To test the phenotypic effect of amino acid substitution, the minimal inhibitory concentrations (MICs) of ampicillin, cephalothin, ceftriaxone, ceftazidime, cefotaxime, cefepime, imipenem, and aztreonam were determined for each clone in the variant library by the agar dilution method using cation-adjusted Mueller-Hinton agar (MHA), following the recommendations of the Clinical and Laboratory Standards Institute.25 ampicillin, cefotaxime, and cephalothin were purchased from Sigma (St. Louis, MO), and imipenem was purchased from U.S. Pharmacopeia (Rockville, MD). The effect of Zn2+ availability was also tested by agar dilution using MH supplemented with 250 μM ZnSO4 or 5 μM EDTA.26 (link) Reported values are the modes of at least three biological replicates.
Cephalothin
Manufactured by Merck Group
Sourced in United States, Sao Tome and Principe
Cephalothin is a cephalosporin antibiotic. It is a type of lab equipment used in research and clinical settings.
Automatically generated - may contain errors
Lab products found in correlation
Ampicillin, by Merck Group (9 mentions)
Ceftriaxone, by Merck Group (6 mentions)
Imipenem, by Merck Group (5 mentions)
Cefoxitin, by Merck Group (4 mentions)
Ceftazidime, by Merck Group (4 mentions)
Ciprofloxacin, by Merck Group (4 mentions)
Cefotaxime, by Merck Group (4 mentions)
Potassium clavulanate, by Merck Group (3 mentions)
Tazobactam, by Chem-Impex (3 mentions)
Chloramphenicol, by Merck Group (3 mentions)
Carbenicillin, by Merck Group (2 mentions)
Nalidixic acid, by Merck Group (2 mentions)
Trimethoprim sulfamethoxazole, by Merck Group (2 mentions)
Rifampin, by Merck Group (2 mentions)
Metronidazole, by Merck Group (2 mentions)
Oxacillin, by Merck Group (2 mentions)
Piperacillin, by Merck Group (2 mentions)
Aztreonam, by Chem-Impex (2 mentions)
Nitrocefin, by Thermo Fisher Scientific (2 mentions)
Angiotensin converting enzyme, by Merck Group (1 mentions)
16 protocols using cephalothin
1
Phenotypic Impact of Amino Acid Substitutions
2
Vibrio Isolates and Antimicrobial Agents
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V. parahemolyticus 0577 was isolated
from food poisoning patients by Dr. Hin-Chung Wong’s laboratory,
Department of Microbiology of Soochow University, Taiwan. V. parahemolyticus 1109O101 and 1109O202
were isolated from oyster by Dr. Hsin-I Hsiao’s laboratory,
Department of Food science of National Taiwan Ocean University, Taiwan. V. cholerae CVD101 was obtain from Dr.
Adrian Robert Walmsley’s laboratory, Department of Biosciences
of Durham University, United Kingdom. Carbenicillin (CAR), cephalothin
(CEP), imipenem (IMI), potassium clavulanate (CLA), hipury-histidyl-leucine
(HHL), and angiotensin-converting enzyme (ACE) were obtained from
Sigma-Aldrich, USA. Aztreonam (ATZ) was obtained from Bionovas, Canada.
Nitrocefin was obtained from Toronto Research Chemicals, Canada. The
antibiotics were purchased with purity levels exceeding 98%, and other
compounds were 95%. Mueller-Hinton (MH) broth was obtained from Formedium,
U.K.
from food poisoning patients by Dr. Hin-Chung Wong’s laboratory,
Department of Microbiology of Soochow University, Taiwan. V. parahemolyticus 1109O101 and 1109O202
were isolated from oyster by Dr. Hsin-I Hsiao’s laboratory,
Department of Food science of National Taiwan Ocean University, Taiwan. V. cholerae CVD101 was obtain from Dr.
Adrian Robert Walmsley’s laboratory, Department of Biosciences
of Durham University, United Kingdom. Carbenicillin (CAR), cephalothin
(CEP), imipenem (IMI), potassium clavulanate (CLA), hipury-histidyl-leucine
(HHL), and angiotensin-converting enzyme (ACE) were obtained from
Sigma-Aldrich, USA. Aztreonam (ATZ) was obtained from Bionovas, Canada.
Nitrocefin was obtained from Toronto Research Chemicals, Canada. The
antibiotics were purchased with purity levels exceeding 98%, and other
compounds were 95%. Mueller-Hinton (MH) broth was obtained from Formedium,
U.K.
3
Synthesis and Characterization of β-Lactamase Inhibitors
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The antibiotics
used in this study were obtained from their respective manufacturers
as indicated: ampicillin, cephalothin, and clavulanic acid, potassium
salt, from Sigma (St. Louis, MO); sulbactam, sodium salt, from Pfizer
(Groton, CT); tazobactam, sodium salt, from Chem-IMPEX (Wood Dale,
IL); and nitrocefin from Becton Dickinson (Franklin Lakes, NJ). The
sodium salt of PSR-3-283A (263 Da) and 6-D,D-sulbactam were synthesized
in the laboratory of John D. Buynak. Chemical structures of β-lactamase
inhibitors are represented in Figure1 .
used in this study were obtained from their respective manufacturers
as indicated: ampicillin, cephalothin, and clavulanic acid, potassium
salt, from Sigma (St. Louis, MO); sulbactam, sodium salt, from Pfizer
(Groton, CT); tazobactam, sodium salt, from Chem-IMPEX (Wood Dale,
IL); and nitrocefin from Becton Dickinson (Franklin Lakes, NJ). The
sodium salt of PSR-3-283A (263 Da) and 6-D,D-sulbactam were synthesized
in the laboratory of John D. Buynak. Chemical structures of β-lactamase
inhibitors are represented in Figure
4
Antimicrobial Susceptibility Testing Protocol
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Antibiotics used for broth microdilution assays (ampicillin, azithromycin, cefazolin, cefoxitin, ceftazidime, ceftriaxone, cephalothin, ciprofloxacin, clarithromycin, enrofloxacin, gentamicin, imipenem, levofloxacin, nalidixic acid, and tetracycline powders) were purchased from the Sigma Chemical Company (St. Louis, MO). Amoxicillin-clavulanic acid and trimethoprim-sulfamethoxazole were obtained as E-test strips from bioMerieux (Durham, NC.). Disks of ampicillin (10 μg), cephalothin (30 μg), enrofloxacin (5 μg), nalidixic acid (30 μg), tetracycline (30 μg), amoxicillin-clavulanic acid (30 μg), and trimethoprim-sulfamethoxazole (25 μg) were obtained from Remel (Lenexa, KS.).
5
Antibiotic Susceptibility of Lactobacilli
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The minimum inhibitory concentrations (MICs) were determined according to the clinical laboratory Standards Institute (CLSI) guideline (2015) using micro titer plate method. In this method, colonies of lactobacilli from TSA were suspended in Muller Hinton broth (Merck, Germany) and the turbidity of suspension was adjusted to 0.5 McFarland and subsequently diluted in Muller Hinton broth (1:100) to reach a final concentration of 1 × 106 CFU/ml. Dilutions of cephalothin (Sigma-Aldrich), cefazolin, amikacin, ceftriaxone and ceftazidime (Exir, Broujerd, Iran) were made in distilled water. The antibiotics were prepared at different concentrations ranged from 0.125 to 512 μg/ml. Each well was filled with 100 μl of each dilution of the antibiotic and 100 μl of bacterial suspension. Each plate included positive controls (bacteria without an antimicrobial), negative controls (medium only). Micro titer plates were incubated at 37°C for 24 h. MIC was determined as the minimum antibiotic concentration that inhibited the visible growth (19 ). All tests were carried out in duplicates.
6
NMR Spectroscopy and Column Chromatography
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NMR spectra were measured with a JEOL Eclipse 500 FT-NMR spectrometer (1H, 500 MHz; 13C 125 MHz). Column chromatography was carried on silica-gel (Kieselgel 60, 70–230 mesh, Merck, Germany), a thin layer chromatography (TLC) on pre-coated Silica-gel F254 (0.25 mm, Merck) and Sephadex LH-20 (25–100 µM, Sigma, U.S.A). Mueller-Hinton broth (MHB) and Mueller-Hinton agar (MHA) (Difco Laboratories, Baltimore, MD, USA). Ampicillin, amoxicillin/clavulanic acid, chloramphenicol, cephalothin, sulfisoxazole, nalidixic acid, norfloxacin, streptomycin, trimethoprim/sulfamethoxazole, ticarcillin, ciprofloxacin, N,N′-Dicyclohexylcarbodiimide, Sodium azide, Tris, Triton X-100 and solvents were purchased from Sigma Aldrich (St. Louis, USA).
7
Antibiotic Susceptibility of Skin Burn Isolates
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A modified Kirby-Bauer single-disk diffusion technique was used to test antibiotic susceptibility. For bacteria and fungi, respectively, Müller-Hinton and SDA agar plates were prepared. For inoculum preparation, the microbial solution was adjusted to a concentration of 1 × 10 7 CFU/ml. In this experiment, 17 commercially available antibacterial discs (amoxicillin, oxacillin, ampicillin, imipenem, cephalothin, cefuroxime, cefotaxime, trimethoprim, levofloxacin, chloramphenicol, erythromycin, rifampin, tetracycline, doxycycline, vancomycin, gentamicin, and nitrofurantoin) and 7 antifungal agents (fluconazole, ketoconazole, amphotericin B, metronidazole, itraconazole, and nystatin -all from Sigma Chemical Co., USA) were tested against the selected bacterial and fungal isolated from skin burn infection. The antibiotic susceptibility test was conducted on Müller-Hinton agar (MHA), and the diameter of the inhibition zone (IZD) surrounding the discs was assessed to evaluate sensitivity. The Clinical and Laboratory Standards Institute recommendations were used to interpret the data (CLSI, 2014) . Resistance to at least three antimicrobial categories was defined as MDR.
8
Antibiotic Susceptibility Testing by MIC
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Mueller-Hinton (MH) agar-dilution minimum inhibitory concentration (MIC) measurements were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines 2014, Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement80 (link),81 (link). The MICs are reported as the concentrations at which bacterial growth was no longer observed. Avibactam was tested at a constant 4 μg/mL in combination with its respective antibiotic partners. All MIC measurements were performed at least three times.
Ampicillin, piperacillin, ceftriaxone, cephalothin, potassium clavulanate, cefotaxime, and chloramphenicol were purchased from Sigma-Aldrich. Ceftazidime was procured from Sigma and Research Products International and used interchangeably throughout the experimentation. Imipenem was obtained from USP and from the commercial source. Sulbactam was bought from Astatech. Tazobactam and aztreonam were purchased from Chem-Impex Int’l. Ceftolozane-Tazobactam, cefepime, meropenem, ertapenem, and doripenem were obtained from their commercial sources. Avibactam was purchased from Advanced ChemBlocks.
Ampicillin, piperacillin, ceftriaxone, cephalothin, potassium clavulanate, cefotaxime, and chloramphenicol were purchased from Sigma-Aldrich. Ceftazidime was procured from Sigma and Research Products International and used interchangeably throughout the experimentation. Imipenem was obtained from USP and from the commercial source. Sulbactam was bought from Astatech. Tazobactam and aztreonam were purchased from Chem-Impex Int’l. Ceftolozane-Tazobactam, cefepime, meropenem, ertapenem, and doripenem were obtained from their commercial sources. Avibactam was purchased from Advanced ChemBlocks.
9
Microbial Susceptibility Testing Agents
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Ampicillin (catalog no. A9518), piperacillin (catalog no. P8396), ceftriaxone (catalog no. C5793), cephalothin (catalog no. C4520), potassium clavulanate (catalog no. 33454), cefotaxime (catalog no. C7912), and chloramphenicol (catalog no. R4405) were purchased from Sigma-Aldrich. Ceftazidime was procured from Sigma (catalog no. C3809) and Research Products International (catalog no. 33527), and the products from the two sources were used interchangeably throughout the experimentation. Imipenem was obtained from USP (catalog no. 1337809) and from the commercial source (pharmacy). Sulbactam was bought from Astatech. Tazobactam (catalog no. 15141) and aztreonam (catalog no. 15151) were purchased from Chem-Impex International. Ceftolozane-Tazobactam, cefepime, meropenem, ertapenem, and doripenem were obtained from their commercial sources. Ceftaroline was provided by Allergan. Nitrocefin (catalog no. BR0063G) was purchased from Oxoid. Avibactam was purchased from Advanced ChemBlocks (catalog no. R16073).
10
Cephalosporin Hydrolysis by EstSTR1
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Antibiotics (cephalothin, cefoxitin, cefotaxime, and cefepime) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The chemical structures of cephalosporins (cephalothin, cefoxitin, cefotaxime, and cefepime) are shown in the Additional file 1 : Figure S1. The hydrolysing activity of EstSTR1 for cephalosporins was measured by the paper disc method as previously described (Jeon et al. 2011 (link)). The enzyme (330 μM) was incubated with antibiotic substrates (3 mM cephalothin, 1 mM cefoxitin, 1 mM cefotaxime, and 1 mM cefepime) in 50 mM Tris–HCl (pH 8.0) for 2 h at 35 °C, and then reaction mixtures were loaded onto small paper discs. After 8 h incubation at 37 °C, the diameters of the inhibition zones around the small paper discs were recorded. For comparison of β-lactam hydrolysing activity, a negative control containing antibiotics without enzyme and a positive control containing antibiotics and the CMY-10, a plasmid-encoded class C extended-spectrum β-lactamase (ESBL) from Enterobacter aerogenes (Kim et al. 2006 (link)), were used.
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