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.