Polymyxin b

Polymyxin B was purchased from Sigma-Aldrich (Sydney, New South Wales, Australia). The stock solution of Polymyxin B (1 mg/ml) was prepared using Milli-Q water (Millipore Australia, North Ryde, NSW, Australia) and filtered through 0.22-μm syringe filters (Sartorius, Melbourne, VIC, Australia).

Supernatants of type strains of the four Achromobacter species were used for further analysis regarding LPS activity and compared to the supernatant of P. aeruginosa strain PAO1. Supernatants were preincubated with or without 40 μg/mL polymyxin B (Sigma-Aldrich) for 20 min at 37°C. P. aeruginosa LPS (Merck) was included as a control (1 μg/mL, final concentration), with or without polymyxin B. Peripheral blood was collected from 3 healthy donors and stimulated with bacterial supernatants, HEPES, LPS, or fMLF according to the flow cytometry protocol above.

Polymyxin B was purchased from Merck (Darmstadt, Hesse) and was prepared by dissolving with Milli-Q water to obtain a final concentration of 512 mg/L. The exogenous metabolites (10 mM PHPYR, 1 mM OROT, 5 mM 3PHP, 10 mM 3PG, 10 mM R5P, and 1 mM UACGAM) were individually examined, alone and in combination with 2 mg/L Polymyxin B against the four K. pneumoniae strains by static time–kill studies. The concentrations of exogenous metabolites were normalized to deliver 60 mM carbon except OROT, 3PHP, and 3PG due to their poor aqueous solubility. All metabolites were purchased from Sigma-Aldrich (Saint Louis, Missouri).

Reference strains are from the German Collection of Microorganisms and Cell Cultures (DSMZ), the American Type Culture Collection (ATCC), the National Collection of Type Cultures (NCTC), and the Medical and Molecular Microbiology, Faculty of Science and Medicine, University of Fribourg, Switzerland. Clinical strains used to determine the MIC₉₀ of NOSO-502 came from hospitals in Warsaw, Poland, Copenhagen, Denmark, Cardiff, UK, and Madrid, Spain. NOSO-502 was synthesized at Nosopharm, Nîmes, France. Ciprofloxacin (Sigma-Aldrich; no. 1134335), gentamicin (Sigma-Aldrich; no. G1397), imipenem (Sigma-Aldrich; no. IO160), polymyxin B (Sigma-Aldrich; no. 92283), and tigecycline (Sigma-Aldrich; no. PZ0021) were provided by the manufacturer as standard powders except for gentamicin and polymyxin B, in solution at 50 and 20 mg/ml, respectively.

Bacteria were grown as described above for AMC binding assays and diluted into filtered 0.1μM potassium phosphate buffer (PPB). After addition of AMCs (250 nM CCL28 diluted in 0.01mg/ml BSA) or BSA (0.01 mg/ml), bacteria were incubated at 21 for 5 h. At the indicated time points, 20 μl of bacteria were removed and put on ice in PBS containing freshly diluted Polybead ® Polystyrene 15 μm Microsphere counting beads diluted 1:62500 (Polysciences Inc). Propidium iodide (PI) (Invitrogen) was added just before reading on the flow cytometer. The number of beads remains constant and the beads can be distinguished from the bacteria on the flow cytometer, which allows the number of viable bacteria to be determined by subtracting PI positive bacteria from the total number of bacteria counted per 30000 beads. Percent survival was calculated by dividing viable bacteria of the sample by viable bacteria in the BSA treated control. Polymyxin B susceptibility assays were carried out in the same manner, except that bacteria were exposed to a range of Polymyxin B (Sigma-Aldrich) concentrations (0–20 μM) for 5 h, and then bacterial survival relative to BSA-treated controls were determined by flow cytometry. The statistical significance of specific comparisons of interest was assessed via two-way ANOVA with Dunnett’s correction using GraphPad Prism software (www.graphpad.com).

According to the established protocol form the kit’s description, the outer membrane permeability of CNMs on Tet-resistant ETEC of stationary phase was determined using N-phenyl-1-naphthylamine (NPN) dye. Tet-resistant ETEC of the log phase was obtained by centrifugation and washed 3 times with HEPES buffer containing 5 mM glucose (pH 7.4). Bacterial cells were resuspended in the same buffer at a final concentration of 10⁶ cfu/ml and treated with NPN dye at a final concentration of 10 μM, and the bacterial cells were stained. In 96-well black plates, bacterial suspensions were treated with CNMs at 0%, 0.025%, 0.05%, 0.1%, and 0.2%. A fluorescence spectrophotometer (Infinite 200 PRO, Tecan, China) at excitation λ = 422 nm and emission λ = 350 nm was applied to record fluorescence. The data are converted to the NPN absorption percentage as follows:
where F_obs is the fluorescence obtained at the concentration indicating CNMs, F₀ is the initial fluorescence of NPN in Tet-resistant ETEC cells without CNMs, F₁₀₀ is added after 10 μg/ml polymyxin B (Sigma, USA) NPN fluorescence, and polymyxin B was used as a positive control to cause outer membrane permeability.

A group of wild-type C3H/HeOuJ background mice were treated with antibiotic containing water ad libitum or control group was given standard drinking water. Antibiotic treatment consisted of neomycin and polymyxin B (final concentrations: 0.6 mg/mL for neomycin and 120 units/mL for polymyxin B, both from Sigma-Aldrich, St. Louis, MO, United States) given in drinking water for 8 weeks as described earlier (Prakash et al., 2015 (link)). This combination of antibiotics was chosen specifically for the ability of both to remain within the gut and not be absorbed into circulation; by using this combination to target the gut microbiome, we could focus primarily on its effects without disruption of other microbiomal niches. Supplementary Figure S6 demonstrates the effects of this combination on the richness, evenness, and diversity of the microbiome after 7 weeks of treatment.