Cation adjusted mueller hinton broth camhb

The PAEs of ciprofloxacin and compounds 1 and 2 were calculated as reported by Huband [31 (link)] with some modifications. Briefly, logarithmically growing cultures of E. coli ATCC 25922, S. aureus ATCC 29213, and S. aureus ATCC BAA-1720, at a starting concentration of 1×10⁶ CFU/ml, were exposed to various concentrations of drugs (i.e., 1×, 4× and 16× the MIC) for 1 hour at 37°C. A drug-free control was included in each experiment. After being exposed to the drug for 1 hour, viable counts were conducted in triplicate by spotting aliquots (10 μl) of suitable dilutions onto MHA plates. The drugs were removed by diluting the bacterial cultures (1:1000) in Cation-Adjusted Mueller-Hinton Broth (CAMHB; Sigma-Aldrich). The diluted cultures were incubated in a shaking bath at 37°C. To monitor the ongoing suppression of bacterial growth over time (5 hours), aliquots from each broth culture were periodically collected, diluted and plated to perform subsequent viable counts (in triplicate). PAE was defined as the difference between the times required for cultures exposed and unexposed to drugs to increase by 1 log₁₀ CFU/ml above the number of CFUs counted immediately after dilution (1:1000).

Clinical isolates of S. aureus were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program (Table 1). clindamycin hydrochloride monohydrate (TCI America, Portland, OR, USA, >98.0% purity) and mupirocin (pure USP) (AppliChem, St. Louis, MO, USA) powders were purchased commercially and dissolved in dimethyl sulfoxide (DMSO) (for clindamycin) or ethanol (for mupirocin) to prepare a stock solution (10 μg/mL). Lipoderm was purchased from the Professional Compounding Centers of America (Houston, TX, USA). Cation-adjusted Mueller-Hinton broth (CAMHB) (Sigma-Aldrich, St. Louis, MO, USA), Tryptic soy broth (TSB) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), mannitol salt agar (Hardy Diagnostics, Santa Maria, CA, USA), phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA), Dulbeco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (FBS) (USA Scientific, Inc., Orlando, FL, USA), petroleum jelly (Equate [Walmart, Inc.], Bentonville, AR, USA), and 96-well plates (CellTreat Scientific Products, Shirley, MA, USA) were all purchased from commercial vendors.

The Mycobacterium abscessus subsp. abscessus CIP104536^T reference strain and all derived mutant strains are listed in Supplementary Table S1. Strains were grown in Middlebrook 7H9 broth (BD Difco) supplemented with 0.05% Tween 80 (Sigma-Aldrich) and 10% oleic acid, albumin, dextrose, catalase (OADC enrichment; BD Difco) (7H9^T/OADC) at 30°C or in Sauton’s medium in the presence of antibiotics, when required. On plates, colonies were selected either on Middlebrook 7H10 agar (BD Difco) supplemented with 10% OADC enrichment (7H10^OADC) or on LB agar. For drug susceptibility testing, cultures were grown in Cation-Adjusted Mueller-Hinton Broth (CaMHB; Sigma-Aldrich). The TAC analogs D6, D15, and D17 were synthesized as reported previously (Coxon et al., 2013 (link)) and dissolved in DMSO. Other antibiotics were purchased from Sigma-Aldrich.