Description
The generation of β-
lactamases by bacteria affords resistance to several classes of β-
lactam antibiotics, including penicillins and cephalosporins. Nitrocefin is a chromogenic cephalosporin substrate commonly used to detect β-
lactamases in bacteria. The presence of β-
lactamase activity is indicated by the appearance of a red color that is proportional in intensity to the original concentration of nitrocefin.
Chemical Properties
Nitrocefin is the chromogenic cephalosporin that acts as an excellent β-lactamase substrate. It exhibits a rapid distinctive color change from yellow (max at pH 7.0 = 390 nm) to red (max at pH 7.0 = 486 nm) as the amide bond in the beta-lactam ring is hydrolyzed by a β-lactamase. It is sensitive to hydrolysis by all known lactamases produced by Gram-positive and Gram-negative bacteria.
Nitrocefin (Yellow) --β-lactamase-->Product (Red) (OD486nm)
Solution preparation and color change before and after β-lactamase exposure
(A) Concentrated nitrocefin (10.0 mg/mL) in DMSO before dilution with PBS buffer. (B) Nitrocefin diluted with PBS buffer
to working concentration (1.0 mg/mL). The yellow color is indicative of intact, undegraded nitrocefin. (C) 25 units of betalactamase dropped on top of nitrocefin (1.0 mg/mL in PBS). The red color is the result of beta-lactamase mediated
cleavage of the nitrocefin. (D) Vortexed mixture of contents shown in picture (C).
Uses
Nitrocefin is a chromogenic β-lactamase substrate that undergoes colour change from yellow to red as the amide bond in the β-Lactam ring is hydrolyzed by β-lactamase. Nitrocefin undergoes colour chang
es induced by lactamases produced by Gram-positive and Gram-negative bacteria. Several studies have utilized the colour changing properties of Nitrocefin for the detection of β-lactamase activity from
bacterial cell extracts by isoelectric focusing and spectroscopy. Nitrocefin has also been used in studies involving β-lactamase resistant antibiotics.
Preparation
Nitrocefin is a key reagent for high and low throughput assays of the activities of penicillin-binding proteins (PBPs) and β-lactamases, the former used for discovery of antibiotics and the latter for inhibitors of resistance determinants for β-lactam antibiotics. This compound is commercially available but is prohibitively expensive because of the circuitous routes to its synthesis. We describe herein a three-step synthesis of nitrocefin that gives an overall yield of 44%. This is a practical route to the synthesis of this key reagent for drug discovery.
A Practical Synthesis of Nitrocefin
Biological Functions
In determination of b-lactamase activity in biological samples.
Nitrocefin is a colorless or faint yellow cephalosporin antimicrobial that is hydrolyzed rapidly by most beta-lactamases.The hydrolysis product is pink.The bacterium to be tested is applied to a paper disk containing nitrocefin. A pink color developing within minutes (positive test) indicates a beta-lactamaseproducing bacterium.
References
[1] C H O’CALLAGHAN. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate.[J]. Antimicrobial Agents and Chemotherapy, 1972, 1 4: 283-288. DOI:
10.1128/aac.1.4.283[2] T R PARR L E B C H Pai. Simple screening method for beta-lactamase-positive and -negative ampicillin-resistant Haemophilus influenzae isolates.[J]. Journal of Clinical Microbiology, 1984, 20 1: 131-132. DOI:
10.1128/jcm.20.1.131-132.1984[3] P E COUDRON C C S E S Moland. Occurrence and detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae at a veterans medical center: seek and you may find.[J]. Journal of Clinical Microbiology, 1997, 35 10: 2593-2597. DOI:
10.1128/jcm.35.10.2593-2597.1997
