Amoxicillin clavulanate

Parental mice were treated with an antibiotic cocktail (ABX) in drinking water 2 weeks prior to mating and subsequent offspring kept being administered ABX for the duration of the experiment. ABX was freshly prepared weekly, kept at 4 °C, and changed every 2–3 days. Antibiotic cocktail (ABX): Ampicillin (A0166-5g, Sigma-Aldrich) (1 g/liter), Amoxicillin-Clavulanate (SMB00607-1G, Sigma-Aldrich) (0.25 g/liter), neomycin (Colivet vet., VetPharma AS) (1 g/liter), gentamicin (G1397-100 ml, Sigma-Aldrich) (1 g/liter), and vancomycin (V2002, Sigma-Aldrich) (0.5 g/liter) in drinking water. Sucralose (sweetener) was added at 0.1 g/liter to make the cocktail palatable.

Amoxicillin/clavulanate, a combination of amoxicillin trihydrate with potassium clavulanate (4:1; Sigma-Aldrich, Saint Louis, MO, United States), a broad-spectrum antibiotic, was used in this study. Prebiotics, 2′-fucosylactose (2’-FL) and galacto-oligosaccharides (GOS) were provided by FrieslandCampina Ingredients (Amersfoort, the Netherlands). Aequival 2’-FL powder is a high-purity human milk oligosaccharide product (94% 2’-FL on a dry matter basis). Purified Vivinal GOS was provided, containing 1.7% monomers, 6.6% dimers (allo-lactose, lactose and lactulose), and 91.7% GOS on a dry matter basis.

The guidelines provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST; https://www.eucast.org) were followed for completion of MIC assays by the broth microdilution method. All MIC assays were conducted in cation-adjusted Mueller–Hinton II broth (Sigma-Aldrich, MO, USA) in 96-well round-bottom polypropylene microplates (Corning, NY, USA) and results determined visually following 18±2 h static incubation at 37 °C. Antibiotics were purchased from Melford Laboratories (amikacin, amoxicillin, amoxicillin-clavulanate, cefotaxime, colistin), Sigma-Aldrich (ceftazidime, ciprofloxacin, doxycycline, erythromycin, meropenem), Carbosynth (imipenem, relebactam), Fisher Scientific (cefepime) and Lonza (gentamicin). Antibiotic stocks were prepared in sterile deionized water, except erythromycin, which was prepared in dimethyl sulfoxide, and stored at −20 °C until use. A 16 : 1 ratio of amoxicillin to relebactam was used, as previously reported [52 (link)]. Classification of the isolate’s susceptibility to antibiotics was determined based on EUCAST breakpoints [53 ] using entries for Pseudomonas spp. as published previously [28, 30, 54 (link)], as well as PK-PD breakpoints which are not species-specific.