Nitrocefin is a

chromogenic In chemistry, the term chromogen refers to a colourless (or faintly coloured) chemical compound that can be converted by chemical reaction into a compound which can be described as "coloured". There is no universally agreed definition of the term. ...

cephalosporin substrate routinely used to detect the presence of

beta-lactamase Beta-lactamases, (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems ( ertapenem), although carbap ...

enzymes produced by various microbes.

Beta-lactamase Beta-lactamases, (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems ( ertapenem), although carbap ...

mediated resistance to

beta-lactam A beta-lactam (β-lactam) ring is a four-membered lactam. A ''lactam'' is a cyclic amide, and ''beta''-lactams are named so because the nitrogen atom is attached to the β-carbon atom relative to the carbonyl. The simplest β-lactam possible i ...

antibiotics such as penicillin is a widespread mechanism of resistance for a number of

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typically a few micrometr ...

including members of the family Enterobacteriaceae, a major group of enteric

Gram-negative bacteria Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall ...

. Other methods for

detection exist including PCR; however, nitrocefin allows for rapid

detection using few materials and inexpensive equipment.

Structure

As a cephalosporin, nitrocefin contains a

ring which is susceptible to

mediated

hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution, elimination, and solvation reactions in which water is the nucleophile. Biological hydrolys ...

. Once hydrolyzed, the degraded nitrocefin compound rapidly changes color from yellow to red. Although nitrocefin is considered a cephalosporin, it does not appear to have antimicrobial properties.

Degradation and chromogenic properties

Intact

antibiotics act by binding to

penicillin binding proteins Penicillin-binding proteins (PBPs) are a group of proteins that are characterized by their affinity for and binding of penicillin. They are a normal constituent of many bacteria; the name just reflects the way by which the protein was discov ...

(PBPs) involved in

peptidoglycan Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like peptidoglycan layer outside the plasma membrane, the rigid cell wall (murein sacculus) characteristic of most ba ...

synthesis.

Beta-lactamases Beta-lactamases, (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems ( ertapenem), although carbapenems ...

hydrolyze the

amide In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a compound with the general formula , where R, R', and R″ represent organic groups or hydrogen atoms. The amide group is called a peptide bond when it i ...

bond between the

carbonyl In organic chemistry, a carbonyl group is a functional group composed of a carbon atom double-bonded to an oxygen atom: C=O. It is common to several classes of organic compounds, as part of many larger functional groups. A compound containi ...

carbon Carbon () is a chemical element with the symbol C and atomic number 6. It is nonmetallic and tetravalent—its atom making four electrons available to form covalent chemical bonds. It belongs to group 14 of the periodic table. Carbon mak ...

and the

nitrogen Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...

in the

ring of susceptible beta-lactams and members of

subclasses (including certain cephalosporins). After hydrolysis of the amide bond, the antibiotic lacks the ability to bind bacterial PBPs and is rendered useless. Visual detection of this process is essentially impossible with most cephalosporins because the shift of

ultraviolet Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30 PHz) to 400 nm (750 THz), shorter than that of visible light, but longer than X-rays. UV radiation ...

absorption from the intact versus hydrolyzed product occurs outside of the

visible spectrum The visible spectrum is the portion of the electromagnetic spectrum that is visible to the human eye. Electromagnetic radiation in this range of wavelengths is called ''visible light'' or simply light. A typical human eye will respond to wa ...

. Hydrolysis of nitrocefin however, produces a shift of

absorption inside the visible light spectrum from intact (yellow) nitrocefin (~380 nm) to degraded (red) nitrocefin (~500 nm) allowing visual detection of

activity on a macroscopic level.

Detection assays

The following assays describe methods in which nitrocefin can be used to detect

enzymes using inexpensive materials and equipment. Working solutions of nitrocefin lie within 0.5 mg/mL to 1.0 mg/mL. Slide Surface Assay # Add one drop of 0.5 mg/ml Nitrocefin to the surface of a clean glass slide. # Select a

colony In modern parlance, a colony is a territory subject to a form of foreign rule. Though dominated by the foreign colonizers, colonies remain separate from the administration of the original country of the colonizers, the '' metropolitan state' ...

from an agar surface using a sterile loop and mix with the drop. # Appearance of red color within 20-30 min. indicates

activity. Direct Contact Assay # Place one drop of 0.5 mg/ml Nitrocefin directly on the surface of an isolated

. # Appearance of red color within 20-30 min. indicates

activity. Broth Suspension Assay # Add 3-5 drops of 0.5 mg/ml Nitrocefin to 1 ml of broth suspension. # Appearance of red color within 20-30 min. indicates

activity. Lysed Cell Assay # Lyse 1ml of cell suspension by sonication. # Add 3-5 drops of 0.5 mg/ml Nitrocefin to lysed cell suspension. # Appearance of red color within 20-30 min. indicates

activity. Filter Paper Assay # Place a small piece of filter paper (~3 x 3 cm) in a clean petri dish or another clean isolated surface and saturate (3-5 ml) with 0.5 mg/ml Nitrocefin # Select an isolated

and smear over the surface of the impregnated filter paper. # Appearance of red color within 20-30 min. indicates

activity

References

{{Reflist Cephalosporin antibiotics

Structure

Degradation and chromogenic properties

Detection assays

See also

References