Defibrinated horse blood

S. pneumoniae strains were grown in liquid cultures in either BBL Trypticase soy broth (TSB) (Becton, Dickinson, USA) or brain heart infusion (BHI) broth (Oxoid, UK). For growth on solid medium, BBL Trypticase soy agar (TSA) or BHI agar (BHIA) supplemented with 3% (vol/vol) defibrinated horse blood (Thermo Scientific, UK) was used. Plates were incubated at 37°C with 5% CO₂ for 16 to 18 h unless otherwise stated. Where appropriate, medium was supplemented with the following antibiotics at the indicated concentrations: ciprofloxacin at 0.25 μg/ml (0.016 μg/ml for recA mutant strains), kanamycin at 500 μg/ml, spectinomycin at 200 μg/ml, chloramphenicol at 10 μg/ml, streptomycin at 500 μg/ml, and erythromycin at 10 μg/ml.

C. jejuni strains were routinely cultured on Mueller Hinton (MH) agar (Oxoid) supplemented with 5% defibrinated horse blood (Thermo Scientific) and 5 μg/ml trimethoprim (Tp). Defined mutants and complemented strains were selected on 10 μg/ml chloramphenicol (Cm) or 50 μg/ml kanamycin (Km), as appropriate. C. jejuni cultures were grown in standard microaerophilic conditions (5% CO₂, 5% H₂, 85% N₂, 5% O₂) at 42 °C, unless otherwise indicated. Electrocompetent Escherichia coli and C. jejuni used in cloning were prepared and transformed as previously described [14] (link). Bacterial strains and plasmids used in this study are detailed in Table 1.

Standard conditions or modifications from CLSI methods by 25% goat serum supplementation were performed to determine the MIC of tildipirosin, gamithromycin, oxytetracycline, and danofloxacin. Minimum inhibitory concentration tests were performed by the microdilution broth technique (Clinical and Laboratory Standards Institute 2009 ) using U-bottom 96-well microtiter plates. Serial two-fold dilutions of the antimicrobial agents were prepared starting from the stock solution. Broth dilutions were made using Mueller–Hinton broth (MHB) (Merck, Madrid, Spain) for CNS, S. aureus, and E. coli. To investigate Streptococcus spp., cation-adjusted Mueller–Hinton broth (Merck, Madrid, Spain) with 5% defibrinated horse blood (Thermo Fisher Scientific, Massachusetts, USA) was used. Concentrations of all antibiotics ranging from 0.03 to 128 mg/l were used. Inocula were prepared by diluting an overnight MHB culture in buffered saline solution to a density of 0.5 on the McFarland Turbidity Scale and finally diluting again 40-fold before testing. The U-bottomed microtiter plates were incubated at 37 °C and observed 24 h later. The MIC was defined as the lowest concentration of antibiotic at which bacterial growth was completely inhibited. The reference strains S. aureus (ATCC 29213) and E. coli (ATCC 25922) were used as controls.