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" (a chromophore). There is no universally agreed definiti ...

cephalosporin The cephalosporins (sg. ) are a class of β-lactam antibiotics originally derived from the fungus '' Acremonium'', which was previously known as ''Cephalosporium''. Together with cephamycins, they constitute a subgroup of β-lactam antibio ...

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 carbapene ...

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 carbapene ...

mediated resistance to beta-lactam antibiotics such as

penicillin Penicillins (P, PCN or PEN) are a group of beta-lactam antibiotic, β-lactam antibiotics originally obtained from ''Penicillium'' Mold (fungus), moulds, principally ''Penicillium chrysogenum, P. chrysogenum'' and ''Penicillium rubens, P. ru ...

is a widespread mechanism of resistance for a number of

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

including members of the family

Enterobacteriaceae Enterobacteriaceae is a large family (biology), family of Gram-negative bacteria. It includes over 30 genera and more than 100 species. Its classification above the level of Family (taxonomy), family is still a subject of debate, but one class ...

, a major group of enteric

Gram-negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the Crystal violet, crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelo ...

. Other methods for

detection exist including PCR; however, nitrocefin allows for rapid

detection using few materials and inexpensive equipment.

Structure

As a

, nitrocefin contains a beta-lactam 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 reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...

. Once hydrolyzed, the degraded nitrocefin compound rapidly changes color from yellow to red. Although nitrocefin is considered a

, it does not appear to have antimicrobial properties.

Degradation and chromogenic properties

Intact beta-lactam 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 layer (sacculus) that surrounds the bacterial cytoplasmic membrane. The sugar component consists of alternating ...

synthesis. Beta-lactamases hydrolyze the

amide In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a chemical compound, compound with the general formula , where R, R', and R″ represent any group, typically organyl functional group, groups or hydrogen at ...

bond between the

carbonyl In organic chemistry, a carbonyl group is a functional group with the formula , composed of a carbon atom double bond, double-bonded to an oxygen atom, and it is divalent at the C atom. It is common to several classes of organic compounds (such a ...

carbon Carbon () is a chemical element; it has chemical symbol, symbol C and atomic number 6. It is nonmetallic and tetravalence, tetravalent—meaning that its atoms are able to form up to four covalent bonds due to its valence shell exhibiting 4 ...

and the

nitrogen Nitrogen is a chemical element; it has Symbol (chemistry), symbol N and atomic number 7. Nitrogen is a Nonmetal (chemistry), nonmetal and the lightest member of pnictogen, group 15 of the periodic table, often called the Pnictogen, pnictogens. ...

in the beta-lactam ring of susceptible beta-lactams and members of beta-lactam 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

s because the shift of

ultraviolet Ultraviolet radiation, also known as simply UV, is electromagnetic radiation of wavelengths of 10–400 nanometers, shorter than that of visible light, but longer than X-rays. UV radiation is present in sunlight and constitutes about 10% of ...

absorption from the intact versus hydrolyzed product occurs outside of the

visible spectrum The visible spectrum is the spectral band, band of the electromagnetic spectrum that is visual perception, visible to the human eye. Electromagnetic radiation in this range of wavelengths is called ''visible light'' (or simply light). The optica ...

. 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 A colony is a territory subject to a form of foreign rule, which rules the territory and its indigenous peoples separated from the foreign rulers, the colonizer, and their ''metropole'' (or "mother country"). This separated rule was often orga ...

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 Biochemistry detection reactions Cephalosporin antibiotics

Structure

Degradation and chromogenic properties

Detection assays

See also

References