Nitrocefin Test (Beta-Lactamase Test) – Principle, Procedure, Uses

The Beta (β) Lactamase test is a rapid test used to detect the production of β-lactamase enzyme by certain bacteria. It is commonly known as Nitrocefin test. This test is used to determine resistance of bacteria against β-lactam antibiotics such as penicillin and cephalosporin. The test is based on the ability of the organism to produce enzyme which breaks the β-lactam ring of antibiotics.

Nitrocefin is a chromogenic cephalosporin substrate containing β-lactam ring. When the organism produces β-lactamase enzyme, the enzyme hydrolyses the amide bond present in the nitrocefin molecule. Due to this hydrolysis, a colour change is produced. Initially the nitrocefin appears yellow, and after enzyme action it changes to red colour. This colour change usually occurs within 5 minutes to one hour, and indicates a positive result.

A positive Nitrocefin test shows that the organism is resistant to penicillinase labile β-lactam antibiotics. This test is commonly used for organisms such as Neisseria gonorrhoeae, Haemophilus influenzae, Enterococcus spp. and Staphylococcus species. However, this test is not recommended for complex Gram negative bacteria like Enterobacteriaceae and Pseudomonas species, as these organisms possess multiple resistance mechanisms which cannot be differentiated by this test.

Principle of Nitrocefin test

The Nitrocefin test is a rapid diagnostic test which is used to detect the presence of β-lactamase enzyme produced by certain bacteria. It is based on the principle that β-lactamase enzyme can break the β-lactam ring present in a chromogenic cephalosporin compound called nitrocefin. Nitrocefin in its intact form absorbs light in the near ultraviolet range and appears yellow in colour.

When the organism produces β-lactamase enzyme, the enzyme hydrolyses the amide bond of the β-lactam ring of nitrocefin. Due to this hydrolysis, the molecular structure of nitrocefin is changed and a shift in electron resonance occurs. This results in a change in light absorption from ultraviolet range to the visible range (approximately 486–500 nm). Because of this change, the colour of nitrocefin changes from yellow to red.

The appearance of red colour within a few minutes to one hour indicates a positive Nitrocefin test. This confirms that the test organism is producing β-lactamase enzyme and is resistant to penicillinase-labile antibiotics such as penicillin, ampicillin and cephalosporins. This test is commonly used for organisms like Neisseria gonorrhoeae, Haemophilus influenzae, Staphylococcus species and Enterococcus species, but it is generally not used for predicting antibiotic susceptibility in complex Gram-negative bacilli due to their multiple resistance mechanisms.

Objectives of Nitrocefin test

The objectives of Nitrocefin test are as follows–

• To detect the presence of β-lactamase enzyme produced by different bacterial strains in a rapid manner.
• To screen β-lactamase production in clinically important organisms such as Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella (Branhamella) catarrhalis, Staphylococcus species and Enterococcus species.
• To detect β-lactamase producing anaerobic bacteria like Bacteroides, Fusobacterium and Clostridium species.
• To predict resistance against penicillinase-labile antibiotics such as penicillin, ampicillin, amoxicillin, carbenicillin, piperacillin and cephalosporins.
• To reduce the time required for antibiotic susceptibility reporting as compared to conventional methods like MIC and disc diffusion test.
• To differentiate enzymatic resistance mechanisms in advanced assays where nitrocefin is used as a reporter molecule for carbapenemase activity.
• To estimate β-lactamase activity in research studies and quantitative assays using different biological samples.

Requirements for Nitrocefin test

The requirements for Nitrocefin test are as follows–

• Nitrocefin reagent in the form of impregnated discs, sticks or liquid solution which acts as the chromogenic substrate.
• Dimethyl sulfoxide (DMSO) and phosphate buffer (pH 7.0) for preparation of nitrocefin solution when powder form is used.
• Sterile distilled or deionized water for moistening the nitrocefin disc or reaction surface.
• Sterile applicator sticks, bacteriological loop or straight wire for picking and smearing bacterial colonies.
• Forceps or disc dispenser for handling nitrocefin discs.
• Clean glass slide or sterile Petri dish to perform the reaction.
• Well isolated colonies from a pure bacterial culture as test specimen.
• Test organisms such as Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus species, Enterococcus species and anaerobic bacteria.
• For Staphylococcus species, colonies taken from the margin of β-lactam antibiotic disc for enzyme induction.
• Positive control strains such as Staphylococcus aureus, Haemophilus influenzae and Neisseria gonorrhoeae.
• Negative control strains such as Escherichia coli and non β-lactamase producing Staphylococcus aureus.
• Proper storage conditions for nitrocefin reagent at low temperature and protection from light to maintain reagent stability.

Procedure of Nitrocefin Test

Paper Disk or Slide Method (Most Common)

1. In the first step, the nitrocefin disks or strips is allowed to reach room temperature.
2. The disk is taken with sterile forceps and it is placed on a clean slide or Petri dish.
3. The disk is moistened with one drop of sterile distilled water. It is not over-saturated because excess water dilute the reagent.
4. In this step, well isolated colonies (1–3 colonies) of similar morphology is taken with sterile loop.
5. The colony mass is now smeared on the moistened disk. These are sometimes applied by sweeping the moistened disk directly on the colony.
6. The colour change is observed. It is the process where yellow colour changes to red at the site of inoculation. Most positive strains develop colour within 5 minutes but some species may take longer time.

Direct Plate Method

1. One drop of nitrocefin solution is placed directly on the isolated colony on agar plate.
2. The colony and surrounding area is observed for red colour development. It is the process which is quick in strong β-lactamase producers and slower in weak producers.

Broth or Lysed Cell Method

1. About 3–5 drops of nitrocefin solution is added into 1 mL grown broth culture.
2. The cell suspension is sometimes sonicated to lyse the cells so that intracellular enzymes is released.
3. It is then observed for red colour formation within 20–30 minutes indicating positive reaction.

Result of Nitrocefin Test

Positive Result

• It is the observation where the yellow colour changes to red or pink at the site of inoculation.
• The reaction is caused because β-lactamase enzyme hydrolyses the β-lactam ring of nitrocefin.
• The colour develops quickly in many organisms within 1–5 minutes, while some species may take up to 60 minutes.
• It indicates that the organism is resistant to penicillinase-labile antibiotics.
• Among the important positive organisms are Staphylococcus aureus, Haemophilus influenzae, Neisseria gonorrhoeae, Moraxella catarrhalis, and Bacteroides species.

Negative Result

• It is the result where no colour change occurs and the reagent remains yellow.
• This is referred to as the absence of detectable β-lactamase activity in the organism.
• It shows susceptibility to penicillin or cephalosporins in those species where β-lactamase is the main resistance factor.
• A negative test does not rule out resistance because other mechanisms like altered PBPs or efflux systems is present in some organisms.
• Some of the main negative controls include Escherichia coli (ATCC 25922) and non-penicillinase producing Staphylococcus aureus (ATCC 25923).

Invalid or Less Predictive Cases

• In Enterobacteriaceae and Pseudomonas species the test is of little diagnostic value because many different enzymes is produced which the reagent cannot differentiate.
• In Staphylococcus species inducible enzymes may give false negative results. It is the process where growth from the edge of inhibition zone is used to confirm enzyme induction.

Uses of Nitrocefin Test

• It is used for rapid detection of β-lactamase enzyme in many bacterial colonies, and this is referred to as the main diagnostic use.
• It is the process that helps in identifying β-lactamase production in Enterococcus species, which is considered one reliable rapid test.
• It is used for predicting resistance towards penicillinase-labile antibiotics (penicillin, ampicillin, amoxicillin, carbenicillin, mezlocillin, piperacillin, ticarcillin).
• It is used in differentiating different carbapenemases when inhibitors are added in advanced assays like NitroSpeed-Carba NP test.
• It is helpful in antimicrobial stewardship as the test reduces the time for detecting resistance and assists in shifting to specific antibiotics.
• It is used in epidemiological monitoring for detecting penicillinase-producing N. gonorrhoeae and related strains.
• Some of the main uses in research include drug discovery assays where nitrocefin acts as a reagent for detecting activities of PBPs and β-lactamases.
• It is used for enzyme kinetics studies (Vmax, Km), because the colour change allows quantitative measurement of β-lactamase activity.
• It is the process used as a colourimetric readout in biosensor systems involving fusion proteins.
• It is used to confirm prochelator activation, where antimicrobial agents are released by β-lactamase-producing bacteria.
• It is applied for detecting β-lactamase-secreting bacteria in biological fluids like saliva, urine, serum and also in different food samples.
• It is used in fermentation monitoring for measuring β-lactamase activity in cultures and media.

Advantages of Nitrocefin Test

• It is used because the result is obtained very rapidly, and most positive reactions develop within a few minutes.
• It is the process that shows very high sensitivity as nitrocefin is hydrolysed by almost all β-lactamases including penicillinases and cephalosporinases.
• It produces a distinct visual colour change (yellow to red) which is easy to interpret without any complicated instrument.
• It is the only reliable rapid method for detecting β-lactamase in Enterococcus species.
• It is highly effective for fastidious organisms like N. gonorrhoeae, H. influenzae, M. catarrhalis and also for staphylococci and anaerobic bacteria.
• It helps in antimicrobial stewardship as rapid enzyme detection supports early switching to specific antibiotics.
• It is used in advanced diagnostic assays where nitrocefin acts as a reporter molecule for differentiating resistance enzymes and for quantitative kinetic measurement.
• It is simple to perform, and the disks or strips required for the test need very minimal equipment.

Limitations of Nitrocefin Test

• It is not useful for predicting susceptibility in many Gram-negative bacilli because different β-lactamase types cannot be differentiated.
• It is the process that only detects enzymatic hydrolysis, so organisms having resistance due to altered PBPs or due to permeability changes will show negative result.
• Some bacteria like staphylococcus produce inducible β-lactamase, and these enzyme is not detected properly unless growth from induced margin is used.
• The reaction time is variable, and some organisms take long time which makes interpretation difficult.
• A negative result is not confirming susceptibility, and it must be verified by standard growth-based tests.
• Nitrocefin reagent is unstable to light and moisture, and strict storage is required, and the cost is also high.
• The test is sensitive to procedure, and excess moisture or degraded reagent may give false-negative or false-positive reactions.