Rifampicin

S. aureus isolates obtained at C1 and C2 were suspended in sterile saline to 0.5 McFarland. Susceptibility testing was performed on Mueller-Hinton II agar 3.8% w/v (BD Diagnostic Systems, Sparks, MD, USA) using the standardized disk diffusion method in accordance with the European Committee on Antimicrobial Susceptibility Testing (www.eucast.org) guidelines for the following antibiotics: cefoxitin (30 µg), fusidic acid (10 µg), erythromycin (15 µg), clindamycin (2 µg), rifampicin (5 µg), gentamicin (10 µg), trimethoprim-sulfamethoxazole (25 µg), tetracycline (30 µg), and norfloxacin (10 µg). All disks were from Oxoid, Basingstoke, UK.

For screening of traI, 83 tetracycline-resistant strains of bacteria isolated from the sediment or seawater of a coastal aquaculture site in Japan were used (Supplementary Table 1). At the site, oxytetracycline and flumequine had been used (Nonaka et al., 2007 (link)). Theses environmental strains were cultured at 25°C in the brain heart infusion (BHI) medium (Becton, Dickinson and Company, Sparks, MD, USA) with 0.2% NaCl and 10 μg/ml of TC (Nakalai tesque, Kyoto, Japan). All of the strains were confirmed by PCR to have tet(M) (Nonaka et al., 2007 (link)), and strains 04Ya001, 04Ya016, 04Ya090, and 04Ya108 have previously been shown to transfer tet(M) to E. coli (Neela et al., 2009 (link)). E. coli W3110 obtained from the National BioResource Project (National Institute of Genetics, Japan) and its rifampicin-resistant derivative, W3110Rif^r (Nonaka et al., 2012 (link)), were cultured at 37°C in the Luria-Bertani medium (BD) with or without 10 μg/ml (for liquid media) or 20 μg/ml (solid media) of TC and 100 μg/ml of rifampicin (Sigma-Aldrich, Saint-Louis, MO).

Bacterial cell suspensions in saline were normalized at 0.5 McFarland standard and swabbed onto MH agar plates. Disks containing ciprofloxacin (5 μg), imipenem (10 μg), gentamicin (10 μg), novobiocin (30 μg), erythromycin (15 μg), rifampicin (5 μg), or tobramycin (10 μg) (Becton Dickinson) were placed on the surface of the inoculated plates, and growth inhibition halo diameters were measured after 24-h incubation at 37°C.

Antimicrobial susceptibility testing was performed by epsilometry (E-test) and agar disk diffusion as described previously with a McFarland value of 4.0 on Columbia agar (Becton Dickinson, Heidelberg, Germany) [34 (link)]. For metronidazole (nitroimidazole), vancomycin (glycopeptide) and moxifloxacin (fluoroquinolone), epsilometry tests were derived from Liofilchem (Roseto degli Abruzzi, Italy) while, for clarithromycin (macrolide) and rifampicin (rifamycin), antibiotic disks originated from Becton Dickinson (Heidelberg, Germany).

Sensitivity to many different antibiotics was assessed by the Kirby–Bauer disc diffusion test. Bacterial cell suspensions in saline were normalized at 0.5 McFarland Standard and swabbed onto MH agar plates, using disks containing ciprofloxacin (5 µg), novobiocin (30 µg), rifampicin (5 µg), erythromycin (15 µg), streptomycin (10 µg), tobramycin (10 µg), imipenem (10 µg), and colistin (10 µg) (Becton Dickinson). Growth inhibition halos were measured after 24 h of growth at 22, 30, or 37 °C.
colistin sensitivity was also assessed with minimum inhibitory concentration (MIC) assay using the broth microdilution method. Briefly, strains were cultured in MH at 37 °C for 8 h, and then refreshed at 5 × 10⁵ cells/ml in the same medium in the presence of increasing concentrations of colistin (up to 16 μg/mL). MIC was defined as the lowest concentration of antibiotics for which no visible growth was observed after 24 h at 37 °C. Each strain was tested in at least three independent experiments.

Resistance to the growth inhibitory activity of several antibiotics was assessed by the Kirby-Bauer disc diffusion test. Bacterial cell suspensions in saline were normalized at 0.5 McFarland Standard and swabbed onto MH agar plates supplemented or not with arabinose at the indicated concentration, using disks containing streptomycin (10 µg), ciprofloxacin (5 µg), imipenem (10 µg), colistin (10 µg), novobiocin (30 µg), erythromycin (15 µg), rifampicin (5 µg) and tobramycin (10 µg) (Becton Dickinson). Growth inhibition halo diameters were measured after 20 h of growth at 37°C.

The antibiotics isoniazid and rifampicin, the efflux inhibitors verapamil and thioridazine, the efflux substrate ethidium bromide (EtBr), Tween 80, phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), and glucose, were obtained from Sigma-Aldrich (St. Louis, MO, USA). rifampicin was prepared in DMSO, while the remaining drugs were prepared in sterile deionized water. The stock solutions were aliquoted and stored at −20 °C and the working solutions freshly prepared on the day of the experiment. The lyophilized drugs (BACTEC MGIT 960 SIRE and PZA kits; SIRE, streptomycin, isoniazid, rifampicin, ethambutol; PZA, pyrazinamide) used in the standard susceptibility testing were purchased from Becton Dickinson (Diagnostic Systems, Sparks, MD, USA) and the stock solutions prepared as per the manufacturer’s instructions. BD Difco Middlebrook 7H9, BD BBL OADC (oleic acid/albumin/dextrose/catalase) supplement and the Mycobacteria Growth Indicator Tubes (MGITs) were from Becton Dickinson.