Ticarcillin

The following antibiotics were obtained as microbiological standard from Sigma-Aldrich, St Louis, MO (sodium salts, potency in brackets): ticarcillin (TIC; 85.25%), piperacillin (PIP; 94.20%), carbenicillin (CAR; 89.16%), and cefoxitin (FOX; potency, 95.11%). The remaining antibiotics were obtained as powder for parenteral use from their corresponding manufacturers (potency in brackets): temocillin (TMO, 78.12%) as Negaban^® from Eumedica (Manage, Belgium), piperacillin-tazobactam (TZP; 97.00%) as Tazocin^® from Wyeth (Louvain-La-Neuve, Belgium), ceftazidime (CAZ, 88.20%) as Glazidim^® from Glaxo-SmithKline (Genval, Belgium), imipenem (IPM, 45.60% [due to the presence of cilastatin in the powder]) as Tienam^® from MSD (Brussels, Belgium), and meropenem (MEM, 74.00%) as Meronem^® from AstraZeneca (Brussels, Belgium).

The antibiotic susceptibility test was performed by the broth dilution method^{69 (link)}, and ticarcillin (Sigma), tobramycin (Sigma), imipenem (Sigma), ciprofloxacin (Fluka), piperacillin (Sigma), and tazobactam (Sigma) for adjuvant of peperacillin were used. PAO1 and the four isolates were precultured and subcultured in MH broth depending on the bacterial strains and growth conditions presented above. Subcultures were incubated at 37 °C and 220 rpm for 3 hr to reach the exponential phase. The bacterial medium was adjusted to optical density (OD) 0.1 (the OD 0.1 of all bacteria is approximately 1 × 10⁸ colony-forming units [CFU]) using fresh MH broth, and this medium was diluted 100-fold and adjusted to a final density of 1 × 10⁶ CFU. All antibiotics were diluted in MH broth to make 2 × concentrations. The final bacterial medium (1 ml) and antibiotic-dissolved media (1 ml) were mixed, and these mixtures were incubated overnight at 37 °C and 230 rpm. The MIC results were determined using the Clinical and Laboratory Standards Institute (CLSI) guidelines.

NMR spectra were measured with a JEOL Eclipse 500 FT-NMR spectrometer (¹H, 500 MHz; ¹³C 125 MHz). Column chromatography was carried on silica-gel (Kieselgel 60, 70–230 mesh, Merck, Germany), a thin layer chromatography (TLC) on pre-coated Silica-gel F254 (0.25 mm, Merck) and Sephadex LH-20 (25–100 µM, Sigma, U.S.A). Mueller-Hinton broth (MHB) and Mueller-Hinton agar (MHA) (Difco Laboratories, Baltimore, MD, USA). Ampicillin, amoxicillin/clavulanic acid, chloramphenicol, cephalothin, sulfisoxazole, nalidixic acid, norfloxacin, streptomycin, trimethoprim/sulfamethoxazole, ticarcillin, ciprofloxacin, N,N′-Dicyclohexylcarbodiimide, Sodium azide, Tris, Triton X-100 and solvents were purchased from Sigma Aldrich (St. Louis, USA).

Plasmid DNA was extracted from both E. hoffmannii WEB-1 and A. hermannii WEB-2 isolates with the Kieser technique as previously described (Kieser, 1984 (link)) and electroporated in E. coli TOP10 and E. cloacae CIP 7933. Recombinant strains were selected on TSA supplemented with 100 mg/L of ticarcillin (Sigma, St-Quentin-Fallavier, France).

Ticarcillin, Thiophene-2-Acetic acid, and Thiophene-3-Acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). LC-MS grade Acetonitrile (ACN) and Ammonium formate (LR grade) were obtained from Supelco (Merck, Darmstadt, Germany) and Fluka (Buchs, Switzerland). Ultrapure Milli-Q-Water was obtained from Milli-Q Academic apparatus (Millipore Corporation, Billerica, MA, USA) and was used for making solutions during analysis.

The selection of mutants and assessment of mutation frequency were performed in triplicates. Overnight cultures inoculated from single colonies were adjusted to an OD_600nm of ~1. 100 µL of suspension were spread on LB-agar containing 50 µg/mL ticarcillin (Sigma-Aldrich, Merck, Darmstadt, Germany), and 100 µL of 10⁻⁶, 10⁻⁷ and 10⁻⁸ dilutions were spread on blood agar for viable cell count. Plates were incubated for 72 h at 35 °C with 5% CO₂ before assessment of growth. Mutants were categorised as true mutants if unrestricted growth was observed after subculture on ticarcillin-containing plates, and if they showed a specific resistance pattern with significant decrease in susceptibility towards piperacillin-tazobactam and meropenem, as evaluated by disk-diffusion tests. Mutation frequency was calculated as the number of ticarcillin-resistant colonies in proportion to the viable cell count, and the reported values correspond to the mean of three replicates.