Clavulanic acid

Polyclonal rabbit antibodies against W. chondrophila were produced in our laboratory as described previously [44 (link)]. Secondary antibody, Alexa Fluor^® 488 donkey anti-rabbit IgG, was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Clavulanic acid, deferoxamine, mecillinam, novobiocin, penicillin, phosphomycin, piperacillin, teicoplanin, and 2,2′-bipyridyl were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vancomycin was obtained from AppliChem (Darmstadt, Germany). MP265 was purchased from American Custom Chemicals Corporation (San Diego, CA, USA). All drugs were diluted in deionized water with the exception of MP265 and 2,2′-bipyridyl, which were diluted in dimethyl sulfoxide (DMSO, AppliChem) and in ethanol, respectively, in order to obtain the specific concentrations described in Table 1. Finally, the solutions were filtered through a 0.22 μm pore filter and stored at −20 °C.

The primary parental strains utilized in this study were the prototypical and well-characterized NaHCO₃-responsive strain, MW2 (10 (link), 20 (link), 30 (link), 38 (link)) and the nonresponsive strain, C36 (15 (link), 30 (link)) (Table 2). Strains MW2 and C36 have previously been identified as having either the “susceptible 2” or “resistant 2” mecA genotypes, respectively (30 (link)), as defined by Harrison et al. (27 (link)). Strains were stored at −80°C and isolated on tryptic soy agar (TSA) at 37°C in ambient air when ready for use. All liquid cultures were grown at 37°C in ambient air with aeration.
For penicillin Etest susceptibility testing of penicillin-clavulanate combinations, Iso-Sensitest Agar (ISA, Oxoid) was prepared as per manufacturer’s instructions, with or without 15 μg/mL clavulanic acid (Sigma-Aldrich; see below for further details). For broth MIC testing, RNA isolation/gene expression studies, GFP reporter assays, and Western blotting, strains were cultured in cation-adjusted Mueller-Hinton Broth (CA-MHB, Difco, Beckton-Dickinson) or CA-MHB buffered with 100 mM Tris (CA-MHB Tris) to maintain pH 7.3 ± 0.1, with or without 44 mM NaHCO₃. Where indicated, 1/2× MIC of OXA (Table 1) was also added to the growth media to stimulate mecA expression. In all experiments in which OXA was added to the growth medium, 2% NaCl was also included.

Meropenem, cefazolin, benzylpenicillin, ampicillin, amoxicillin, amoxicillin/clavulanic acid and ampicillin/sulbactam, were acquired from Pharma investi and Laboratorios (Chile); clavulanic acid, sulbactam, BSA and all other chemical agents were acquired from Sigma-Aldrich; Biosensor: PenP E166C to a concentration of 5 × 10⁻⁸ M was prepared in PBS with 1% of BSA.