Clavulanate

The standard used was Ciprofloxacin, pharmaceutical secondary standard; certified reference material purchased from the supplier Sigma-Aldrich, with CAS number: 855721–33-1. Amoxicillin and clavulanate potassium buffer standards were also provided from Sigma-Aldrich.
The solvents used were methanol, acetonitrile, sodium dihydrogen phosphate, triethylamine and phosphoric acid, all being of analytical grade. Ultra-pure water was prepared on a milli-Q purification system from Millipore (18 MΩ.cm). The uncertainty of the weighing balance was ±1 mg.

M. tuberculosis reference strain H₃₇Rv (ATCC 25618) was used in this study. Cultures of this strain were grown in Middlebrook 7H9 complete broth (Difco) supplemented with 0.5% glycerol, 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.05% Tween-80 as described previously [18 ] at 37°C with constant shaking. Organ homogenates were inoculated and M. tuberculosis colony-forming units (CFUs) were grown in Middlebrook selective 7H11 agar plates supplemented with 0.5% glycerol, 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.05% Tween-80 at 37°C. Rifampin, meropenem, and clavulanate were obtained from Sigma-Aldrich.

Mab, Msm, and Mtb were grown to mid-log phase and diluted to an OD₆₀₀=0.003 in each well of non-treated 96-well plates (Genesee Scientific) containing 100 μl of antibiotic serially diluted in 7H9+OADC + 5 μg/ml clavulanate (Sigma-Aldrich). For MICs on Mab knockdown cells, cultures were induced for knockdown 18 hr prior with 500 ng/μl ATc. Wells with knockdown bacteria also contained 500 ng/μl ATc. Msm media contained ADC rather than OADC. Cells were incubated with drug at 37°C with shaking for 1 day (Mab, Msm) or 7 days (Mtb). Afterward, 0.002% resazurin diluted in ddH₂O (Sigma-Aldrich) was added to each well. Plates were then incubated for 24 hr. MICs were determined by the concentration of antibiotic that turned wells blue signifying no metabolic activity (Kieser et al., 2015b (link)).

MIC determination by microbroth dilution (HTM, Oxoid Ltd, Basingstoke, UK) was carried out according to CLSI guidelines [36 ], except that testing of penicillin-beta-lactamase inhibitor combinations was performed with fixed inhibitor concentrations [37 ]. Beta-lactam agents tested were ampicillin, amoxicillin, piperacillin, cefuroxime, cefotaxime (Sigma-Aldrich, St. Louis, MO, USA) and meropenem (Sequoia, Pangbourne, UK). For beta-lactamase positive isolates, ampicillin, amoxicillin and piperacillin MICs were determined in the presence of sulbactam 4 mg/L (Sequoia), clavulanate 2 mg/L and tazobactam 4 mg/L (Sigma-Aldrich), respectively. MICs were within accepted ranges for H. influenzae ATCC 49247 (rPBP3) and H. influenzae ATCC 49766 (sPBP3), and within the wild type range (http://www.eucast.org/MIC_distributions) for H. influenzae ATCC 35056 (TEM-1 positive).
MICs were interpreted according to EUCAST clinical breakpoints, except for piperacillin and piperacillin-tazobactam where breakpoints are not defined [37 ]. Meningitis breakpoints were used for susceptibility categorization of meropenem to allow quantification of low-level resistance. Data from this study are included in the EUCAST database for MIC distributions of clinical isolates.