Flumequine

Twenty investigated antibiotics—enrofloxacin (ENR), erythromycin (ERY), levofloxacin (LVX), norfloxacin (NFX), oxytetracycline (OTC), sulfamethazine (SMZ), sulfamethoxazole (SMX), sulfaquinoxaline (SQX), trimethoprim (TMP), ciprofloxacin (CPR), metronidazole (MTZ), roxithromycin (RXM), clarithromycin (CLR), flumequine (FMQ), enoxacin (ENX), tylosin tartrate (T-T), midecamycin (MED), spiramycin (SPR), josamycin (JOS), azithromycin (AZM) (Supplementary Table 1) were purchased from Sigma Aldrich (Buchs, Switzerland). LC/MS-grade methanol, acetonitrile and formic acid were purchased from Carlo Erba Reagents (Val-de-Reuil, France), ultrapure water (>18.2 MΩ cm^–1) was prepared with the Milli-Q IQ 7000 system (Millipore SAS, Molsheim, France). Individual raw solutions (200 mg/L) were prepared for each antibiotic by dissolving the standard powder in methanol and stored in the dark at −20°C. The standard solution mixtures were prepared at three concentrations (10, 5, 1 mg/L) by diluting the raw solutions with water and then stored in the dark at 4°C. Antibiotics were measured in all the collected biofilms by the procedure described in Aubertheau et al. (2017) (link).

The antimicrobial agents used to perform the susceptibility assays of F. psychrophilum isolates were those currently licensed for treatment of Gram-negative bacterial pathogens in the Chilean salmonid farm industry. In addition, due to its worldwide use in fish aquaculture, enrofloxacin was included in the study. The antimicrobials used were amoxicillin, florfenicol, oxytetracycline, oxolinic acid, flumequine and enrofloxacin, and were commercially obtained from Sigma (Sigma, Poole, UK). Antimicrobial solutions were prepared according to CLSI guidelines (CLSI, 2006 ) and solvents used were deionized distilled water for oxytetracycline-HCl, 95% ethanol for amoxicillin and florfenicol and 0.1 M NaOH for oxolinic acid, flumequine, and enrofloxacin. All solutions were filter-sterilized (0.22 μm) and used immediately.

The following solvents, reagents, and antimicrobial agent analytical standards were purchased from Merck (Darmstadt, Germany):

Methanol and n-hexane (hypergrade for LC-MS LiChrosolv^®);

Trichloroacetic acid (TCA) crystals, disodium hydrogen phosphate dihydrate, citric acid monohydrate, and EDTA (for preparing EDTA-McIlvaine buffer solution, pH 4);

Ampicillin, penicillin G, cloxacillin, amoxicillin, penicillin V, oxacillin, dicloxacillin, nafcillin, enrofloxacin, ciprofloxacin, danofloxacin, marbofloxacin, flumequine, tetracycline, 4-epitetracycline, oxytetracycline, 4-epioxytetracycline, chlortetracycline, 4-epichloretracycline, doxycycline, sulfadiazine, sulfathiazole, sulfadimethoxine, sulfadimidine, and enrofloxacin d5 as the internal standards (IS).

Formic acid (98–100%) was provided from Riedel-de Haën (Sigma-Aldrich, St. Louis, MO, USA).
Water was purified using a Milli-Q system (Millipore, Merck KGaA, Darmstadt, Germany).
The Oasis HLB cartridges (3 mL, 60 mg) were supplied by Waters (Milford, MA, USA).

Flumequine (98%) and nonylphenol (PESTANAL^®, analytical standard) are products sold by Sigma-Aldrich (St. Louis, MO, USA). Flumequine is an antibiotic molecule of the quinolone class. It inhibits the enzymatic action of bacterial DNA gyrases. nonylphenol is a synthetic organic compound belonging to the alkylphenol family. It is used as a precursor in the manufacture of polyethoxylated nonylphenols.

All reagents were of analytical grade. Antibiotic standards of 4 different families, sulfonamides (sulfacetamide, sulfapyridine, sulfamerazine, sulfamethazine, sulfadimidine, sulfameter; sulfamethoxypyridazine, sulfachloropyridazine, sulfadimethoxine, sulfadoxine, sulfamethoxazole, sulfabenzamide, and sulfaquinoxaline), quinolones (nalidixic acid, oxolinic acid, ciprofloxacin, and norfloxacin, ofloxacin, flumequine, danofloxacin, enrofloxacin, lomefloxacin, and sarafloxacin), tetracyclines (tetracycline, oxytetracycline, and doxycycline), and beta-lactam antibiotics (amoxicillin, ampicillin, cloxacillin, and penicillin G), were purchased from (Sigma-Aldrich, St louis, MO, United States). All the standards were of high purity grades (>99%). Individual stock solutions were prepared at 1 g/L in acetonitrile and stored at −18 °C.
The working standard solutions containing all analytes except the lactams group with variable concentrations, according to their MRLs, was prepared as dilution of the stock solution in water /acetonitrile ratio of 70:30 (v/v), and the working standard solutions were kept at −20 °C in brown glass and were used for 1 week.
HPLC-grade water, HPLC-grade acetonitrile, trifluoroacetic acid (TFA; 0.2%), disodium ethylenediaminetetraacetate dihydrate (Na₂EDTA), and magnesium sulfate (MgSO₄) were also supplied by Sigma-Aldrich.

Overnight cultures were first diluted 1:6 or 1:600 in fresh MHB for 10⁷–10⁸ CFU/ml inocula and 10⁵–10⁶ CFU/ml inocula, respectively. Initial CFU counts were determined by serial dilution and plating as described above. To overcome resistance phenotypes, time-kill assays were performed at antibiotic concentrations much higher than the MICs (128- or 256-fold higher) for each strain. Antibiotic used were ciprofloxacin, enrofloxacin, norfloxacin, nalidixic acid, flumequine, tetracycline, and gentamicin (Sigma and Fluka). The survival fraction was calculated as the ratio of bacterial numeration at a given time of the treatment versus the bacterial numeration before treatment.