Streptomycin

The c-myc-immortalized mouse microglial cell line, MG6 (RIKEN Cell Bank, Tsukuba, Japan), was maintained in DMEM containing 10% fetal bovine serum (FBS, ICN Biomedicals Japan Co., Tokyo, Japan) supplemented with 10 g ml⁻¹ insulin, 100 g ml⁻¹ streptomycin, and 100 U ml⁻¹ penicillin (BD Falcon, Franklin Lakes, NJ, USA)38 (link)39 (link). For the infection with various bacteria, MG6 microglial cells were cultured in DMEM without FBS or penicillin.

Skeletal muscle stem cells were isolated from hind limb muscles of 6- to 8-week-old mice (kindly provided by P. Muñoz Cánoves of the Pompeu Fabra University, Barcelona) as described in [75 (link)]. After mechanical and enzymatic dissociation with 1 % Pronase protease (Calbiochem), the filtered digest was centrifuged through an isotonic Percoll (Amersham) gradient (60 % overlaid with 20 %) and cells were collected from the gradient interface, resuspended in growth medium (GM: Ham’s F-10 plus 20 % FBS, 5 ng/ml bFGF, 100 U/ml penicillin and 100 μg/ml streptomycin) and grown on tissue culture dishes coated with 0.05 mg/ml collagen I from rat tail (Becton and Dickinson) to amplify the myoblast population. To induce myotube formation, confluent proliferating primary myoblasts were grown on matrigel coated culture dishes (Basement Membrane Matrix, Becton and Dickinson) and switched to differentiation medium the next day (DM: DMEM plus 2 % horse serum, 100 U/ml penicillin and100 μg/ml streptomycin (Life Technologies)) for 4 days. Myofibres directly isolated from EDL muscles were kindly provided by S. Gutarra. Two biological replicates of each experiment were performed and analysed. Mouse protocols were approved by the Animal Care and Use Committee of the PRBB, and the Ethical Committee for Animal Experimentation of the Government of Catalonia.

Antimicrobial susceptibility testing was performed using the disk-diffusion method and the antibiotics ampicillin (AM), amoxicillin-clavulanic acid (AMC), cefotaxime (CTX), nalidixic acid (Na), ciprofloxacin (CIP), gentamicin (GM), kanamycin (K), streptomycin (S), sulfamethoxazole (Su), trimethoprim (TMP), tetracycline (T), and chloramphenicol (C) (Becton Dickinson, Heidelberg, Germany). Results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) performance standards (CLSI, 2016 ). For sulfamethoxazole, for which breakpoints are not listed separately from trimethoprim, an inhibition zone of ≤10 mm was interpreted as resistant. Isolates displaying resistance to three or more classes of antimicrobials (counting β-lactams as one class) were defined as multidrug-resistant (MDR). Synergistic effects between AMC and CTX were regarded as an indication of the presence of an ESBL producer (Kaur et al., 2013 (link)).

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.

The antimicrobial agents were selected among those commonly carried by integrons. Antimicrobial susceptibility testing was performed using disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for each bacterial species (15 , 16 ), for ampicillin (10 µg), chloramphenicol (30 µg), streptomycin (10 µg) (Becton Dickinson, Sparks, Maryland, USA), cotrimoxazole (25 µg), tetracycline (30 µg), ciprofloxacin (5 µg) (Difco Laboratories, Detroit, Michigan, USA), and trimethoprim (Mast Diagnostics Ltd, Bootle, Merseyside, UK) (5 µg) (15 , 16 ). The antimicrobials were classified as aminoglycosides (STR, AMP), tetracyclines (TE), amphenicols (CHL), fluoroquinolones (CIP), trimethoprim alone (TMP), or in combination with sulfonamides (SXT).