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.
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 (