Beta (β) Lactamase Test – Principle, Procedure, Results

Beta-lactamase

Beta-lactamases, (β-lactamases) are enzymes (EC 3.5.2.6) produced by bacteria that confer multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems (ertapenem). Beta-lactamase provides antibiotic resistance by destroying the structure of antibiotics. In their molecular structure, all of these antibiotics share a four-atom ring known as a beta-lactam (β-lactam) ring. The lactamase enzyme hydrolyzes the β-lactam ring, deactivating the antibacterial properties of the molecule.

Typically, beta-lactam antibiotics are employed to combat a wide range of gram-positive and gram-negative microorganisms.

Gram-negative bacteria typically secrete beta-lactamases, particularly when antibiotics are present in the environment.

Structure

• The structure of a Streptomyces serine β-lactamase (SBLs), as represented by 1BSG, exhibits an alpha-beta fold, as classified by InterPro with the identifier IPR012338. This folding pattern is similar to that of a DD-transpeptidase, suggesting that the enzyme may have evolved from this type of protein. DD-transpeptidases are involved in bacterial cell wall biosynthesis, and β-lactam antibiotics bind to them to inhibit this process.
• Serine β-lactamases are categorized into different types based on their sequence similarity, namely types A, C, and D. These types share structural and functional characteristics and play a role in bacterial resistance to β-lactam antibiotics.
• In contrast to the serine type, another class of β-lactamase is the metallo type, also known as “type B” β-lactamases. Metallo-beta-lactamases (MBLs) require one or two metal ions, typically Zn2+ ions, at their active site for their catalytic activities. The presence of these metal ions enables MBLs to carry out their function effectively. The structure of one specific metallo-beta-lactamase, the New Delhi metallo-beta-lactamase 1, is represented by 6C89. Interestingly, it shares structural similarities with a protein called RNase Z, suggesting a possible evolutionary relationship between the two.
• Overall, β-lactamases play a crucial role in bacterial resistance by inactivating β-lactam antibiotics, which are widely used in clinical settings. Understanding the structure of different types of β-lactamases provides valuable insights into their mechanism of action and aids in the development of strategies to combat antibiotic resistance.

Mechanism of Beta-lactamase

• Beta-lactamases operate through two primary mechanisms that involve the cleavage or hydrolysis of the β-lactam ring.
• The serine β-lactamases (SBLs) share structural and mechanistic similarities with the penicillin-binding proteins (PBPs), which are essential for the construction and modification of the bacterial cell wall. Both SBLs and PBPs undergo a covalent modification of a serine residue within their active sites. However, there is a key distinction between PBPs and SBLs: SBLs rapidly hydrolyze the acyl-enzyme intermediate, resulting in the formation of a free enzyme and rendering the antibiotic inactive. This quick hydrolysis of the acyl-enzyme intermediate by SBLs leads to the inactivation of β-lactam antibiotics.
• On the other hand, metallo-beta-lactamases (MBLs) employ Zn2+ ions to activate a water molecule within their binding site. This activated water molecule then carries out the hydrolysis of the β-lactam ring. The presence of the Zn2+ ions is crucial for the catalytic activity of MBLs, as they facilitate the activation of the water molecule, enabling efficient cleavage of the β-lactam ring.
• These mechanisms employed by beta-lactamases allow them to counteract the activity of β-lactam antibiotics. By either rapidly hydrolyzing the acyl-enzyme intermediate or activating a water molecule for β-lactam ring hydrolysis, beta-lactamases contribute to bacterial resistance by rendering these antibiotics ineffective in inhibiting bacterial cell wall synthesis. Understanding the mechanisms of beta-lactamases is vital in developing strategies to overcome antibiotic resistance and enhance the efficacy of β-lactam antibiotics.

Penicillinase is a specific form of β-lactamase that hydrolyzes the β-lactam ring with specificity for penicillins. The average molecular weight of penicillinases is approximately 50 kiloDaltons.

The first identified β-lactamase was penicillinase. In 1940, Abraham and Chain were the first to isolate penicillinase from Gram-negative E. coli. However, penicillinase production rapidly spread to bacteria that previously did not produce it or produced it infrequently. Even with the development of penicillinase-resistant beta-lactams, such as methicillin, there is now ubiquitous resistance.

What is Beta (β) Lactamase Test?

• The beta-lactamase test is a diagnostic method used to detect the presence of beta-lactamase enzymes produced by certain types of bacteria. These enzymes are responsible for inactivating penicillin and conferring resistance to antibiotics in the β-lactam group, including cephalosporins.
• The development of acidimetric methods for beta-lactamase detection has been significant in the history of this test. In 1977, Slack et al. introduced a rapid acidimetric method to detect beta-lactamase in specific strains of Staphylococcus aureus. This method utilized benzylpenicillin as the substrate and cresol red as the indicator. The acidimetric principle relies on the change in pH that occurs when the beta-lactamase hydrolyzes the benzylpenicillin substrate, resulting in a color change of the indicator.
• Subsequently, Wheldon and Slack further expanded the application of acidimetric methods by detecting beta-lactamase production in ampicillin-resistant strains of Haemophilus influenzae. They employed a rapid acidimetric method using benzylpenicillin as the substrate and bromocresol purple as the indicator.
• In 1976, Percival et al. identified beta-lactamase-producing strains of Neisseria gonorrhoeae using a rapid acidimetric method. This test played a crucial role in identifying clinically relevant strains of N. gonorrhoeae that exhibited resistance to beta-lactam antibiotics.
• The Clinical and Laboratory Standards Institute (CLSI) recognizes the importance of rapid beta-lactamase tests in the detection of clinically relevant strains of Haemophilus spp. and N. gonorrhoeae. These tests offer faster results compared to traditional disk diffusion tests, enabling prompt identification of resistance mechanisms and aiding in the selection of appropriate treatment options.
• In summary, the beta-lactamase test is a valuable diagnostic tool for detecting the presence of beta-lactamase enzymes in bacteria. The development of acidimetric methods using various substrates and indicators has significantly contributed to the rapid and accurate detection of beta-lactamase-producing strains of Staphylococcus aureus, Haemophilus influenzae, and Neisseria gonorrhoeae. These tests have played a critical role in identifying antibiotic resistance and guiding appropriate treatment strategies.

Purpose of Beta (β)-Lactamase Test

• For the detection of the enzyme beta-lactamase that gives penicillin resistance to a variety of bacteria.

Principle of Beta (β)-Lactamase Test

The principle of the beta-lactamase test is based on the detection of the beta-lactamase enzyme produced by certain bacteria. Beta-lactamases are a class of enzymes that can be encoded by genes on plasmids or chromosomes within bacterial cells. These enzymes can be constitutively produced or induced in response to exposure to antimicrobials.

Beta-lactamases function by hydrolyzing the beta-lactam ring present in various susceptible penicillins and cephalosporins. This hydrolysis leads to the inactivation of these antibiotics, rendering them ineffective against the bacteria producing beta-lactamase. The beta-lactamase test aims to rapidly identify the presence of this enzyme in bacterial strains, particularly in Staphylococcus aureus, Neisseria gonorrhoeae, Branhamella catarrhalis, and Haemophilus influenzae.

The test is based on the reaction of the beta-lactamase enzyme with a specific substrate. One common substrate used is nitrocefin, which is a chromogenic cephalosporin. Nitrocefin is impregnated onto Nitrocef Disks used in the test. As the beta-lactamase enzyme hydrolyzes the amide bond within the beta-lactam ring of nitrocefin, a color change occurs from yellow to red. This color change is visible and indicates the presence of significant amounts of beta-lactamase activity in the tested bacteria.

The beta-lactamase test is valuable because it can provide clinically relevant information in a rapid manner, often faster than other susceptibility testing methods such as minimum inhibitory concentration (MIC) or disk diffusion tests. Different methods, including the iodometric method, acidometric method, and chromogenic substrates, have been developed for the detection of beta-lactamases. However, the Nitrocef Disk test using nitrocefin is widely used due to its broad spectrum of susceptibility and sensitivity to commercially available beta-lactam antibiotics.

By identifying the presence of beta-lactamase, the test helps determine the resistance profile of bacteria to penicillin antibiotics, including amoxicillin, ampicillin, penicillin, carbenicillin, mezlocillin, and piperacillin, which are susceptible to inactivation by beta-lactamases.

In summary, the principle of the beta-lactamase test is based on the detection of the beta-lactamase enzyme produced by bacteria, which inactivates certain penicillin antibiotics by hydrolyzing the beta-lactam ring. The test utilizes a substrate, such as nitrocefin, that undergoes a visible color change upon hydrolysis by beta-lactamase, providing a quick and reliable means of detecting the presence of this enzyme in bacterial strains.

or

The Beta Lactam Disk is impregnated with benzylpenicillin containing a β-lactam ring. When an organism produces the β-lactamase enzyme, the β-lactam ring of benzylpenicillin is hydrolyzed into penicilloic acid. This cleavage of the β-lactam ring renders the antibiotic inactive. The pH decrease is indicated by the color change of the brom cresol purple indicator from purple to yellow.

Requirement for Beta (β)-Lactamase Test

To run a β-lactamase test, you need some essential materials, varying slightly depending on the specific method used. Here’s a clear, no-nonsense rundown:

For Nitrocefin Test

1. Nitrocefin Disks: Get them commercially. Store at 2-8°C.
2. Sterile Distilled Water: For moistening the disks.
3. Glass Slide or Petri Dish: Use these to hold the test materials.
4. Sterile Pasteur Pipette: For transferring liquids without contamination.
5. Sterile Wooden Stick and Inoculating Loop: For handling and spreading the bacterial colonies.
6. Test Organism Colony: Grow it overnight (18-24 hours) on non-selective media.

For Acidimetric Method

• Disk or Strip Test:
  1. Acidimetric Disks: Store at 2-8°C.
  2. Sterile Distilled Water: For moistening the disks.
  3. Glass Slide or Petri Dish: Hold the test materials.
  4. Sterile Pasteur Pipettes: For liquid transfer.
  5. Sterile Wooden Stick and Inoculating Loop: For handling colonies.
  6. Test Organism Colony: Grow overnight on non-selective media.
• Tube Test:
  1. 0.5% Phenol Red Solution: Prepare by dissolving 0.5g of phenol red in water, heat if needed. Store at 25°C (shelf life: 6 months).
  2. Crystalline Potassium Penicillin G: Follow storage instructions from the manufacturer.
  3. 1 N NaOH: Prepare by dissolving 4g of NaOH in 100ml of water, store at 25°C.
  4. Sterile Pipettes (1ml and 10ml) and Pipette Bulb: For accurate measurement and transfer.
  5. Sterile Polystyrene Capped Tubes (12x75mm): For the reaction.
  6. Sterile Wooden Applicator Sticks or Inoculating Loops: For handling colonies.
  • Preparation of Penicillin-Phenol Red Substrate Reagent:
    1. Mix 2ml of 5% phenol red solution with 16.6ml sterile distilled water.
    2. Add this to a vial of crystalline benzylpenicillin G.
    3. Adjust the pH to 8.5 using 1 N NaOH until it turns violet.
    4. Dispense in 0.1ml aliquots and freeze at –20°C or lower.

For Iodometric Method

1. Penicillin: Dissolve at 6,000 µg/ml in phosphate buffer (pH 6.0, 0.05 to 1 M). Store at 2-8°C (shelf life: 24 hours).
2. Starch Reagent: Dissolve 1g of soluble starch in 100ml of water, heat to dissolve. Store at 2-8°C (shelf life: 1 week).
3. Iodine Reagents: Dissolve 2.03g iodine and 53.2g potassium iodide in water, adjust to 100ml. Store at 2-8°C in a dark bottle (shelf life: 2 months).
4. Sterile Microdilution Tray or Small Test Tube: For the reaction.
5. Sterile 1ml Pipettes and Pipette Bulb: For accurate measurement and transfer.
6. Sterile Wooden Applicator Sticks or Inoculating Loops: For handling colonies.

Procedure of Beta (β)-Lactamase Test

A. Procedure of Nitrocefin Test for Beta (β)-Lactamase

1. Set Up the Disks: Use sterile forceps to place the nitrocefin disks on a clean microscope slide or an empty petri dish. Let the disks come to room temperature.
2. Moisten the Disks: Add a drop of sterile distilled water to each disk.
3. Inoculate the Disks: Use a sterile loop or applicator stick to smear several colonies of the test organism onto the disk. Also, include positive and negative control strain colonies on separate disks.
4. Observe for Color Change: Watch for a shift from yellow to red. A positive result usually shows up within 15 seconds to 5 minutes. If no change happens within 5 minutes, the result is negative. For some staphylococci, positive results may take up to 1 hour.

B. Procedure of Acidimetric Method for Beta (β)-Lactamase

Acidimetric Method (Disk Test)

1. Setup: Dispense the necessary number of acidimetric disks onto a clean slide or dish using sterile forceps. Immediately return any unused disks to the freezer.
2. Preparation: Let the disks come to room temperature and moisten each with a drop of sterile distilled water.
3. Inoculation: Apply several colonies of the test organism onto each disk using a sterile loop or applicator stick. Include positive and negative control strains.
4. Color Change Observation: Check for a color change within 10 minutes. A positive result shifts from violet or red to yellow. No change indicates a negative result. For dry disks, color changes can remain visible for up to 24 hours.

Acidimetric Method (Tube Test)

1. Thawing Reagent Tubes: Remove the desired number of tubes from the freezer and allow them to thaw at room temperature (one tube per organism).
2. Inoculation: Add four or five colonies of the test organism to each tube using a sterile loop or stick, creating a milky suspension.
3. Observation: Monitor for a color change. A positive reaction appears in less than 15 minutes. If there’s no change after 15 minutes, consider the result negative. Note: Positive reactions for some staphylococci may take up to 1 hour, but changes occurring after 15 minutes might not reliably indicate β-lactamase presence.

C. Procedure of Iodometric Method for Beta (β)-Lactamase

1. Prepare the Solution: Add 0.1 ml of penicillin solution to a well of a microdilution tray or a small test tube.
2. Add the Test Organism: Mix the test organism to create an opaque, milky suspension.
3. Add Starch Solution: Add two drops of starch solution and mix. Let it sit at room temperature (around 25°C) for 30 to 60 minutes.
4. Add Iodine Reagent: Add one drop of iodine reagent, then shake or stir the mixture for one minute.
5. Observe for Color Change: Check for decolorization (turning white) within 10 minutes. If no color change occurs within 10 minutes, the test is negative. For some staphylococci, positive results may take up to 1 hour.

Result Interpretation of Beta Lactamase Test

When interpreting the results of β-lactamase tests, clarity is key:

Nitrocefin Test

• Positive Result: The yellow color of the disk changes to red after inoculation with the culture.
• Negative Result: No change in the color of the disk observed.

Acidimetric Method

• Positive Result: A violet or red color changes to yellow.
• Negative Result: No color change occurs.

Iodometric Method

• Positive Result: The blue color fades to colorless.
• Negative Result: The solution remains blue or purple in color.

Method	Positive Result	Negative Result
Nitrocefin Test	Yellow color changes to red	No change in color
Acidimetric Method	Violet or red color changes to yellow	No color change
Iodometric Method	Blue color fades to colorless	Blue or purple color

Organisms	Result	Approximate reaction time	Interpretation
Staphylococcus aureus	Positive	1 hour	Resistant to penicillin, ampicillin, carbenicillin. Probably susceptible to cephalothin, methicillin, oxacillin, naficillin and other penicillinaseresistant penicillins.
Enterococcus faecalis	Positive	5 min	Resistant to penicillin and ampicillin
Hameophilus influenzae	Positive	1 min	Resistant to ampicillin Susceptible to cephalosporins
Neisseria gonorrhoeae and Branhamella catarrhalis	Positive	1 min	Resistant to penicillin

Note: For the majority of bacteria, a positive result can be seen within five minutes. However positive reactions for certain staphylococci can take up to 1 hour to manifest and color changes do typically not occur across an entire disc.

Applications of Beta Lactamase Test

Use of Nitrocefin Test

• Detection Range: The Nitrocefin test efficiently identifies beta-lactamase-producing strains of N. gonorrhoeae, H. influenzae, Staphylococcus spp., Enterococcus spp., and Moraxella catarrhalis.
• Reliability: It is particularly reliable for detecting beta-lactamase production by Enterococcus spp., making it a crucial tool in clinical microbiology.
• Speed and Convenience: This chromogenic method is known for its ease of use and provides results quicker than traditional methods like acidimetric and iodometric tests.
• Broad Sensitivity: It detects both penicillinase and cephalosporinase enzymes, ensuring high sensitivity and efficiency in identifying beta-lactamase activity.

Use of Acidimetric Method

• Effectiveness: The acidimetric method proves effective for detecting beta-lactamase in coagulase-positive Staphylococci and is commonly used for Haemophilus spp., Neisseria gonorrhoeae, and various Staphylococcus spp.
• Ease of Use: It offers a rapid and straightforward procedure, facilitating swift testing and interpretation in clinical laboratories.

Use of Iodometric Method

• Versatility: The iodometric method serves as an alternative when chromogenic methods are unavailable. It provides reliable results comparable to the Nitrocefin test for detecting Staphylococcal beta-lactamase.
• Accuracy: This method is known for its high sensitivity and accuracy, especially in detecting beta-lactamase production by Staphylococcus spp.
• Specific Applications: It remains the most reliable method for testing beta-lactamase production by Neisseria gonorrhoeae, ensuring accurate diagnosis and treatment guidance in clinical practice.

Limitations of Beta Lactamase Test

Beta lactamase tests, while valuable, have specific limitations that impact their utility in clinical and research settings:

Limitations of Acidimetric Method

• Limited Applicability: The acidimetric method is applicable only to aerobic bacteria, restricting its use in anaerobic environments.
• Enzyme Differentiation: It does not distinguish between acylase and β-lactamase activity, limiting its specificity in enzyme detection.
• Enzyme Detection: This method primarily detects penicillinase and may miss cephalosporinase enzyme activity, influencing comprehensive enzyme profiling.

Limitations of Nitrocefin Test

• Moisture Sensitivity: Proper moisture content of the nitrocefin disk is crucial for accurate color development. Drying of the disk necessitates rehydration, affecting test reliability.
• Variable Reactivity: Some strains grown on blood agar plates may exhibit weak or indistinct reactions, reducing the test’s sensitivity in certain conditions.
• Supplementary Testing Needed: It should not replace conventional susceptibility testing methods entirely, as factors beyond beta-lactamase production influence antimicrobial susceptibility.
• Specific Organism Limitations: Inapplicable to Enterobacteriaceae, Pseudomonas species, and aerobic gram-negative bacilli, limiting its predictive value for therapy susceptibility.
• Organism Specificity: It cannot detect resistance mechanisms unrelated to beta-lactamase production in organisms like Streptococcus pneumoniae and Streptococci.

Limitations of Iodometric Method

• Enzyme Specificity: While specific for β-lactamase detection, the iodometric method does not employ a chromogenic approach to detect cephalosporinase activity, limiting its enzyme profiling capabilities.
• Resource Dependency: Routine use may be hindered by the need for freshly prepared starch and iodine solutions, affecting practicality in busy clinical laboratories.

Quality Control of Beta (β)-Lactamase Test

Quality control measures are essential in ensuring the reliability and accuracy of beta-lactamase tests, such as the Nitrocefin and Acidimetric methods:

Nitrocefin Test

• Positive Control: Staphylococcus aureus ATCC 29213 serves as a standard positive control, reliably producing beta-lactamase activity.
• Negative Control: Haemophilus influenzae ATCC 10211 and Staphylococcus aureus ATCC 25923 are used as negative controls, confirming absence of beta-lactamase activity.

Acidimetric Method

• Positive Result: Indicates a color change from violet or red to yellow, confirming beta-lactamase activity in the test organism.
• Negative Result: No color change observed, indicating absence of beta-lactamase activity in the organism being tested.

Method	Positive Control	Negative Control	Result Interpretation
Nitrocefin Test	Staphylococcus aureus ATCC 29213	Haemophilus influenzae ATCC 10211<br>Staphylococcus aureus ATCC 25923	Positive: Color change to red indicates beta-lactamase activity.<br>Negative: No color change indicates absence of beta-lactamase activity.
Acidimetric Method	Not applicable	Not applicable	Positive: Violet or red changes to yellow.<br>Negative: No color change.

Recent Studies about Beta (β)-Lactamase Test

• A Prospective Evaluation of the Accuracy of the VITEK MS for Detection of Extended-Spectrum β-Lactamase-Producing Escherichia coli in Clinical Urine Samples. doi: 10.1128/AAC.01407-22
• Evaluation of the BD Phoenix β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.03342-21
• Comparison of the BD Phoenix β-Lactamase Test and the VITEK MS for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1111/cmi.13867
• Comparison of the BD Phoenix β-Lactamase Test and the MicroScan β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.05281-21
• Evaluation of the BD Phoenix β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Respiratory Samples. doi: 10.1128/JCM.02723-22
• Comparison of the BD Phoenix β-Lactamase Test and the BD Phoenix β-Lactamase Disc Test for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.05703-21
• Comparison of the BD Phoenix β-Lactamase Test and the Etest for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.03394-21
• Evaluation of the BD Phoenix β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Blood Cultures. doi: 10.1128/JCM.05306-21
• Comparison of the BD Phoenix β-Lactamase Test and the Sensititre™ β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Clinical Samples. doi: 10.1128/JCM.04730-21
• Evaluation of the BD Phoenix β-Lactamase Test for Detection of β-Lactamase-Producing Bacteria in Wound Swabs. doi: 10.1128/JCM.02722-22

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