Enrofloxacin

Salmonella isolates were evaluated for antimicrobial resistance using the Kirby–Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines (Clinical and Laboratory Standards Institute [Clsi], 2018 ). Susceptibility to the following 22 antibiotics was tested: ampicillin, amoxicillin-clavulanic acid, cefazolin, cefoxitin, ceftriaxone, ceftazidime, cefotaxime, ceftiofur, cefepime, aztreonam, imipenem, gentamicin, kanamycin, amikacin, streptomycin, tetracycline, ciprofloxacin, enrofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, chloramphenicol, and florfenicol (Oxoid, Basingstoke, United Kingdom).

Disk diffusion antimicrobial susceptibility testing was performed to assess MDR, as described by EUCAST (EUCAST, 2021). Briefly, 90 mm circular MHA plates were prepared and stored at 4–8 °C for no more than 7 days prior to testing. The inoculum was prepared from a single colony selected from an MHA plate, the same colony used for MIC testing, and resuspended in saline to an optical density equivalent to 0.5 McFarland (Thermo Scientific, Basingstoke, UK). The inoculum was applied to the MHA agar plate and pre-dried within 15 min of preparation, to remove residual moisture, and even coverage over the entire surface area was then ensured. Antimicrobial discs were applied using an antimicrobial susceptibility disk dispenser (Oxoid, Basingstoke, UK) within 15 min of inoculation. Antibiotics tested, representing six different antimicrobial families, were ampicillin, tetracycline, enrofloxacin, ceftriaxone, gentamicin, and trimethoprim/sulfamethoxazole (Oxoid™, UK). Incubation was performed statically at 37 °C overnight (18 ± 2 h). Zone of inhibition was measured using digital callipers (Thermo Scientific, Basingstoke, UK), and classification of susceptibility was determined with EUCAST or CLSI (when data were not available using EUCAST) cut-offs (Appendix B).

The ESBL-producing E. coli strains were tested for susceptibility to antimicrobial agents using the disc agar diffusion method [11 ]. The antimicrobial agents tested included amikacin 30 μg, ampicillin 10 μg, amoxyclav 30 μg, ceftiofur 30 μg, cephalothin 30 μg, ciprofloxacin 10 μg, doxycycline 30 μg, enrofloxacin 5 μg, florfenicol 30 μg, gentamicin 30 μg, nalidixic acid 30 μg, streptomycin 10 μg, co-trimoxazole 25 μg, tetracycline 10 μg; all of the discs were purchased from Oxoid (Oxoid, Hampshire, UK).

Antimicrobial susceptibility testing of E. coli isolates was performed using the standard disk-diffusion method recommended by the Clinical and Laboratory Standards Institute [24 , 25 ], with the following antimicrobials: ciprofloxacin (5 μg), gentamicin (10 μg), norfloxacin (10 μg), enrofloxacin (10 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), tetracycline (30 μg), nalidixic acid (30 μg), chloramphenicol (30 μg), nitrofurantoin (300 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg) and amoxicillin-clavulanic acid (20/10 μg) (Oxoid Ltd., Basingstoke, Hants, UK). For the negative control, we used E. coli strain ATCC 25922. All strains resistant to 3rd generation cephalosporins were tested for phenotypic confirmation of ESBL production by standard ceftazidime and cefotaxime disks combined with clavulanic acid [25 ] and by the double-disk diffusion method with disks containing cefepime, cefotaxime, ceftazidime and aztreonam placed 25 mm apart (center to center) to a disk containing a beta-lactamase inhibitor (amoxicillin-clavulanic acid) [26 (link)].

All isolates were tested using the disk diffusion method, and the zone of inhibition for each antimicrobial was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) documents, M100-Ed31 [29 ], and VET01S-Ed5 [47 ]. The antimicrobials tested in the present study were selected based on a previous work [15 (link)] and aiming to cover the most frequent antimicrobial classes used in veterinary medicine. The following antimicrobials were tested: oxacillin (OXA, 1 µg), cefoxitin (FOX, 30 µg), penicillin (PEN, 10 IU), gentamicin (GEN, 10 µg), erythromycin (ERY, 15 µg), clindamycin (CLI, 2 µg), tetracycline (TET, 30 µg), ciprofloxacin (CIP, 5 µg), nitrofurantoin (NIT, 300 µg), trimethoprim-sulfamethoxazole (SXT, 1.25/23.75 µg), chloramphenicol (CHL, 30 µg), enrofloxacin (5 µg), and rifampicin (RIF, 5 µg) (Oxoid, Basingstoke, United Kingdom). Staphylococcus aureus ATCC^® 25923 was used as the control. Strains were considered multidrug resistant (MDR) when they were resistant to three or more classes of antimicrobials [48 (link)].