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

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Nitrocefin test is a rapid diagnostic test used to detect the presence of beta lactamase enzyme produced by different bacteria. It is also known as beta lactamase test. These enzyme are responsible for bacterial resistance to beta lactam antibiotics like penicillins and cephalosporins because the drugs are hydrolyzed and inactivated by the enzyme. In this test nitrocefin is used which is a chromogenic cephalosporin substrate and it appears yellow in colour. When a beta lactamase producing bacteria is exposed to nitrocefin, the enzyme breaks the amide bond present within the beta lactam ring of nitrocefin molecule. The hydrolysis reaction causes a visible colour change from yellow to red or deep pink. Thus development of red or pink colour indicates positive result and it confirms the bacteria are resistant to certain beta lactam antibiotics while no colour change (yellow colour remains) indicates negative result.

Principle of Nitrocefin test

Principle of nitrocefin test is based on visual detection of beta lactamase enzyme produced by some bacteria to resist beta lactam antibiotics. In this test nitrocefin is used which is a chromogenic cephalosporin compound and it appears yellow in colour. If the tested bacteria produces beta lactamase, this enzyme hydrolyzes the amide bond present within the beta lactam ring of nitrocefin. The beta lactam ring is opened and an electronic shift is produced which changes the light absorption and the colour changes from yellow to deep red or pink. Thus rapid development of red colour indicates positive test for beta lactamase activity while persistence of yellow colour indicates negative result.

Objectives of Nitrocefin test

Objectives of nitrocefin test are–

  • To rapidly detect the presence of beta lactamase enzyme produced by isolated bacterial colonies like Neisseria gonorrhoeae, Moraxella catarrhalis, Staphylococcus species, Haemophilus influenzae, and some anaerobic bacteria.
  • To identify bacterial resistance to beta lactam antibiotics and it confirms that tested bacteria can inactivate drugs like penicillins and cephalosporins.
  • To predict specific clinical resistance pattern like penicillin resistance in Neisseria gonorrhoeae and Staphylococcus species or ampicillin resistance in Haemophilus influenzae.
  • To provide a reliable detection method for Enterococcus species as nitrocefin test is the only dependable method for identifying beta lactamase production in these bacteria.

Requirements for Nitrocefin test

  • Nitrocefin disks (commercially prepared) or Nitrocefin powder with sterile filter paper disks.
  • Sterile distilled water or deionized water, or 0.1 M phosphate buffer (pH 7.0) for moistening the disk before inoculation.
  • Clean glass microscope slide or empty Petri dish to hold the disk during testing.
  • Sterile inoculating loop or wooden applicator sticks for picking and smearing the bacterial colonies onto the disk.
  • Forceps for dispensing and handling the disks.
  • Sterile Pasteur pipettes for dropping water or buffer onto the disk.
  • Bacterial test colonies grown overnight (18–24 hours) on non-selective culture media.
  • Quality control strains- known positive strain (e.g. Staphylococcus aureus) and known negative strain (e.g. Haemophilus influenzae) for ensuring the accuracy of test.

Procedure of Nitrocefin test (Step by step)

  1. Using sterile forceps or disk dispenser, required number of Nitrocefin disks are placed on a clean microscope slide or in an empty sterile Petri dish.
  2. The Nitrocefin disks are allowed to reach room temperature before starting the inoculation (equilibration step).
  3. The disk is moistened by adding exactly one drop of sterile distilled water or deionized water to hydrate the disk.
  4. It is important that disk is not over moistened or oversaturated because excess water can dilute the reagent and reaction becomes weak.
  5. Using sterile inoculating loop or wooden applicator stick, several well isolated bacterial colonies (18–24 hours growth on non-selective media) are picked and smeared directly on the surface of moistened disk.
  6. Alternatively, the moistened disk can be grasped with sterile forceps and it is wiped directly across a bacterial colony on agar plate.
  7. Control strains are tested- a known positive control strain (e.g. Staphylococcus aureus ATCC 29213) and a known negative control strain (e.g. Haemophilus influenzae ATCC 10211) are smeared on separate disks along with test sample.
  8. The inoculated disk is observed at room temperature for colour change.
  9. Rapid development of red, deep pink, or orange colour indicates positive result and it is seen within 15 seconds to 5 minutes.
  10. If the disk remains yellow after 5 minutes, the result is negative, but some Staphylococcus and anaerobic species may take up to 60 minutes to show positive reaction.

Result of Nitrocefin Test

Positive result

It is indicated by a rapid colour change from yellow to red, deep pink, or orange colour.

This confirms that the tested bacteria is producing beta-lactamase enzyme.

A positive result predicts resistance to penicillinase-labile beta lactam antibiotics such as penicillin, ampicillin, and amoxicillin.

The colour change is usually developed within 5 minutes but certain Staphylococcus species may take up to 60 minutes.

The red colour may not develop on the whole disk, colour change only in the area where culture is smeared is sufficient for positive interpretation.

Negative result

It is indicated when the disk remains yellow and no colour change is seen within the observation time.

This indicates absence of beta-lactamase enzyme production.

Negative result suggests that organism may be susceptible to beta-lactam antibiotics but it does not guarantee susceptibility because resistance can also be due to other mechanisms such as altered penicillin-binding proteins.

Nitrocefin test
Nitrocefin test

Organisms for Nitrocefin test result

Positive result organisms

These are the organisms which gives positive result when beta-lactamase enzyme is produced-

Neisseria gonorrhoeae.

Moraxella (Branhamella) catarrhalis.

Staphylococcus species (beta-lactamase producing strains such as Staphylococcus aureus ATCC 11632, ATCC 29213, ATCC 43300).

Haemophilus influenzae (beta-lactamase producing strain such as ATCC 33533).

Enterococcus species.

Anaerobic bacteria- Bacteroides, Clostridium, Porphyromonas, Fusobacterium, Prevotella.

Achromobacter.

Beta-lactamase producing strains (ESBL, AmpC, or carbapenemase) of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Proteus mirabilis, Pseudomonas aeruginosa, Hafnia alvei, and Acinetobacter baumannii.

Negative result organisms

These are the organisms which gives negative result due to absence of beta-lactamase enzyme or resistance is due to other mechanism-

Streptococcus pneumoniae and viridans streptococci.

MRSA (Methicillin Resistant Staphylococcus aureus) strains where resistance is due to target site modification (mecA) without beta-lactamase production.

Haemophilus influenzae ATCC 10211 (negative control strain).

Escherichia coli ATCC 25922 (negative control strain).

Staphylococcus aureus ATCC 25923 (negative control strain).

Moraxella (Branhamella) catarrhalis ATCC 25240 (negative control strain).

It is noted that Nitrocefin test is not used to predict therapeutic susceptibility for Enterobacteriaceae, Pseudomonas species, and other aerobic gram negative bacilli because negative result does not guarantee susceptibility to beta-lactam drugs.

Uses of Nitrocefin Test

Some of its uses include:

  • Rapid detection of beta-lactamase enzyme from isolated bacterial colonies such as Neisseria gonorrhoeae, Moraxella catarrhalis, Staphylococcus species, Haemophilus influenzae, and anaerobic bacteria.
  • Interpretation of bacterial resistance to beta-lactam antibiotics mainly penicillin and cephalosporins.
  • Predicting penicillin resistance in Neisseria gonorrhoeae and Staphylococcus species.
  • Predicting ampicillin resistance in Haemophilus influenzae.
  • Nitrocefin test is referred to as the most reliable method for detecting beta-lactamase production in Enterococcus species.
  • Detection of beta-lactamase enzyme produced by Achromobacter species.
  • Simultaneous detection of both penicillinase and cephalosporinase enzyme due to high sensitivity of Nitrocefin reagent.

Advantages of Nitrocefin Test

  • It gives rapid result as compared to traditional methods such as MIC and disk diffusion test.
  • It is simple and easy to perform, only smearing of bacterial colony on a moistened disk is required.
  • It does not require secondary chemical reaction therefore it is more convenient than acidimetric or iodometric methods.
  • It gives clear visual end point, a distinct colour change from yellow to red or pink is seen.
  • It is highly sensitive and efficient as it can detect both penicillinase and cephalosporinase enzyme.
  • It is recognized as a reliable phenotypic method for detecting beta-lactamase production in certain bacteria like Enterococcus species.
  • It is cost effective and self contained, no additional reagent preparation is required.

Limitations of Nitrocefin Test

Some of the limitations are-

  • It should not be used as a complete substitute for conventional growth dependent susceptibility testing because intrinsic resistance and other factors can affect actual susceptibility.
  • Negative result only indicates absence of beta-lactamase enzyme but it does not guarantee susceptibility as resistance can also be due to other mechanisms such as altered penicillin-binding proteins.
  • It is not suitable for testing Enterobacteriaceae family, Pseudomonas species, and other aerobic gram negative bacilli because rapid result is not reliably predictive of therapeutic susceptibility to beta-lactam drugs.
  • It cannot detect non enzymatic resistance mechanisms therefore it is not useful for organisms like Streptococcus pneumoniae, Streptococci, and MRSA (Methicillin Resistant Staphylococcus aureus).
  • In some Staphylococcus and anaerobic species, reaction may be delayed and it can take up to 60 minutes and staphylococci may also require prior enzyme induction with a beta-lactam antibiotic for giving positive result.
  • Bacterial strains grown on blood agar plates may produce weak or indistinct colour reaction and result can be obscured.
  • Moisture level of disk is critical, disk should not dry out and it should not be over saturated because excess water can dilute reagent and reaction becomes weak.
  • Reading result after recommended observation time (15 to 60 minutes) can give late false positive because spontaneous substrate deterioration can cause late colour change not correlating with true clinical resistance.

Precautions of Nitrocefin test

Some of the precautions are-

  • Nitrocefin disks are stored in freezer (below -10°C) or in refrigerator (2–8°C) as per manufacturer instruction and it should be protected from direct light moisture and excess heat.
  • Before opening the container and before inoculation, the disks are allowed to equilibrate at room temperature.
  • The disks should not be kept at room temperature for long time, unused disks are returned immediately to freezer storage in an air tight seal.
  • The disks should not be used if there is deterioration, discoloration, or expiry date is passed.
  • While hydrating the disk, only one drop of sterile water is added, over moistening is avoided because reagent can be diluted and reaction becomes weak.
  • The disk should not be allowed to dry during observation, if drying starts then it is rehydrated with small amount of water.
  • Quality control is done routinely using known positive and negative strains, and batch is not used if control organisms gives incorrect reaction.
  • It is for in vitro diagnostic use by trained personnel only, all specimens and materials are treated as potentially infectious and aseptic technique is followed.
  • Reagent should not be ingested or inhaled and direct contact with skin is avoided.
  • All biohazard waste is sterilized properly before disposal.
  • This test should not be used for Enterobacteriaceae, Pseudomonas species, and other aerobic gram negative bacilli because result will not accurately predict therapeutic susceptibility to beta-lactams.
  • It should not be used for organisms where penicillin resistance is due to other mechanism than beta-lactamase production (e.g. Streptococcus pneumoniae and viridans streptococci).
  • Negative result only implies susceptibility but it does not guarantee it because other resistance mechanism can be present.

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