Ofloxacin

Sarafloxacin (SAR), ofloxacin (OFL), gatifloxacin (GAT), enrofloxacin (ENR), ciprofloxacin (CIP), and tetracycline (TC) were purchased from Meilun Biological Technology Corp. (Dalian, China). Dopamine (DA), tris(hydroxymethyl) aminomethane hydrochloride (Tris-HCl), and phthalic acid (PA) were obtained from Aladdin (Shanghai, China). Graphene oxide (GO) power was obtained from Nanjing XFNANO Materials Tech Co., Ltd (Nanjing, China). Deionized water (DI) used was prepared by a Milli-Q® ultrapure water system (Bedford, MA USA).

MICs of nalidixic acid (Aladdin, USA), ciprofloxacin (Aladdin, USA), enrofloxacin (Aladdin, USA) and ofloxacin (Meilunbio, China) for 162 R. anatipestifer isolates were determined using the standard microscale broth dilution method, performed according to Clinical and Laboratory Standard Institute (CLSI) criteria [23 ], to analyze the antimicrobial susceptibility of randomly collected isolates between 1932 and 2018.
To explore whether the R. anatipestifer site-directed in vivo mutants showed cross-resistance phenotypes with other nonquinolone antibiotics, the MICs of several other nonquinolone antibiotics such as ampicillin (Aladdin, USA), ceftiofur (Meilunbio, China), amikacin (Meilunbio, China), florfenicol (Aladdin, USA), doxycycline (Meilunbio, China) and lincomycin (Aladdin, USA) were determined for these strains.
The concentration of these antimicrobial agents in 96-well plates ranged from 0.25 to 1024 μg/mL, except that of nalidixic acid, which ranged from 0.5 to 2048 μg/mL. E. coli ATCC 25922 was used for quality control. The experiments were repeated in triplicate.

Susceptibility to antimicrobials was determined in triplicate using the standard agar dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2020) [66 ]. The following eighteen antimicrobials were tested: kanamycin (KAN), gentamicin (GEN), erythromycin (ERY), azithromycin (AZM), norfloxacin (NOR), ofloxacin (OFX), ciprofloxacin (CIP), lomefloxacin (LOM), levofloxacin (LEV), sulfadiazine (SD), trimethoprim (TMP), ceftriaxone (CRO), cefixime (CFM), ampicillin (AMP), amoxicillin (AML), aztreonam (ATM), imipenem (IPM) and tetracycline (TET) (Meilun Biotechnology Co., LTD, Dalian, China). The isolates were grown on TSA plates, suspended in stroke-physiological saline solution to a turbidity equivalent to 0.5 McFarland Standard, and inoculated onto Mueller-Hinton agar plates using a multipoint inoculator (MIT-60P; Sakuma Seisakusyo, Tokyo, Japan). The final inoculum was approximately 10⁴ CFU per spot. Plates were incubated at 37 °C for 16–18 h. The range of 2-fold concentrations used to determine the susceptibility was determined by CLSI criteria. In addition, the results were interpreted in accordance with CLSI criteria (Supplementary Table S3). E. coli ATCC25922 and E. faecalis ATCC29212 were used as quality control strains.