Polymyxin

Manufactured by Merck Group

Sourced in United States

Polymyxin is a type of antibiotic used in various laboratory applications. It functions as an antimicrobial agent, primarily against Gram-negative bacteria. The core function of Polymyxin is to disrupt the cell membranes of targeted microorganisms.

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Lab products found in correlation

9 protocols using polymyxin

Synthesis of peptidoglycan precursors

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Polymyxin, UDP-GlcNAc, D-Ala-D-Ala, PEP, ATP and Hepes were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). UDP-MurNAc-L-Ala-D-Glu-m-DAP was obtained from BACWAN facility, School of Life Sciences, The University of Warwick, UK. MurA from E. coli and S. aureus were recombinant, expressed in E. coli [10 (link)], while MurF from E. coli and S. aureus were recombinant expressed in E. coli [40 (link),41 (link)].

Funes Chabán M., Hrast M., Frlan R., Graikioti D.G., Athanassopoulos C.M, & Carpinella M.C. (2021). Inhibition of MurA Enzyme from Escherichia coli and Staphylococcus aureus by Diterpenes from Lepechinia meyenii and Their Synthetic Analogs. Antibiotics, 10(12), 1535.

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Recombinant Paracoccin Purification and Endotoxin Removal

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Recombinant paracoccin was expressed in Escherichia coli, as previously described [20] (link). It was purified by affinity chromatography on an N-acetylglucosamine column. Before use, endotoxins were removed with an immobilized polymyxin B agarose column (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions. rPCN was then resuspended in endotoxin-free PBS (sterile PBS). For in vitro assays, rPCN aliquots were incubated for 1 h at room temperature (RT) with polymyxin (50 µg/mL; Sigma-Aldrich, St. Louis, MO, USA) to neutralize any potential contamination with bacterial lipopolysaccharides (LPS). rPCN preparations contained less than 0.05 ng/mL of bacterial endotoxin, as determined by the Endpoint Chromogenic LAL assay kit (Walkersville, Maryland, USA).

Alegre-Maller A.C., Mendonça F.C., da Silva T.A., Oliveira A.F., Freitas M.S., Hanna E.S., Almeida I.C., Gay N.J, & Roque-Barreira M.C. (2014). Therapeutic Administration of Recombinant Paracoccin Confers Protection against Paracoccidioides brasiliensis Infection: Involvement of TLRs. PLoS Neglected Tropical Diseases, 8(12), e3317.

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Decellularization and Bioburden Reduction

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The decellularisation procedure for Study 2 was as described for Study 1 but with the acetone treatment included as a fat reduction step. The following changes were then subsequently introduced. A 3 h bioburden reduction step was included immediately after the acetone treatment using either peracetic acid (0.1%; v/v [Sigma]) or an antibiotic solution (PBS containing 0.05 mg ml⁻¹ vancomycin hydrochloride, 0.5 mg ml⁻¹ gentamicin sulphate, 0.2 mg ml⁻¹ polymyxin [all from Sigma]), both at room temperature. In addition, each bioburden reduction step was investigated with and without the terminal PAA treatment described in Study 1. This was to determine any interacting effects it may have with the bioburden steps on the mechanical properties of the tissue.
Hence, seven groups were investigated in Study 2:

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Native (untreated)

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DC2+TPAA

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DC2−TPAA

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DC2+PAAbio+TPAA

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DC2+PAAbio−TPAA

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DC2+ABbio+TPAA

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DC2+ABbio−TPAA.

DC2: decellularisation process with acetone permanently included (i.e. DC2=DC1+ACE), TPAA: terminal peracetic acid treatment, PAAbio: peracetic acid bioburden treatment, ABbio: antibiotic bioburden treatment. + and − denote ‘with’ and ‘without’ respectively.
The steps investigated and the processes involved are shown in Fig. 1(b).

Herbert A., Jones G.L., Ingham E, & Fisher J. (2015). A biomechanical characterisation of acellular porcine super flexor tendons for use in anterior cruciate ligament replacement: Investigation into the effects of fat reduction and bioburden reduction bioprocesses. Journal of Biomechanics, 48(1), 22-29.

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Bone Marrow Transplantation in LDLR-/- Mice

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C57BL/6 LDLR^-/- CD45.2 acceptor mice (in house breeding colony, Maastricht University) were lethally irradiated with two doses of 6 Gy, 24 h apart. C57BL/6 LDLR^-/- CD45.1 donor mice (in house breeding colony, Maastricht University) were sacrificed by carbon dioxide inhalation followed by cervical dislocation. Subsequently, bone marrow was isolated from femur and tibia, pooled and dissolved in RPMI medium (Gibco^® 1640, Carlsbad, CA, USA) supplemented with heparin (Leo Pharma, Ballerup, Denmark) at a concentration of 50 × 10⁶ cells/mL. Ten million donor bone marrow cells were injected in the tail vein of each acceptor mouse. Acceptor mice received 100 mg/mL Neomycin (Gibco, Carlsbad, CA, USA) and 60 000 units/mL Polymyxin (p4932, Sigma-Aldrich, St. Louis, MO, USA) antibiotics in their drinking water 1 week before and 3 weeks after bone marrow transplantation. After 6 weeks recovery, mice were given either a HFD for 10 days (n = 6) or 5 weeks (n = 7), or a control diet for 5 weeks (n = 6). Subsequent to the diet, the mice were sacrificed by carbon dioxide inhalation followed by cardiac puncture and vAT was isolated for flow cytometry analysis.

Wetzels S., Bijnen M., Wijnands E., van de Gaar J., Tan A., Coort S., Biessen E.A., Schalkwijk C.G, & Wouters K. (2020). CD11c−MHC2low Macrophages Are a New Inflammatory and Dynamic Subset in Murine Adipose Tissue. Immunometabolism, 2(2), e200015.

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Brucella Strain Culturing and Metabolic Mutant Phenotyping

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Brucella strains were routinely grown in peptone-glucose broth (tryptic soy broth [TSB], Biomerieux) or in TSB-agar (TSA). When appropriate, the following antibiotics (all from Sigma) were used: kanamycin (Km; 50 μg/mL), ampicillin (Amp; 200 μg/mL), polymyxin (Pmx; 1.5 μg/mL), and/or chloramphenicol (Cm; 20 μg/mL). When needed, media were supplemented with 5% sucrose. The media used to study the phenotype of the metabolic mutants were peptone-glucose, Gerhardt’s defined medium (glutamate-lactate-glycerol) (16 (link)), or Plommet’s vitamin-mineral salts (15 (link)) modified by Barbier et al. (18 (link)) (Supplementary Table 3). BL21 cells were grown in Luria-Broth (LB) or in M9 minimal medium (Sigma) supplemented with glucose (1 mM), MgSO₄ (1 mM) and CaCl₂ (1 mM).

Lázaro-Antón L., Veiga-da-Cunha M., Elizalde-Bielsa A., Chevalier N., Conde-Álvarez R., Iriarte M., Letesson J.J., Moriyón I., Van Schaftingen E, & Zúñiga-Ripa A. (2024). A novel gluconeogenic route enables efficient use of erythritol in zoonotic Brucella. Frontiers in Veterinary Science, 11, 1328293.

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Bone Marrow Transplant in Transgenic Mice

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This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at the University of Illinois at Chicago (Protocol approval #12–152).
Six week old C57BL/6-Tg-(UBC-GFP) (TLR4+/+) and C57BL/10ScN (TLR4-/-) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Green fluorescent protein (GFP) is under transcriptional control of the human ubiquitin C promoter resulting in high-level expression in most tissues.
Following one week acclimatization on regular chow and water ad libitum at a constant temperature (22°C) with humidity of 45% to 55% in a 12-hour light/dark cycle, all recipient mice for bone marrow transplant received water containing polymyxin 13mg/L (Sigma, St. Louis, MO), and neomycin 100mg/L (Sigma, St. Louis, MO) for 5 days. Mice were fed a low fat (LF) diet (12.3 kcal % fat, Research Diets, Inc., New Brunswick, NJ) (S1 Table) throughout the study.

Ilievski V., Cho Y., Katwala P., Rodriguez H., Tulowiecka M., Kurian D., Leoni L., Christman J.W., Unterman T.G, & Watanabe K. (2015). TLR4 Expression by Liver Resident Cells Mediates the Development of Glucose Intolerance and Insulin Resistance in Experimental Periodontitis. PLoS ONE, 10(8), e0136502.

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HIV+ Serum Induces Tissue Factor Expression

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Column-purified CD14⁺ monocytes were obtained from
healthy blood donors and resuspended in RPMI-1640 media supplemented with
10% of indicated serum (commercialized human AB serum or pooled
0.22μm filtered sera from 12 healthy controls, 20 ART-naïve
HIV⁺ patients and at week 12 and 48 of ART, and 16
HCV⁺ persons) at 10⁶ cells/well in 96-well
plates. Cells were left overnight (18h) at 37^oC in 5%
CO₂. Cells were then washed and lysed for measurement of TF
protein expression in cell lysates. In additional experiments, cells were
incubated with 10μg/mL of anti-IL1R1 (polyclonal, R&D Systems),
anti-IL6Ra (clone 17506, R&D Systems), anti-IFNγR (clone 92101,
R&D Systems), anti-IFNAR (polyclonal, ab10719, Abcam) and anti-TNFR1
(clone 16803R, R&D Systems) for 1h before incubation with
HIV⁺ serum and persisted in cultures for additional 18h.
HIV⁺ serum was also treated with Polymyxin
(0.5μg/μL, Sigma-Aldrich) for 1h and filtered before incubating
for 18h.

Schechter M.E., Andrade B.B., He T., Richter G.H., Tosh K.W., Policicchio B.B., Singh A., Raehtz K.D., Sheikh V., Ma D., Brocca-Cofano E., Apetrei C., Tracy R., Ribeiro R.M., Sher A., Francischetti I.M., Pandrea I, & Sereti I. (2017). Inflammatory monocytes expressing tissue factor drive SIV and HIV-coagulopathy. Science translational medicine, 9(405), eaam5441.

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Bone Marrow Transplantation in LDLR-/- Mice

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RAW264.7 cell treatment responses

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Cell culture and treatments RAW264.7 cells were purchased from and grown according to ATCC methods, in DMEM supplemented with 10% FBS. No antibiotics or any other antimicrobial reagents were added to the culture medium, as they were found to interfere with the LPS inhibitory effects on lysozyme expression. Cells were treated for 3 h with filtered fecal slurry (in PBS) obtained from euthanized mice, or with LPS (L4391; Sigma, St. Louis, MO, USA), sodium butyrate (B5887; Sigma) or Desulfovibrio vulgaris (DSV) for 24 h. Polymyxin (P4932; Sigma) was added to a final concentration of 0.1 mg/ml 1 h before the LPS challenge. Rapamycin (R8781; Sigma) at 100 nM was added to the cells 1 h prior to challenge with test reagents. DMSO-treated cells (volume equivalent to what was used for 100 nM Rapamycin) were used as controls. Rapamycin was also added to the cells 24 h after LPS challenge. In this case, Rapamycin was added for 24 h in the continued presence of LPS. Cells were harvested and processed for preparation of protein and RNA samples.

Singh S.B, & Lin H.C. (2017). Autophagy counters LPS-mediated suppression of lysozyme. Innate immunity, 23(6).

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