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Nitrocefin

Nitrocefin is a chromogenic cephalosporin compound used to detect the presence of beta-lactamase enzymes in bacterial cultures.
When exposed to beta-lactamase, nitrocefin undergoes a color change from yellow to red, allowing for rapid identification of beta-lactam resistant strains.
This simple, rapid assay is commonly employed in clinical microbiology laboratories to screen for antibiotic resistance and guide appropriate treatment options.
Nitrocefin's sensitivity and ease of use make it an invaluable tool for researchers and clinicians monitoring the spread of antimicroial resistance worlwide.

Most cited protocols related to «Nitrocefin»

1
Antibiotic Regimens for Deep Neck Infections
Cited 8 times
This study retrospectively reviewed the medical records of 981 patients diagnosed with deep neck infection who were admitted to Chang Gung Memorial Hospital in Keelung, Taiwan between April 2001 and October 2006. Diagnostic imaging procedures included computed tomography (CT), ultrasonography (US), and plain radiography. Patients with superficial skin infection or abscess, deep neck infection without evidence of abscess formation, or limited intraoral space infection were excluded. The study enrolled 100 patients. All these patients received abscess drainage via US-guided needle aspiration, or via a surgical incision, or both, with sterilization of intact skin.
Aerobic and anaerobic bacterial cultures were performed for all cases under aseptic field and technique. Patients were treated with empiric antibiotic therapy covering aerobic and anaerobic bacteria. Intravenous broad-spectrum antibiotics were administered before the results of abscess culture became available. Pathogen-directed antibiotic therapy was done when the final results of bacterial cultures were obtained. Abscess specimens were taken and sent by sterile transport swab (Copan, Italy). Then every specimen was inoculated onto 5% sheep’s blood agar, chocolate agar, and eosin-methylene blue (EMB) agar plates for the culture of aerobic and facultative organisms. The plates were incubated at 37 °C aerobically, under 5% carbon dioxide, and were examined at 48 and 72 hours. For anaerobes, the material was inoculated onto anaerobic blood agar (CDC) (BBL, USA), whose nutrition base was tryptic soy agar supplemented with yeast extract, vitamin K3, hemin, and 5% sheep blood. The anaerobic plates were incubated in anaerobic chambers (Concept Plus, Anaerobic Workstation; Ruskinn technology, UK) and examined at 72 and 120 hours. β-lactamase activity was measured for all isolates with a cefinase disc (chromogenic cephalosporin nitrocefin).
The antimicrobial susceptibility testing was determined by disk diffusion with Mueller-Hinton (MH) agar plates. Additionally, M100-S18 was used for aerobic bacteria. The disks used for gram-positive aerobic bacteria included penicillin, oxacillin, erythromycin, trimethoprim/sulfamethoxazole, clindamycin, chloramphenicol, teicoplanin, and vancomycin. The disks for gram-negative bacteria comprised ampicillin, piperacillin, cefazolin, cefuroxime, ceftriaxone, ceftazidime, imipenem, aztreonam, gentamicin, amikacin trimethoprim/sulfamethoxazole, and ciprofloxacin. In some situations, cefepime, flomoxef, and meropenem were added to the tests for gram-negative aerobes. As for anaerobic pathogens, M11-A7 was the method used for antimicrobial susceptibility testing of anaerobic bacteria. The antibiotic test consisted of penicillin, piperacillin, clindamycin, chloramphenicol, and metronidazole.
Various empiric antibiotics for deep neck infection have been proposed previously (Table 1) (Brondbo et al 1983 (link); Gates 1983 (link); Nagy et al 1997 (link); Sakaguchi et al 1997 (link); Chen et al 1998 (link), 2000 (link); Simo et al 1998 (link); Parhiscar and Har-El 2001 (link); Plaza Mayor et al 2001 (link); Sichel et al 2002 (link); McClay et al 2003 (link)). Five different empiric antibiotics were determined: regimen 1 (penicillin G and clindamycin and gentamicin), regimen 2 (ceftriaxone and clindamycin), regimen 3 (ceftriaxone and metronidazole), regimen 4 (cefuroxime and clindamycin), and regimen 5 (penicillin and metronidazole), and were analyzed with descriptive statistics (Chi square, degree of freedom, probability value). The cultured bacteria that were susceptible to the antibiotic regimen were defined as sensitive, while those resistant to antibiotics were defined as resistant. Regimen coverage rate is defined as the number of cases with sensitive results divided by the total number of cases.
Descriptive statistics, for example frequency and percentage, were used. Since abscess specimens were all tested with these five regimens, the McNemar test was made to compare the coverage among different antimicrobial regimens. The level of significance was 0.005 due to Bonferrori adjustment (alpha divided by number of pairwise comparison) for multiple comparisons in order to maintain type I error of 0.05 (Bland and Altman 1995 (link)).
Yang S.W., Lee M.H., See L.C., Huang S.H., Chen T.M, & Chen T.A. (2008). Deep neck abscess: an analysis of microbial etiology and the effectiveness of antibiotics. Infection and drug resistance, 1, 1-8.
Publication 2008
2
Sulfazecin Production and Purification Protocol
Cited 6 times

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Publication 2016
Acetone Amides Bacteria Biological Assay Carbon Charcoal Chromatography Dowex Fermentation High-Performance Liquid Chromatographies nitrocefin Plasmids Resins, Plant Sodium Chloride Strains sulfazecin
3
Amplification and Expression of MBL Variants
Cited 5 times
In this study, we amplified and expressed MBL variants (β‐lactamase N1, BAA1717‐BLA‐2 and NDM‐1) and TEM‐1 (class A β‐lactamase) from S aureus, E coli strains and pET‐21a plasmid for use in further experiments. The protein‐coding genes were cloned into pET‐21a using the primers shown in Table 2. The combined plasmids were tested by next‐generation sequencing and then transformed into BL21 (DE3) competent cells. The BL21 (DE3) cells with the recombinant gene were cultured to OD600 nm = 0.6 at 37°C and then were cultured with 0.2 mmol/L IPTG with shaking at 16°C for 8 hours. After centrifugation, the bacteria were resuspended in sterile phosphate buffer (pH = 7.2) and broken by ultrasound in an ice bath. Then, the mixtures were centrifuged at 4°C, and the supernatants were collected for the subsequent protein purification as described by Liu.15 The Gln242Ala and Ser369Ala mutants of β‐lactamase N1 were expressed and purified as described above, and the primers used for mutation are shown in Table 2.
A nitrocefin assay was used for the screening of potential effective inhibitors and further the determination of the inhibitory effect of TFDG on the hydrolysis activities of MBLs. Nitrocefin serves as an indicator whose colour changes from yellow to red with increased hydrolysis. β‐Lactamase N1 (500 ng/mL), BAA1717‐BLA‐2 (500 ng/mL) and NDM‐1 (250 ng/mL) were incubated with various concentrations of TFDG (0, 4, 8, 16 and 32 μg/mL) in phosphate buffer (pH = 7.2) at 37°C for 5 minutes, and then, 50 μg/mL of nitrocefin was added to the mixture. After 10 minutes of incubation, the samples were read at OD492 nm to determine the level of nitrocefin hydrolysis. Additionally, the inhibitory effect of TFDG against β‐lactamase N1 in the presence of excess zinc ion was further evaluated as described above.
Teng Z., Guo Y., Liu X., Zhang J., Niu X., Yu Q., Deng X, & Wang J. (2019). Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity. Journal of Cellular and Molecular Medicine, 23(10), 6955-6964.
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Publication 2019
Bacteria Bath beta-Lactamase Biological Assay Buffers Cells Centrifugation Escherichia coli Gene Products, Protein Genes Hydrolysis inhibitors Isopropyl Thiogalactoside MBL2 protein, human Mutation nitrocefin Oligonucleotide Primers Phosphates Plasmids Proteins Psychological Inhibition Staphylococcus aureus Sterility, Reproductive Strains Ultrasonics Zinc
4
Antibiotic Resistance Evaluation Protocol
Cited 5 times
Antibacterials used were aztreonam (ATM; Sigma-Aldrich, Tokyo, Japan), ciprofloxacin (CIP; Tokyo Chemical Industry co., Ltd., Tokyo, Japan), erythromycin (ERY; Nacalai Tesque, Inc., Kyoto, Japan), and polymyxin B (PMB; MERCK KGaA, Darmstadt, Germany). D13-9001 (Daiichi Sankyo Co., Tokyo, Japan; Figure 1) and PAβN (Sigma-Aldrich; Figure 1) were used as efflux pump inhibitors. Polymyxin B nonapeptide (PMBN; Sigma-Aldrich) was used as an outer membrane permeabilizer. Ampicillin (AMP; Sigma-Aldrich) was added to cultures to ensure retention of the plasmid. Lactose (Sigma-Aldrich) or isopropyl-β-d-galactopyranoside (IPTG; Sigma-Aldrich) was added to the medium for β-galactosidase and plasmid-mediated pump induction. FDG (Marker Gene Technologies, Inc., Eugene, USA; Figure 1) and SYTOX Green (Lonza Walkersville, Inc., USA) were used for fluorometry. Nitrocefin (CalBiochem, San Diego, USA) was used as a substrate of β-lactamase in nitrocefin permeabilizing assay.
Matsumoto Y., Hayama K., Sakakihara S., Nishino K., Noji H., Iino R, & Yamaguchi A. (2011). Evaluation of Multidrug Efflux Pump Inhibitors by a New Method Using Microfluidic Channels. PLoS ONE, 6(4), e18547.
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Publication 2011
Ampicillin Anti-Bacterial Agents Aztreonam beta-Lactamase Biological Assay Ciprofloxacin D13-9001 Erythromycin Fluorometry Galactose Genes GLB1 protein, human inhibitors Isopropyl Thiogalactoside Lactose nitrocefin Plasmids Polymyxin B polymyxin B nonapeptide Retention (Psychology) SYTOX Green Tissue, Membrane
5
Assaying β-Lactamase and dUTPase Activities
Cited 5 times
β-Lactamase assays, using nitrocefin as substrate, were performed with cells in exponential growth phase as described13 (link), using a Thermomax (Molecular Devices) microtiter plate reader. β-Lactamase activities were recorded as initial slopes divided by cell density (maximum velocity (Vmax))/OD650nm.
dUTPase activity was assayed using His6–dUTPase proteins purified after expression in E. coli. Enzyme assays were performed using the EnzCheck Pyrophosphate Assay Kit (Molecular Probes), as previously reported14 (link).
Tormo-Más M.Á., Mir I., Shrestha A., Tallent S.M., Campoy S., Lasa Í., Barbé J., Novick R.P., Christie G.E, & Penadés J.R. (2010). Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands. Nature, 465(7299), 779-782.
Publication 2010
beta-Lactamase Biological Assay Cells Diphosphates dUTP pyrophosphatase Enzyme Assays Escherichia coli Medical Devices Molecular Probes nitrocefin Proteins

Most recents protocols related to «Nitrocefin»

1
Nitrocefin Permeability Assay for E. coli
Overnight cultures of luciferase-expressing E. coli were grown, and luciferase protein expression
was induced as above to mimic conditions of the cells used in the
bioluminescence assay. Cells were then washed twice and resuspended
in 1× PBS and then incubated with the different concentrations
of PMBN (same as those concentrations used in the bioluminescence
assays) with 50 μg/mL nitrocefin for 30 min. As a negative control,
cells were treated with PBS, and as a positive control, cells were
treated with 10 mM EDTA for 30 min. At the end of the incubation period,
the absorbance of the cells was read at 486 nm using the BioTek Synergy
H1Microplate Reader. Untreated cells with no nitrocefin were treated
as the blank. Wherever presented, the absorbances were blank-subtracted.
Dash R., Holsinger K.A., Chordia M.D., Gh. M.S, & Pires M.M. (2024). Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli. ACS Infectious Diseases, 10(5), 1602-1611.
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Publication 2024
2
Rapid Screening of Bacterial Antibiotic Resistance
Not available on PMC !
Overnight cultures of the isolates were normalised to an optical density at 600 nm (OD600) of 0.1 in 10 ml of LB broth and centrifuged at 4,500 xg for 15 minutes. The supernatant was discarded, and the pellet resuspended in 5ml of phosphate-buffered saline (PBS). The resuspension was then sonicated on ice, for three intervals of 15 seconds, with a 30 second break between each sonication, using a Soniprep 150 plus (MSE centrifuges, UK). 90 μl of the supernatant was added to 10 μl of a 0.5 mg/ml nitrocefin solution in a 96 well clear, flat bottom plate. The absorbance of the plate was read at OD450 every 39 seconds for 9 minutes and 45 seconds, using a FLUOstar OMEGA spectrophotometer (BMG lab systems, Germany). Triplicate overnight liquid cultures were grown from the selection of three distinct colonies, each liquid culture was then assayed in triplicate.
Fraser A.J., Ball R.T., Cantillon D., Brettell L.E., F G., Lewis J.M., Aartsen J.v., Parry C.M., Heinz E., & Edwards T. (2024). A high-resolution genomic and phenotypic analysis of resistance evolution of anEscherichia colistrain from a critical care patient treated with piperacillin/tazobactam.
Publication 2024
3
Kinetic Analysis of β-Lactamase Activity
The β-lactamase plasmid (pExp-Bla, Addgene plasmid #112561) was expressed in E. coli and isolated using nickel agarose beads (Goldbio, Cat# H-350-5). 5 µM of HaBlap was mixed with 50 µM nitrocefin in 5 mM phosphate buffer pH 7. Perchlorate salt concentration varied by experiment. Nitrocefin hydrolysis was monitored on a SpectraMax 340PC384 plate reader (using SoftMax Pro software V 5.4; Molecular Devices) measuring absorbance at 486 nm and blanked to identical solutions lacking nitrocefin. Kinetic assays were performed the same way but with 5 nM HaBlap.
High-salt concentrations affected the extinction coefficient of hydrolyzed nitrocefin, which distorted the absorbance readings. We corrected the absorbance readings by incubating nitrocefin with 5 nM HaBlap in buffered solution for 30 min (to fully hydrolyze the nitrocefin) and then diluting that reaction into an array of sodium chloride/perchlorate solutions (nitrocefin final concentration: 50 µM). The extinction coefficient was calculated for each salt solution based on the Beer-Lambert law and graphed (Supplementary Fig. 11).
Hoog T.G., Pawlak M.R., Gaut N.J., Baxter G.C., Bethel T.A., Adamala K.P, & Engelhart A.E. (2024). Emergent ribozyme behaviors in oxychlorine brines indicate a unique niche for molecular evolution on Mars. Nature Communications, 15, 3863.
Full text: Click here
Publication 2024
4
Spectrophotometric Assay for β-Lactamase Inhibition
β-lactamase activity was assessed spectrophotometrically by measuring the hydrolysis of nitrocefin as previously described with little modification [47 (link)]. Briefly, the reaction mixture consisted of 83 μg of nitrocefin (EMD Millipore Corp., Burlington, MA, USA), 10% glycerol (Bio Basic Inc., Ontario, Canada), 167 mg of bovine serum albumin (Sigma-Aldrich Corp, Burlington, MA, USA), and 0.02 mg/mL of β-lactamase obtained from Bacillus cereus 569H (EMD Millipore Corp., Burlington, MA, USA). The activity of β-lactamase was determined by kinetically measuring the absorbance at 490 nm for 60 sec using UV-Vis spectrophotometer (Shimadzu Corp., Kyoto, Japan). To evaluate the inhibitory effects, inhibitors including tBLIP-II, tBLIP-II-CPP, and CPP-tBLIP-II were pre-incubated with the assay mixture without nitrocefin for 15 min at 30°C before adding nitrocefin as the substrate. The concentrations of the inhibitors ranged from 0.0078 μM to 2 μM. The IC50 values were determined through direct competition between nitrocefin and protein inhibitors under the aforementioned conditions. The IC50 values were obtained by plotting the percent residual enzyme activity on nitrocefin against the inhibitor concentration. The IC50 was defined as the concentration of the inhibitor that inhibited the hydrolytic activity of the enzyme by 50%.
Chatupheeraphat C., Peamchai J., Kaewsai N., Anuwongcharoen N, & Eiamphungporn W. (2024). Enhancing the activity of β-lactamase inhibitory protein-II with cell-penetrating peptide against KPC-2-carrying Klebsiella pneumoniae. PLOS ONE, 19(1), e0296727.
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Publication 2024
5
Nitrocefin-Based β-Lactamase Activity Assay
β-lactamase activity was assessed by following the hydrolysis of the chromogenic β-lactamase substrate nitrocefin. The assay mixture contained 500 μM nitrocefin (Sigma-Aldrich) and 50 μg protein extract in a final volume of 50 μL of 10 mM phosphate buffer (pH 6.5). Hydrolysis of nitrocefin was monitored spectrophotometrically at 486 nm for 30 min at room temperature (Δε = 15,200 M−1 cm−1). Protein extracts of C. jeikeium CIP82.51 and Mycobacterium chelonae #2445 (generous gift from B. Heym, Institut Pasteur, France) were used as negative and positive controls, respectively.
Lavollay M., Buon C., Le Moigne V., Compain F., Guyonvarch A, & Fonvielle M. (2024). Exploration of the role of the penicillin binding protein 2c (Pbp2c) in inducible β-lactam resistance in Corynebacteriaceae. Frontiers in Microbiology, 15, 1327723.
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Publication 2024

Top products related to «Nitrocefin»

1
Nitrocefin by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe
Nitrocefin is a chromogenic cephalosporin substrate used to detect the presence of beta-lactamase enzymes in bacterial cells. It undergoes a color change from yellow to red upon hydrolysis by beta-lactamase, providing a simple and rapid method for identifying beta-lactamase-producing organisms.
2
Nitrocefin by Thermo Fisher Scientific
Sourced in United Kingdom
Nitrocefin is a chromogenic cephalosporin compound used in various laboratory procedures. It undergoes a color change when exposed to bacterial beta-lactamase enzymes, allowing for the detection and identification of these enzymes.
3
Etest by bioMérieux
Sourced in France, Sweden, United States, Germany, United Kingdom, Denmark, Italy, Australia, Spain, Switzerland, Japan
Etest is a quantitative antimicrobial susceptibility testing (AST) method developed by bioMérieux. It provides minimum inhibitory concentration (MIC) values for specific antimicrobial agents. Etest utilizes a predefined antimicrobial gradient on a plastic strip to determine the MIC of a tested microorganism.
4
Nitrocefin by Abcam
Sourced in United Kingdom, United States
Nitrocefin is a chromogenic cephalosporin compound used as a substrate for the detection of beta-lactamase activity. When hydrolyzed by beta-lactamases, Nitrocefin undergoes a color change from yellow to red, allowing for the rapid identification of beta-lactamase-producing organisms.
5
Cefotaxime by Merck Group
Sourced in United States, Germany, United Kingdom, Switzerland, Sao Tome and Principe, France, Belgium
Cefotaxime is a third-generation cephalosporin antibiotic used in laboratory settings. It is a broad-spectrum antibiotic effective against a variety of Gram-positive and Gram-negative bacteria.
6
Ampicillin by Merck Group
Sourced in United States, Germany, United Kingdom, France, China, Switzerland, Sao Tome and Principe, Spain, Ireland, India, Italy, Japan, Brazil, Australia, Canada, Macao, Czechia, New Zealand, Belgium, Cameroon, Austria, Israel, Norway, Denmark, Netherlands
Ampicillin is a broad-spectrum antibiotic used in laboratory settings. It is a penicillin-based compound effective against a variety of gram-positive and gram-negative bacteria. Ampicillin functions by inhibiting cell wall synthesis, leading to bacterial cell lysis and death.
7
Imipenem by Merck Group
Sourced in United States, Germany, France, China, Sao Tome and Principe, United Kingdom, Italy, India, Spain, Brazil, Canada
Imipenem is a broad-spectrum antibiotic medication used to treat various bacterial infections. It is a member of the carbapenem class of antibiotics and functions by inhibiting bacterial cell wall synthesis, leading to cell death.
8
Nitrocefin test by Thermo Fisher Scientific
Sourced in United Kingdom
Nitrocefin test is a biochemical assay used to detect the presence of beta-lactamase enzymes. It is a colorimetric test that changes color from yellow to red in the presence of beta-lactamase activity. The test is commonly used in microbiology laboratories to quickly identify bacteria that produce beta-lactamase, an enzyme that can confer resistance to certain antibiotics.
9
Bovine serum albumin (bsa) by Merck Group
Sourced in United States, Germany, United Kingdom, China, Italy, Japan, France, Sao Tome and Principe, Canada, Macao, Spain, Switzerland, Australia, India, Israel, Belgium, Poland, Sweden, Denmark, Ireland, Hungary, Netherlands, Czechia, Brazil, Austria, Singapore, Portugal, Panama, Chile, Senegal, Morocco, Slovenia, New Zealand, Finland, Thailand, Uruguay, Argentina, Saudi Arabia, Romania, Greece, Mexico
Bovine serum albumin (BSA) is a common laboratory reagent derived from bovine blood plasma. It is a protein that serves as a stabilizer and blocking agent in various biochemical and immunological applications. BSA is widely used to maintain the activity and solubility of enzymes, proteins, and other biomolecules in experimental settings.
10
Elx808 microplate reader by Agilent Technologies
Sourced in United States, Germany, United Kingdom
The ELx808 microplate reader is a compact and versatile instrument designed for absorbance-based assays. It can accurately measure optical densities of samples in standard 96-well microplates. The ELx808 is equipped with a tungsten-halogen lamp and a high-quality monochromator, allowing for wavelength selection from 400 to 750 nm.

More about "Nitrocefin"

Nitrocefin is a chromogenic cephalosporin compound that is widely used in clinical microbiology laboratories to detect the presence of beta-lactamase enzymes in bacterial cultures.
When exposed to beta-lactamase, nitrocefin undergoes a color change from yellow to red, allowing for rapid identification of beta-lactam resistant strains.
This simple, rapid assay is an invaluable tool for researchers and clinicians monitoring the spread of antimicrobial resistance worldwide.
Nitrocefin is commonly employed alongside other antibiotic resistance testing methods, such as the Etest and disc diffusion assays.
The Etest, for example, uses a predefined gradient of antibiotic concentrations on a plastic strip to determine the minimum inhibitory concentration (MIC) of a particular antibiotic, such as cefotaxime, ampicillin, or imipenem, against a bacterial isolate.
Similarly, the nitrocefin test can be used to screen for the presence of beta-lactamases, which confer resistance to beta-lactam antibiotics like those mentioned.
The nitrocefin test is known for its sensitivity and ease of use, making it a popular choice for both research and clinical applications.
Researchers may utilize nitrocefin to monitor the spread of antibiotic-resistant strains, while clinicians can use it to guide appropriate treatment options for patients with suspected bacterial infections.
The test is often performed alongside the use of an ELx808 microplate reader, which can quantify the colorimetric change in the nitrocefin solution, further enhancing the precision and objectivity of the results.
To prepare the nitrocefin solution, the compound is typically dissolved in a buffer containing bovine serum albumin (BSA), which helps to stabilize the solution and prevent non-specific color changes.
This simple, yet effective, assay continues to be an invaluable tool in the fight against the growing threat of antimicrobial resistance.