Cefuroxime

The wild type strain used in this study was L. monocytogenes serotype 1/2c strain LO28 (27 (link)). Isogenic mutant derivatives of this strain were constructed as described previously (24 (link)). Primers used for in-frame deletions are listed in Supplementary Table S1. All strains used in the study are listed in Supplementary Table S2. In order to grow L. monocytogenes strains, overnight cultures were diluted 100-fold into Brain Heart Infusion medium (BHI, Oxoid) and vigorously shaken at 37°C; when appropriate, kanamycin (50 μg/ml) was added. For northern blot experiments, the following concentrations were used to induce cell envelope stress: 4 μg/ml cefuroxime (Sigma-Aldrich), 0.03% bile salts (DIFCO, No. 3), 4% NaCl, pH5 adjusted with HCl and 2% ethanol. In stress tolerance assays overnight cultures were diluted 1000-fold into BHI adjusted with cefuroxime (4 μg/ml), bile salts (0.07%), NaCl (8%), pH5 and ethanol (5%); growth was monitored for 24 h.
For cloning purposes and green fluorescent protein (GFP) reporter experiments, Escherichia coli TOP10 and E. coli DH5α (Invitrogen) were used and grown in Luria Broth (LB, Oxoid) at 37°C. When appropriate, LB was supplemented with kanamycin (50 μg/ml), ampicillin (30 μg/ml), chloramphenicol (25 μg/ml) or erythromycin (150 μg/ml).

Minimum inhibitory concentration determinations were carried out in triplicate in 96 well microtitre plates as described previously (Field et al., 2010 (link), 2012 (link), 2015b (link)). Briefly, target strains were grown overnight in the appropriate conditions and medium, subcultured into fresh broth and allowed to grow to an OD₆₀₀ of ∼0.5, diluted to a final concentration of 10⁵ cfu ml^-1 in a volume of 0.2 ml. Chloramphenicol, penicillin G, ampicillin, vancomycin, streptomycin, tetracycline, erythromycin, ceftazidime, and cefuroxime (Sigma) were resuspended in CA-MH media to a stock concentration of 128 or 256 μg/ml. The antibiotics were adjusted to 16, 32, or 64 μg/ml starting concentration and twofold serial dilutions of each compound were made in 96 well plates for a total of 12 dilutions. The target strain was then added and after incubation for 16 h at 37°C and the MIC was read as the lowest peptide concentration causing inhibition of visible growth.

Antibiotics, including doxycycline, cefuroxime, miconazole, metronidazole, rifampin, and fluconazole, were purchased from Sigma Aldrich and were dissolved in appropriate solvents [18 ] to form stock solutions. All the antibiotic stocks were filter-sterilized by a 0.2 μm filter except the DMSO stocks. Then the stocks were diluted into 500 μM and stored at −20 °C. The JHCCL FDA-approved drug library was prepared in 10 mM stock solutions with DMSO. Drugs in the master plates were diluted with PBS to make 500 μM pre-diluted working plates. The pre-diluted drug plates were sealed and stored at −20 °C.

Antibiotics including amikacin, azithromycin, cefuroxime, ciprofloxacin, clofazimine, daptomycin, disulfiram, doxycycline, gentamicin, methylene blue, miconazole, nitrofurantoin, rifampin and trimethoprim/sulfamethoxazole (SXT), were purchased from Sigma & Aldrich and were dissolved in appropriate solvents [37 ] to form stock solutions. All the antibiotic stocks were filter-sterilized by 0.2 μm filter except the DMSO stocks.

Bacterial isolates were subjected to broth microdilution susceptibility testing using panels prepared in-house and performed according to Clinical and Laboratory Standards Institute (CLSI) methods (26 , 27 ). Cation-adjusted Mueller-Hinton broth (CAMHB; Becton, Dickinson, Sparks, MD) was used as a test medium. Tebipenem and ceftibuten were from BOC Sciences (Shirley, NY), cefixime, cefdinir, cefuroxime, cephalexin, cefditoren, and amdinocillin were from Sigma-Aldrich (St. Louis, MO), and cefpodoxime was from Carbosynth (San Diego, CA).

Nutrient agar and broth as well as Saboraud dextrose agar and broth were purchased from Oxoid Limited, Basingstoke, United Kingdom. Ciprofloxacin, cefuroxime, amoxycillin, and clotrimazole were obtained from Sigma Aldrich™ (Michigan, USA).

Stock solutions of ampicillin, amoxicillin, and cefuroxime (all from Sigma-Aldrich Inc., St. Louis, MO, USA) were prepared as follows: ampicillin trihydrate and amoxicillin were dissolved in pure DMSO, sterilized using a 0.22 µm syringe filter with nylon membrane (Acrodisc, Pall Corporation, Port Washington, NY, USA), and aliquoted into sterile cryovials for storage at −20°C. cefuroxime sodium salt was dissolved in BHI broth and sterilized as above. All antibiotic stock solutions were frozen and thawed once before use. All were used immediately after thawing and then discarded.