Trimethoprim

The recombinant flagellin FliC_Δ174−400 came from S. enterica serovar Typhimurium FliC and was produced with an histidine tag, as described previously (20 (link), 32 (link)). The protein FliC_Δ174−400 was certified to be immunologically active in reporter cells and in mouse assays, and the residual lipopolysaccharide concentration was determined to be < 20 pg per μg of flagellin (20 (link)). For flagellin treatment, FliC_Δ174−400 (1 ng to 25 μg in 30 μl PBS) was administrated intranasally under light anesthesia via isoflurane inhalation (Axience, Pantin, France). Control animals received intranasal PBS alone. Mice were treated either intragastrically with AMX [5–350 μg of amoxicillin trihydrate (Sigma-Aldrich) in 200 μl water per animal] or intraperitoneally with SXT—a combination of the antibiotics sulfamethoxazole and trimethoprim (Bactrim® Roche, Basel, Switzerland) at total doses of 1 mg (0.84 mg sulfamethoxazole and 0.16 mg trimethoprim) or 4 mg (3.34 mg sulfamethoxazole and 0.66 mg trimethoprim) in 200 μl PBS per animal.

The antibiotic susceptibility profiles of the S. aureus isolates were determined by the Minimum Inhibitory Concentration (MIC) technique with the microdilution method in Mueller-Hinton broth (MH; Becton Dickinson, East Rutherford, NJ, USA), as recommended by the Clinical and Laboratory Standards Institute (2014) . The MIC tests were conducted with vancomycin, ciprofloxacin, erythromycin (MP Biomedicals, Solon, OH, USA), clarithromycin (Grünenthal Gmbh, Aachen, Germany), oxacillin, clindamycin, linezolid (Sigma-Aldrich, St. Louis, MO, USA), meropenem (AstraZeneca Pharmaceuticals LP, Wilmington, DE, USA), trimethoprim, sulfamethoxazole (Roche, Basel, Switzerland), and gentamicin (Schering-Plough Pharmaceuticals, Kenilworth, NJ, USA). To identify methicillin-resistant S. aureus clinical isolates, the bacteria were tested for oxacillin resistance by the oxacillin-salt screening method. oxacillin is a more stable antibiotic than methicillin, although they are chemically identical. S. aureus strain ATCC 29213 (American Type Culture Collection, Manassas, VA, USA) was used as a positive control.