Colistin sulfate

Analytical grade colistin sulfate, netilmicin sulfate, 9-fluorenylmethyl chloroformate (FMOC-Cl), trichloroacetic acid, sodium hydroxide, acetone, sodium hydrogen carbonate, and boric acid, and HPLC grade methanol, acetonitrile, tetrahydrofuran, and distilled water were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The serum employed for quality control (QC) was purchased from Alfresa Pharma Corporation (Osaka, Japan).
Stock solutions of colistin sulfate (100 μg/mL) and netilmicin sulfate (5 μg/mL) internal standard were prepared by dissolving 1.0 and 0.05 mg of the respective substances in 10 mL of distilled water. A 100 mM FMOC-Cl stock solution was prepared by dissolving 258.7 mg of FMOC-Cl in 10 mL of acetonitrile. The carbonate buffer (1 wt%, pH 10) was prepared by dissolving the sodium hydrogen carbonate (1 g) in distilled water (100 mL) and the pH of the solution was adjusted to 10 using sodium hydroxide. All solutions were stable for at least 2months when stored in a refrigerator at 4 °C.

P. aeruginosa (NBRC 13275 and PAO1) was purchased from Biological Resource Center in the National Institute of Technology and Evaluation, Chiba, Japan. A. baumannii (JCM 6841) was purchased from RIKEN BioResource Research Center, Ibaraki, Japan. MRSA (IID 1677) was purchased from the Institute of Medical Science in the University of Tokyo, Japan. Lysozyme (Lysozyme BIO; purified from fresh chicken egg white) was purchased from Biocon Japan Ltd., Aichi, Japan. Chitosan oligosaccharide (COS) (made from shells of the crab) was purchased from Kimika Co., Aichi, Japan. COS presented a deacetylation degree of over 98% and a molecular weight (MW) of 5 kDa. Galactomannan was purchased from Taiyo Kagaku, Tokyo, Japan. Galactomannan presented an MW of 15 kDa. Precast sodium dodecyl sulfate (SDS) gel (SuperSep Ace 10–20%), the Wako Silver Stain II kit, colistin sulfate, vancomycin hydrochloride, N-phenyl-1-naphthylamine (NPN), 2-nitrophenyl β-D-galactopyranoside (ONPG), and Micrococcus luteus were purchased from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan. Defibrinated rabbit blood was purchased from Cosmo Bio Co., Tokyo, Japan. The LIVE/DEAD BacLight Bacterial Viability Kit (L7012) was purchased from Thermo Fisher Scientific Corporation, Massachusetts, USA.

The type strain A. baumannii ATCC 19606 and ATCC BAA-1605 were purchased from ATCC (Manassas, VA, USA). Bacteria were cultured for 18 h in Luria-Bertani (LB) broth (BD Biosciences, San Diego, CA, USA) at 37°C with constant shaking (135 rpm). Colistin-resistant strains of A. baumannii were isolated by directly plating the parent strain onto LB agar (BD Biosciences) containing 10 μg/mL (5× MIC) of colistin sulfate (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) for 24 h at 37°C (29 (link)). LPS-deficient strains were cultured for 24 h in LB broth at 37°C with constant shaking (180 rpm).