Polymyxin b sulfate

Manufactured by Merck Group

Sourced in United States, Australia, United Kingdom

Polymyxin B sulfate is a broad-spectrum antibiotic used in laboratory settings. It functions as a bactericidal agent, targeting and disrupting the cell membranes of Gram-negative bacteria.

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Lab products found in correlation

70 protocols using polymyxin b sulfate

Polymyxin B Susceptibility in Pseudomonas and E. coli

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P. aeruginosa cultures were grown to an OD₆₀₀ of 0.6 in high or low phosphate minimal media A (see above). Cultures were then diluted 1:100 in prewarmed minimal media A containing 4 µg mL⁻¹ polymyxin B sulfate (Sigma). Samples were incubated at 37 °C, 180 rpm, and assayed for survivors at specified time points by serial dilution plating onto LB. E. coli cultures containing pET-28a-Agt1 or pET-28a-Agt2 were grown to an OD₆₀₀ of 0.6 in LB broth, and the expression of Agt1 and Agt2 was induced by incubation with 0.4 mM IPTG overnight at 25 °C. A negative control of E. coli containing the pET-28a vector only was also set up. Overnight cultures were diluted 1:100 in prewarmed LB broth containing 0.4 mM IPTG and 20 µg mL⁻¹ polymyxin B sulfate (Sigma). Samples were incubated at 37 °C, 180 rpm, and assayed for survivors at specified time points by serial dilution plating onto LB agar + kanamycin 25 µg mL⁻¹.

Jones R.A., Shropshire H., Zhao C., Murphy A., Lidbury I., Wei T., Scanlan D.J, & Chen Y. (2021). Phosphorus stress induces the synthesis of novel glycolipids in Pseudomonas aeruginosa that confer protection against a last-resort antibiotic. The ISME Journal, 15(11), 3303-3314.

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Synthesis and Characterization of Antimicrobial Nanoparticles

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Bacterial strains of E. coli (DH5α) and S. typhi (serovar enterica) were obtained from the School of Biological Sciences at IIT Delhi. The S3 sushi peptide sequence (HAEHKVKIGVEQKYGQFPQGTEVTYTCSGNYFLMC, M.W. 4,083 Da, purity > 95%) was synthesized by Genemed Synthesis (San Antonio, USA). Gold(III) chloride trihydrate (HAuCl₄.3H₂O), sodium citrate dihydrate (C₆H₅Na₃O₇.2H₂O), tannic acid (C₇₆H₆₂O₄₈), polymyxin B sulfate (PMB), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ortho-pthaldehyde (OPA), Luria broth (LB), folic acid (FA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and boric acid were purchased from Sigma Aldrich (India). Dithiolalkane aromatic PEG6 hydrazide (DTH) was obtained from Sensopath Technologies (USA). Neutral red and gluteraldehyde (GLA) were obtained from CDH (India). Dulbecco’s modified eagle medium (DMEM, high glucose), fetal bovine serum (FBS), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s phosphate-buffered saline (PBS), antibiotic-antimycotic solution and calcein AM were purchased from Invitrogen (India). All solutions were prepared using ultrapure deionised (DI) Milli-Q water (∼18.8 mΩ.cm resistivity) (CDUFBI001, Millipore, USA). HeLa cell line was obtained from the National Centre for Cell Science (NCCS) (Pune, India).

Singh R., Patil S., Singh N, & Gupta S. (2017). Dual functionality nanobioconjugates targeting intracellular bacteria in cancer cells with enhanced antimicrobial activity. Scientific Reports, 7, 5792.

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Intranasal M. bovis BCG Infection in Mice

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M. bovis bacillus Calmette-Guérin (M. bovis BCG) Pasteur strain was cultured in 7H9 media (Sigma) plus OADC (Fisher) containing 0.05% Tween-80 (Sigma) at 37°C, shaking at ~50 r.p.m. M. bovis BCG bacilli were washed twice with sterile PBS and passed through a 40 μm nylon mesh strainer prior to use. M. bovis BCG concentration was determined by measuring absorbance at A₆₀₀ and adjusted to appropriate concentrations in sterile PBS. Mice were anesthetized with isoflurane, and M. bovis BCG was administered via intranasal inoculation in 50 μL containing ~1.0 to 5.0 × 10⁶ bacilli. Blood was collected from euthanized mice, and lungs and spleens were removed, weighed, and homogenized in sterile PBS. Homogenate dilutions were plated on 7H10 agar (Becton Dickinson) containing OADC and antimicrobials: polymyxin B sulfate (26 μg/mL, Sigma), trimethoprim lactate (20 μg/mL, Sigma), carbenicillin disodium (50 μg/mL, Sigma), and amphotericin B (2.5 μg/mL, Sigma). Plates were incubated at 37°C for 14–21 days prior to counting. Colony forming units (CFU) from the left lung, or spleen (spleen CFU were determine per gram of tissue as half of the spleen was used for additional assays) were determined. For histology, alternate lobes of the lungs were inflated with 10% formalin and processed for H & E staining.

Lange S.M., McKell M.C., Schmidt S.M., Zhao J., Crowther R.R., Green L.C., Bricker R.L., Arnett E., Köhler S.E., Schlesinger L.S., Setchell K.D, & Qualls J.E. (2019). L-arginine synthesis from L-citrulline in myeloid cells drives host defense against mycobacteria in vivo. Journal of immunology (Baltimore, Md. : 1950), 202(6), 1747-1754.

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Antimicrobial Susceptibility Evaluation

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In vitro susceptibility to polymyxin B sulfate (Sigma), colistin methanesulfate (Sigma), cecropin A (Sigma), Spodoptera frugiperda cecropin B was evaluated by determination of minimal inhibition concentration as previously described [53] (link).

Mouammine A., Lanois A., Pagès S., Lafay B., Molle V., Canova M., Girard P.A., Duvic B., Givaudan A, & Gaudriault S. (2014). Ail and PagC-Related Proteins in the Entomopathogenic Bacteria of Photorhabdus Genus. PLoS ONE, 9(10), e110060.

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Biocide Susceptibility Assay Protocol

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Polymyxin B sulfate (Sigma) was dispensed into polypropylene microtiter plates (Greiner Bio-One, cat. no 650261) together with benzalkonium chloride (BAC). Each row in a plate contained serial two-fold dilutions of a biocide solution and each column contained serial two-fold dilutions of Polymyxin B sulfate. The following ranges of compounds were tested in a single plate: Polymyxin B: 1–0.03125 mg/L and 0.125–0.0039 mg/L, BAC: 64–0.125 mg/L. Freshly restreaked colonies of strain E. coli CFT073 were resuspended in 0.9% NaCl and cell density adjusted to 1–2 × 10⁸ CFU/mL using McFarland reagent 0.5. The culture was added to the microtiter plate, resulting in 5 × 10⁵ CFU/mL in each well. Plates were sealed and incubated at 37 °C in a static incubator. Results were read after 18 h. The same procedure was applied to the Polymyxin resistant strain E. coli 2009-710-65-10, but here the Polymyxin B range on the plates was from 4 to 0.125 mg/L. All strains were tested in three biological replicates.

Ligowska-Marzęta M., Hancock V., Ingmer H, & M. Aarestrup F. (2019). Comparison of Gene Expression Profiles of Uropathogenic Escherichia Coli CFT073 after Prolonged Exposure to Subinhibitory Concentrations of Different Biocides. Antibiotics, 8(4), 167.

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Enumerating Intracellular Bacteria in Nasal Tissue

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Infected BALB/c mice were euthanized at different times post challenge (3 h.p.c. and 7, 28, and 56 d.p.c.). NWs were collected prior to nose harvest to assess extracellular CFU in the nasal fluid. Nasal tissue was scraped from the nasal cavity, cut into small pieces, and digested with collagenase D (0.6 mg/ml; Roche) and DNase I (20 U/ml; Sigma-Aldrich) for 40 min at 37 °C. The nasal tissue was flushed several times and then passed through a cell strainer to obtain a single-cell suspension. Cells were pelleted and the supernatant was conserved to assess extracellular CFU. Cells were washed twice and then incubated for 1 h with 100 µg/ml polymyxin B sulfate (Sigma) to eliminate extracellular bacteria^{46 (link)}. Cells were washed twice and then lysed with 0.1% saponin (Sigma). CFU counting was performed on cell lysates by plating 10-fold serial dilutions onto BG agar plates containing 10 μg/ml gentamicin to estimate the number of intracellular bacteria.

Dubois V., Chatagnon J., Thiriard A., Bauderlique-Le Roy H., Debrie A.S., Coutte L, & Locht C. (2021). Suppression of mucosal Th17 memory responses by acellular pertussis vaccines enhances nasal Bordetella pertussis carriage. NPJ Vaccines, 6, 6.

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Purification of Holotoxin Proteins

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Protein expression and purification of holotoxins was performed as described previously^{31 (link)}. Briefly, the genes for human LT, CT, H1 hybrid toxin, and H2 hybrid toxin were expressed in OverExpress™ C43 (DE3) cells (Sigma-Aldrich). Cells were grown at 37 °C in TB medium containing chloramphenicol until an OD_{600 nm} of 2.0 was reached. Cells were then induced with l-arabinose and harvested after 3 h. Holotoxins were extracted from the bacterial pellet by inducing periplasmic lysis with polymyxin B sulfate (Sigma-Aldrich). Holotoxins were purified by TALON affinity chromatography using a HiTrap TALON crude column (GE Healthcare, Chicago, IL) and size exclusion chromatography with a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) equilibrated with phosphate-buffered saline. To reduce B-pentamer contamination due to partially overlapping peaks, only size exclusion fractions prior to the holotoxin peak maximum were pooled, filtered and stored at 4 °C. The hybrid toxins were characterized by SDS-PAGE analysis, tryptic digestion and mass spectrometry.

Serrano A., Guyette J.L., Heim J.B., Taylor M., Cherubin P., Krengel U., Teter K, & Tatulian S.A. (2022). Holotoxin disassembly by protein disulfide isomerase is less efficient for Escherichia coli heat-labile enterotoxin than cholera toxin. Scientific Reports, 12, 34.

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Kallistatin Modulates TGF-β1-Induced Endothelial Cell Responses

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Human umbilical vein endothelial cells (HUVECs) were kindly provided by Dr. Robin Muise-Helmericks (Medical University of South Carolina, Charleston, SC, USA). HUVECs were cultured in endothelial cell basal medium (EBM)-2 supplemented with EGM-2 SingleQuots (Lonza, Walkersville, Maryland), and maintained in a humidified atmosphere of 5% CO₂ in air at 37 °C two passages weekly. HUVECs were incubated in the presence or absence of kallistatin (1 μM) for 30 min, followed by the addition of 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, Minnesota) for another 24 or 72 h. Culture medium containing TGF-β1 or kallistatin was replaced every 24 h. For real-time polymerase chain reaction (PCR) experiments, cells were treated with TGF-β1 with or without kallistatin for 24 h. For NADPH oxidase activity, western blot, immunostaining and gelatin zymography, cells were treated with TGF-β1 with or without kallistatin for 72 h. In another set of experiments, cells were pretreated with 5 μM genistein (Sigma, Saint Louis, Missouri) for 30 min, and then further incubated with 2 μM WT-KS, HM-KS or AM-KS in the presence of 10 μg/ml polymyxin B sulfate (Sigma, Saint Louis, Missouri) for 30 min followed by stimulation with TGF-β1 for another 24 h.

Guo Y., Li P., Bledsoe G., Yang Z.R., Chao L, & Chao J. (2015). Kallistatin inhibits TGF-β-induced endothelial–mesenchymal transition by differential regulation of microRNA-21 and eNOS expression. Experimental cell research, 337(1), 103-110.

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Synthesis of Polymyxin Lipopeptide Antibody

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Anti-polymyxin B mouse IgM antibody (clone 45) was obtained from Thermo
Fisher Scientific (Rockford, Illinois, USA). Polymyxin B (sulfate), colistin
(sulfate), triisopropylsilane (TIPS), trifluoroacetic acid, (TFA) and
diphenylphosphorylazide (DPPA), Streptavidin were purchased from Sigma-Aldrich
(Sydney, New South Wales, Australia). Piperidine and diisopropylethylamine
(DIPEA) were obtained from Auspep (Melbourne, Victoria, Australia).
Fmoc-Dab(Boc)-OH was purchased from Try-lead Chem (Hangzhou, Zhejiang,China).
Fmoc-Thr(tBu)-OH and Fmoc-Leu-OH were from Mimotopes (Melbourne, Australia).
Fmoc-Dab(ivDde)-OH, Fmoc-D-Phe-OH, Fmoc-Dap-OH, Fmoc-D-Dap-OH and
1H-Benzotriazolium-1-[bis(dimethylamino)methylene]-5-chloro
hexafluoro-phosphate-(1-),3-oxide (HCTU) were purchased from Chem-Impex
International (Wood Dale, Illinois, USA). Fmoc-Thr(tBu)-TCP-Resin was obtained
from Intavis Bioanyltical Instruments (Köln, Cologne,Germany).
Dimethylformamide (DMF), methanol (MeOH), diethyl ether, dichloromethane (DCM),
hydrochloric acid and acetonitrile were purchased from Merck (Melbourne,
Victoria, Australia). Polymyxin lipopeptide stock solutions were prepared with
Milli-Q water (Millipore, North Ryde, New South Wales, Australia) and filtered
using 0.22-µm syringe filters (Sartorius, Melbourne, Victoria,
Australia). Solutions were stored at 4°C for up to one month.^{57 (link)}

Velkov T., Yun B., Schneider E.K., Azad M.A., Dolezal O., Morris F.C., Nation R.L., Wang J., Chen K., Yu H.H., Chen L., Thompson P.E., Roberts K.D, & Li J. (2016). A novel chemical biology approach for mapping of polymyxin-lipopeptide antibody binding epitopes. ACS infectious diseases, 2(5), 341-351.

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Mitochondrial Function Assays

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5,5′,6,6′-tetrachloro-1,1′,3,3″-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), calcein-AM, propidium iodide (PI), and rhodamine 123 were from Invitrogen (Carlsbad, CA, USA). The ATP Biomass Kit HS was from BioThema AB (Haninge, Sweden). Alamethicin A4665, ascorbic acid, bovine serum albumin (BSA), carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP), cyclosporin A (CsA), 7- dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine- 3-one (MCLA), dithionite, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), glutamate, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), mannitol, malate, polymyxin B sulfate, rotenone, sucrose, succinate, Trizma Base, tetraphenylphosphonium chloride (TPP+), N,N,N’N’-tetramethyl-p-phenylenediamide (TMPD), valinomycin, and polyethyleneglycols of different molecular weights were obtained from the Sigma-Aldrich Corporation (St Louis, MO, USA). Other chemicals were of analytical grade and were purchased from local suppliers.

Mikkola R., Andersson M., Kharechkina E., Kruglova S, & Kruglov A. (2019). Fusaricidin-Type Compounds Create Pores in Mitochondrial and Plasma Membranes of Mammalian Cells. Biomolecules, 9(9), 433.

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