Polymyxin b sulfate pmb

All bacterial strains used in this study are listed in Table 1. L. monocytogenes Scott A was obtained from the International Life Sciences Institute (ILSI) North America [36 (link)] and used as the wild-type (WT) reference strain and as the parental strain for the generation of mutants. L. monocytogenes strains were grown at 30 °C in brain heart infusion (BHI; Oxoid, Hampshire, UK). E. coli DH5α [37 (link)] and S17-1λpir [38 (link)] were used as cloning hosts and as donor strains for conjugational transfer of plasmid DNA to L. monocytogenes, respectively, and were grown at 37 °C in Luria–Bertani medium (LB; 10 g/L tryptone (Thermo Fisher Scientific, Waltham, MA, USA), 5 g/L yeast extract (Thermo Fisher Scientific), 5 g/L NaCl). The growth media were supplemented with 50 µg/mL kanamycin (Km; AppliChem GmbH, Darmstadt, Germany), 100 µg/mL ampicillin (Amp; Thermo Fisher Scientific), 5–50 µg/mL polymyxin B sulfate (PMB; AppliChem GmbH), or 10 µg/mL erythromycin (Ery; Acros Organics, Geel, Belgium) as required.

Lipopolysaccharide (LPS) from E. coli O55:B5 (≥ 500,000 EU/mg) was purchased from Sigma-Aldrich Co. LLC (Vienna, AT). Polymyxin B- sulfate (PMB), an antibiotic, which is known to bind and inactivate endotoxins, was obtained from AppliChem (Darmstadt, D). Two bentonites (Bentonite 1 – main part smektite), one being a natural sodium-bentonite (Bentonite 2 – main part consist of smectite, feldspar and gypsum), and two clays treated with amines (organophilised) were used: a smectite (Organoclay 1 – fully organophilised: whole surface coated with amines) consisting of montmorillonite and an attapulgite (magnesium phylosillicate) consisting of smectite and palygorskite (Organoclay 2 – part of the surface organophilised).
Reagents used for the Limulus amebocyte lysate (LAL) test were obtained from Charles River Laboratories (CRIVER), Inc. Charleston, US: Limulus amebocyte lysate (Endochrome K; Charge: C4452E), Endotoxin-free (< 0.005 EU/mL) LAL reagent water (LRW; Charge: 99732088) and Endosafe control Standard Endotoxin from E. coli O55:B5 1.000 EU/mL (Charge: EX 14392 and EM11302 – RSE/CSE ratio 10 EU/ng; EX01022 – RSE/CSE ratio 12 EU/ng; EM11302 – RSE/CSE ratio 7 EU/ng). Potassium phosphate monobasic, sodium hydroxide and pancreatin for the preparation of the artificial intestinal fluid were obtained from Merck KGaA (Darmstadt, D).