The bacterial strain used for SELEX selection was P. aeruginosa 692 (PA692) (ATCC 14502) obtained from New Zealand Culture Collection (Porirua, NZ). Other bacterial strains used were: P.aeruginosa PAO1 (ATCC 15692), Salmonella enterica serovar Typhi (ATCC 19430), Klebsiella pneumoniae (ATCC 13883), Enterobacter cloacae (ATCC 13047), Listeria innocua (ATCC 33090) and Escherichia coli DH5α. Additional P. aeruginosa strains used in binding studies were clinical isolates PA1024 (catheter, urine), PA1205 (left thigh), PA1323 (cystic fibrosis sputum), PA1079 (tracheal aspirate), and PA1236 (blood) obtained from Environmental Science and Research Culture Collection (Porirua, NZ). All bacterial strains were cultured in standard Luria Broth (LB) medium at 37°C for 16 hours with aeration. After growth, the cells were diluted 1:100 in fresh LB and grown for 4 hours to allow them to reach exponential phase.
P aeruginosa pao1
Manufactured by American Type Culture Collection
Sourced in United States
P. aeruginosa PAO1 is a reference strain of the bacterium Pseudomonas aeruginosa. It is a well-characterized, wild-type strain that serves as a model organism for research on this opportunistic pathogen.
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Lab products found in correlation
S aureus, by American Type Culture Collection (4 mentions)
K pneumoniae, by American Type Culture Collection (2 mentions)
S epidermidis, by American Type Culture Collection (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Enterobacter cloacae, by American Type Culture Collection (1 mentions)
Klebsiella pneumoniae, by American Type Culture Collection (1 mentions)
Listeria innocua, by American Type Culture Collection (1 mentions)
Ethanol, by Carl Roth (1 mentions)
Tris hydroxymethyl aminomethane, by Merck Group (1 mentions)
Hydrofluoric acid, by Avantor (1 mentions)
Hexane, by Merck Group (1 mentions)
Isopropanol, by Merck Group (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Ethylene glycol, by Merck Group (1 mentions)
Chloroform, by Carl Roth (1 mentions)
Proteinase k from tritirachium album, by Merck Group (1 mentions)
Methanol, by Carl Roth (1 mentions)
E coli bl21 de3, by Thermo Fisher Scientific (1 mentions)
P aeruginosa xen41, by PerkinElmer (1 mentions)
16 protocols using p aeruginosa pao1
1
Bacterial Strains for SELEX Selection
2
Comprehensive Silicon Wafer Characterization and Bacterial Culturing
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p-type silicon wafers with an <100> orientation (resistivity 0.01–0.02 mΩ cm) were purchased from Siegert Wafer, Aachen, Germany. Hydrofluoric acid (48% aqueous) was obtained from VWR Chemicals, Darmstadt, Germany. PLA (Mw = 18,000–24,000 g mol−1), proteinase K from tritirachium album (≥30 units mg−1), acetone (99.8%), ethyl acetate (99.7%), hexane (99%), isopropanol (99.5%), ethylene glycol (99%) and tris(hydroxymethyl)aminomethane were purchased from Sigma Aldrich, Steinheim, Germany. Sodium hydroxide (98.8%) was purchased from ChemSolute, Renningen, Germany. P. aeruginosa PAO1 (ATCC 15692, isolated from infected wounds) [42 (link),43 (link)]). Luria–Bertani medium (LB broth), LB agar, methanol (99.9%), chloroform (99%) and ethanol (96%) were purchased from Carl Roth, Karlsruhe, Germany. Milli-Q water was obtained from Millipore Direct Q 8 system (MILLIPORE, Schwalbach, Germany) with a resistivity of 18 MΩ cm.
3
Bacterial culture and macrophage maintenance
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P. aeruginosa PAO1 (ATCC, Manassas, VA) and E. coli BL21 (DE3) (Thermo Fisher Scientific, Waltham, MA) were cultured in Luria-Bertani broth at 37 °C. MH-S (ATCC), a murine alveolar macrophage cell line, was maintained in RPMI 1640 medium (HyClone, Logan, UT) containing 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were cultured at 37 °C in an atmosphere of 5% CO2.
4
Cultivation of Bacterial Strains for Bioluminescent Imaging
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Bacteria were cultured on Todd Hewitt (TH) broth (BBLTM, BD, Sparks, MD, USA) solidified by 15 g/L of BactoAgar (Saveen Werner AB, Limnhamn, Sweden) at 37 °C overnight, and then kept in 4 °C until cultivation. Single bacterial colonies were taken from the cultures and inoculated in 5 mL of TH broth at 37 °C on a shaking incubator overnight. The following day, the bacterial culture was refreshed in 5 mL TH-broth and grown to logarithmic phase at OD600 = 0.4 at 37 °C on a shaking incubator. After washing of the bacterial pellet in 10 mM Tris (pH 7.4), a bacterial solution with 1 × 109 colony forming units (CFU)/mL was prepared using 10 mM Tris pH 7.4. The bacterial strains used in this study include S. aureus (ATCC 29213; American type culture collection, Manassas, VA, USA) and P. aeruginosa PAO1 (ATCC 27853, Manassas, VA, USA). The bioluminescent strains P. aeruginosa Xen41 (PerkinElmer, Waltham, MA, USA) and S. aureus SAP229 (provided by Dr. Roger Plaut, Division of Bacterial, Parasitic, and Allergenic Products, FDA, Bethesda, MD, USA) were also used in experiments studying bioluminescent imaging.
5
Antimicrobial Assay Protocol for Diverse Pathogens
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Methanol and acetonitrile of LCMS grade and all other chemicals and solvents of analytical grade used in this study were purchased from Sigma–Aldrich, unless otherwise indicated. Bacterial strains S. aureus (ATCC 6538), P. aeruginosa (ATCC 9027), B. subtilis (ATCC 6633), C. sporogenes (ATCC 19404), C. albicans (ATCC 10231), A. brasiliensis (ATCC 16404), S. epidermidis (ATCC 12228), E. coli K12 (ATCC 25404), K. pneumoniae (ATCC KP1), P. aeruginosa PAO1 (ATCC BAA-47), S. aureus (ATCC 25923), and A. baumannii (ATCC BAA-2801) were obtained from the American Type Culture Collection. A. brasiliensis ATCC 16404 was obtained from EZ Accu ShotTM Select (Microbiologics, Saint Cloud, MN, USA).
6
Anti-Biofilm Assay with Drug-Resistant Bacteria
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The anti-biofilm assay was performed using P. aeruginosa PAO1 (ATCC 15692) and S. aureus (ATCC 25923) obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Drug-resistant P. aeruginosa (CCARM 2073 and CCARM 2075) and S. aureus (CCARM 3125 and 3709) were purchased from the Culture Collection of Antibiotic Resistant Microbes (CCARM, Seoul, Korea). Drug-resistant P. aeruginosa ((DRPa)-4007 and DRPa-3241) and drug-resistant S. aureus ((DRSa)-3399 and DRSa-3518) were clinically isolated from patients with otitis media. All strains were grown in Mueller–Hinton (MH) broth under aerobic conditions at 37 °C.
7
Bacterial Strains and Growth Conditions
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E. coli DH10B was purchased from Invitrogen. A. baylyi (ATCC 33305) and P. aeruginosa PAO1 (ATCC BAA-47) were purchased from ATCC. Unless stated otherwise, all the strains were grown at 37 °C in Luria Broth (LB) liquid media with agitation at 200 rpm or on LB plates with 1.5% agar. For sacB counter selection, we used LBNS plates (LB n o s alt: 1% Tryptone, 0.5% yeast extract and 1.5% agar) supplemented with 10% sucrose (Fisher Scientific). Appropriate antibiotics and concentrations were used to select for bacterial cells that are antibiotic resistant. E. coli DH10B: chloramphenicol (Cam; Gold Biotechnology), 25 μg/ml; kanamycin (Kan; Fisher Scientific), 50 μg/ml. A. baylyi: Cam, 10 μg/ml; Kan, 10 μg/ml. P. aeruginosa PAO1: Cam, 250 μg/ml; Kan, 500 μg/ml.
8
Synthesis and Characterization of PEG-based Biopolymer
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Poly(ethylene glycol) methacrylate (PEG, Mn 360 g/mol), (3-mercaptopropyl) trimethoxysilane (MPS), TBNO, sodium dodecyl sulfate (SDS), IPA, ethanol, acetone and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich and used without further purification. Ferrous ammonium sulfate hexahydrate and 4% paraformaldehyde were purchased from Macklin Inc. and Santa Cruz Biotechnology, Inc. respectively. Deionized (DI) water was from a Milli-Q water system. Antibiotic/Antimycotic solution (10,000 U/mL Penic illin, 10,000 μg/mL Streptomycin, 25 μg/mL Amphotericin B) and PBS was from GE Healthcare Life Science. 00.25% Trypsin-EDTA, fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were from Thermo Fisher Scientific. Bacterial growth media and agar were all purchased from BD. Strains used, MRSA BAA38, MRSE 35984, VRE 583, A. baumannii AB-1, E. coli UTI89 and P. aeruginosa PAO1, were obtained from ATCC.
9
Antimicrobial Efficacy of Peptides
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The antimicrobial efficacy of peptides was tested using a serial dilution titration, as previously described 13 on a panel of clinically relevant bacterial strains; i.e. E. coli (ATCC 25922), P. aeruginosa PAO1 (ATCC 15692), S. aureus (ATCC 29213). Conventional antibiotics used throughout the study include Ampicillin (A9518), Ciprofloxacin (17850), Tetracycline (T7660), Tobramycin (T4014) and Polymyxin B (92301), all purchased from Sigma. In short, overnight cultures were grown to mid-log phase before diluting to ~5×10 5 CFU/mL in Mueller Hinton broth (Becton Dickinson) and 90 µL was used to inoculate each well of a 96-well polypropylene plate (Cat. No. 3879, COSTAR). Then, 10 µL aliquots of a 2-fold dilution series of the peptides were added and the minimal inhibitory concentration (MIC) was scored as the lowest concentration that inhibited visible growth after 48 hours of incubation at 37°C.
10
Antimicrobial Peptide-Loaded Polymer Nanoparticles
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