cx, 30 µg); one macrolide: erythromycin (Er, 15 µg); one quinolone: nalidixic acid (Na, 30 µg); one sulfonamide: sulfamethoxazole/trimethoprim (ST, 23.75/1.25 µg); one aminoglycosides: neomycin (Ne, 30 µg); one glycopeptide: vancomycin (Va, 30 µg); two fluoroquinolones: ofloxacin (Of, 5 µg) and norfloxacin (No, 10 µg); two tetracyclines: doxycycline (Dx, 30 µg) and oxytetracycline (OTC, 30 µg); and one amphenicol: florfenicol (Fl, 30 µg).
E. ictaluri isolates were grown in Mueller Hinton (MH) broth and adjusted to a McFarland turbidity of 0.5. The suspension was then spread onto MH agar using a sterilized cotton swab. Antibiotic discs were placed on the inoculated plates and incubated at 28 °C for 48 h. The inhibition zone diameters were recorded and classified as susceptible, intermediate, or resistant according to the standard CLSI (2020) method valid for Enterobacterales. In the case of amoxicillin and neomycin, for which CLSI (2020) assessment does not exist, the clinical breakpoints according to the European Committee on Antimicrobial Susceptibility Testing standard (EUCAST, 2021) were applied. The multiple antibiotic resistance (MAR) index of the isolates was calculated as described by Krumperman (1983), in which MAR = a/b, where ‘a’ represents the number of antibiotics to which the isolate is resistant, and ‘b’ represents the total number of antibiotics to which the isolates are exposed for susceptibility testing.