Beta Lactamase Test: Principle, Types, Procedure, Results, Notes

Beta (β) Lactamase Test Introduction

The Beta (β) Lactamase Test is a crucial diagnostic tool employed in microbiology to identify the production of beta-lactamase enzymes by various bacteria. Here are some detailed insights into this test:

Beta-lactamases: Unveiling the Enzymatic Defense

• Plasmid or Chromosomal Origin: Beta-lactamases can be encoded either on plasmids or chromosomes within bacterial cells.
• Hydrolysis of Beta-Lactam Ring: These enzymes function by hydrolyzing the beta-lactam ring present in antibiotics, causing their inactivation. This process is a major mechanism of bacterial resistance against beta-lactam antibiotics, including penicillins and cephalosporins.
• Integron Association: The beta-lactam gene is often located on integrons, genetic elements that facilitate the capture and expression of gene cassettes.

Historical Context: Pioneering Beta-Lactamase Discovery

• 1940 Discovery: The first beta-lactamase, initially termed “penicillinase,” was described in 1940. This enzyme was identified in Escherichia coli (E. coli) and demonstrated the ability to hydrolyze penicillin.

Regulation of Beta-Lactamase Production

• Constative or Induced: Bacteria may constitutively produce beta-lactamases, or their production may be induced upon exposure to antimicrobial agents.

Bush-Medeiros Classification System

• Molecular Classes: According to the Bush-Medeiros Classification System, beta-lactamases are categorized into four molecular classes (A-D). This classification is based on the amino acid structure of the enzymes.

Role of Beta-Lactamase Test

• Early Detection: The Beta Lactamase Test serves as a rapid method for the early detection of beta-lactamase enzyme production.
• Prompt Results: Compared to other susceptibility testing methods like Minimum Inhibitory Concentration (MIC) or Disk Diffusion, the beta-lactamase test provides quicker results.

Clinical Significance

• Informed Treatment: Identification of beta-lactamase production is crucial for guiding clinicians in choosing appropriate antibiotic therapies. It helps in tailoring treatment strategies based on the resistance profile of bacteria.

In the context of rising antibiotic resistance, the Beta Lactamase Test plays a pivotal role in facilitating timely and informed decisions for effective antibacterial therapies.

Objective

The primary objective of the Chromogenic Cephalosporin Test (Nitrocefin Test) within the Beta (β) Lactamase Test is to detect the presence of the beta-lactamase enzyme through chromogenic, iodometric, and acidometric tests. This enzyme is responsible for conferring resistance to beta-lactam antibiotics in various bacterial organisms.

Principle of Nitrocefin Test:

• Nitrocefin Disks: Nitrocefin disks are impregnated with nitrocefin, a chromogenic cephalosporin.
• Detection Mechanism: Bacteria producing a significant amount of β-lactamase enzyme lead to the hydrolysis of the amide bond in the β-lactam ring of nitrocefin.
• Color Change: The color of the nitrocefin disk changes from yellow to red upon hydrolysis, serving as a positive indication. No color change indicates a negative result.
• Target Antibiotics: The beta-lactamases detected by this test have the capability to inactivate “penicillinase-labile-penicillins” such as amoxicillin, ampicillin, penicillin, carbenicillin, mezlocillin, and piperacillin.

Requirements:

1. Nitrocefin disk (commercially available, follow manufacturer instructions) stored at 2-8 ℃.
2. Sterile distilled water.
3. Glass slide or empty petri dish.
4. Sterile Pasteur pipette.
5. Sterile wooden stick and inoculating loop.
6. Overnight (18-24 hrs) grown colony of the test organism on non-selective media.

Procedure:

1. Using sterile forceps, dispense the required number of nitrocefin disks onto a clean microscope slide or an empty petri dish.
2. Allow the nitrocefin disk to reach room temperature before inoculation.
3. Moisten each disk with 1 drop of sterile distilled water.
4. With a sterile loop or applicator stick, smear several colonies onto the disk surface. Include positive and negative control strain colonies in the smearing process.
5. Observe the disk for color change. Positive reactions usually occur within 15 s to 5 min. A lack of color change within 5 min indicates a negative result. However, positive reactions for some staphylococci may take up to 1 h.

The Chromogenic Cephalosporin Test is a valuable tool in the Beta (β) Lactamase Test battery, providing rapid and visual detection of beta-lactamase activity in bacterial organisms.

Results and Interpretation of Nitrocefin Test:

• Positive Result:
  • Observation: The yellow color changes to red when the culture is inoculated onto the nitrocefin disk.
  • Interpretation: This indicates the presence of beta-lactamase enzyme activity in the tested bacterial organism. The beta-lactamase has successfully hydrolyzed the nitrocefin, causing the color change.
• Negative Result:
  • Observation: No change in the color of the nitrocefin disk.
  • Interpretation: This suggests the absence of beta-lactamase enzyme activity in the tested bacterial organism. The nitrocefin remains unchanged, indicating that the beta-lactam ring has not been hydrolyzed.

Positive and Negative Controls:

• Positive Control (Bacteria giving positive results):
  • Strain: Staphylococcus aureus ATCC 29213 (Quality control).
  • Result: This strain is expected to yield a positive result, serving as a quality control measure for the functionality of the test.
• Negative Controls (Bacteria giving negative results):
  • Strains:
    1. Haemophilus influenza ATCC 10211 (Quality control)
    2. Staphylococcus aureus ATCC 25923
  • Results: These strains are expected to yield negative results, confirming that the test is not generating false positives and ensuring the reliability of the test.

The positive and negative controls are essential for validating the accuracy and consistency of the Nitrocefin Test results. The inclusion of quality control strains helps in verifying that the test is functioning correctly and providing reliable information regarding the presence or absence of beta-lactamase enzyme activity.

Applications of Nitrocefin Test:

1. Detection of Beta-Lactamase:
   • The Nitrocefin test is a sensitive method for detecting beta-lactamase-producing strains of various bacteria, including:
     • Neisseria gonorrhoeae
     • Haemophilus influenzae
     • Staphylococcus spp.
     • Enterococcus spp.
     • Moraxella catarrhalis
   • It is particularly reliable for detecting beta-lactamase production by Enterococcus spp.
2. Quick Results:
   • The test is easy to perform and provides results earlier than other methods, making it a rapid and convenient option in a laboratory setting.
3. Quicker and More Convenient Method:
   • The chromogenic method using Nitrocefin is quicker and more convenient compared to acidimetric and iodometric methods for detecting beta-lactamase activity.
4. High Sensitivity and Efficiency:
   • The chromogenic method is highly sensitive and efficient, detecting both penicillinase and cephalosporinase enzymes produced by isolates.
5. Widespread Applicability:
   • Nitrocefin has a wide susceptibility and sensitivity to commercially available beta-lactams, making it applicable to a broad range of bacterial isolates.

Limitations of Nitrocefin Test:

1. Moistening Disk Critical:
   • The moistening of the disk is critical for color development. If the disk dries out, rehydration with a small amount of water may be necessary.
2. Indistinct and Weak Reactions:
   • Strains grown on blood agar plates may yield indistinct and weak reactions, affecting the reliability of the results.
3. Cannot Replace Conventional Methods:
   • Beta-lactamase detection with Nitrocefin should not entirely replace conventional susceptibility test methods, as other factors can influence the results.
4. Over-saturation Warning:
   • Over-saturation of the tip should be avoided, as it could dilute the reagent and affect the accuracy of the test.
5. Time Considerations:
   • Detection of beta-lactamase activity in staphylococci may take up to one hour, and induction of the enzyme may be required.
6. Negative Result Limitation:
   • A negative result does not rule out resistance due to other mechanisms, and the test is not applicable to organisms where penicillin resistance is not due to beta-lactamase production.
7. Limited Applicability:
   • The Nitrocefin disk method cannot be used for certain bacterial groups, including Enterobacteriaceae, Pseudomonas species, and other aerobic, gram-negative bacilli, as the results may not predict susceptibility to commonly used beta-lactams.
8. Not Suitable for Certain Organisms:
   • The Nitrocefin disk cannot be used for organisms like Streptococcus pneumoniae and other Streptococci where penicillin resistance is not due to beta-lactamase production.

Acidimetric Method of Beta (β) Lactamase Test:

Principle: In the acidimetric method, the penicillin-phenol red substrate reacts with the beta-lactamase enzyme, leading to the production of penicilloic acid. This reaction results in a change in color from violet or red to yellow due to a decrease in pH. A positive test is indicated by the color change to yellow, while no change in color indicates a negative test.

Requirements for Disk or Strip Test:

1. Acidimetric disk (commercially available, following manufacturer instructions), stored at 2-8 ℃.
2. Sterile distilled water
3. Glass slide or empty petri dish
4. Sterile Pasteur pipettes
5. Sterile wooden stick and inoculating loop
6. Test organism colony grown overnight (18-24 hrs) on non-selective media.

Requirements for Tube Test:

1. 0.5% Phenol red solution
2. Crystalline potassium penicillin G
3. 1 N NaOH
4. Sterile 1- and 10-ml pipettes and pipette bulb
5. Sterile polystyrene capped tubes (12 by 75 mm)
6. Sterile wooden applicator sticks or inoculating loops.

Preparation of Penicillin-Phenol Red Substrate Reagent:

1. Mix 2 ml of 5% phenol red solution with 16.6 ml of sterile distilled water.
2. Add the phenol red-water solution (18.6 ml) to the vial of crystalline benzylpenicillin G.
3. Adjust the pH to 8.5 with 1 N NaOH.
4. Dispense in 0.1-ml aliquots into sterile tubes and freeze at –20°C or lower.

Procedures (Disk Test):

1. Dispense the required number of disks onto a clean microscope slide or an empty petri dish.
2. Moisten each disk with 1 drop of sterile distilled water.
3. Smear several colonies onto the disk surface using a sterile loop or applicator stick.
4. Observe the disk for color change. Positive results usually appear within 10 min.

Procedures (Tube Test):

1. Thaw the desired number of reagent tubes.
2. Add four or five colonies to the test solution to make an opaque, milky suspension.
3. Observe for color change. A positive reaction will occur in less than 15 min.

Results and Interpretation:

• Positive: Violet or red color changes to yellow.
• Negative: No change in color occurs.

Applications:

• More effective than iodometric method for detecting coagulase-positive Staphylococcal β-lactamase.
• Rapid acidimetric method used for performing a beta-lactamase test on Haemophilus spp, Neisseria gonorrhoeae, and Staphylococcus spp.
• Easy to perform and interpret.

Limitations:

• Applies only to aerobic bacteria.
• Does not differentiate between acylase and β-lactamase activity.
• Only detects penicillinase, not cephalosporinase enzyme.

Iodometric Method of Beta (β) Lactamase Test:

Principle: The iodometric method is based on the ability of β-lactamase to hydrolyze penicillin G, releasing a reducing product (penicilloic acid). This reducing product can reduce iodine and prevent it from combining with starch. A discoloration of the dark blue iodine-starch complex indicates positive results.

Requirements:

1. Penicillin (6,000 µg/ml) dissolved in phosphate buffer (pH 6.0, 0.05 to 1 M), stored at 2 to 8°C with a shelf life of 24 hours.
2. Starch reagent: 1 g of soluble starch in 100 ml of distilled water, stored at 2 to 8°C with a shelf life of 1 week.
3. Iodine reagents: Dissolve 2.03 g of iodine and 53.2 g of potassium iodide in distilled water to make the final volume 100 ml. Store at 2 to 8°C in a dark bottle with a shelf life of 2 months.
4. Empty sterile microdilution tray or small test tube
5. Sterile 1 ml pipettes and pipette bulb
6. Sterile wooden applicator sticks or inoculating loops

Procedure:

1. Dispense 0.1 ml of the penicillin solution into a well of a microdilution tray or a small test tube.
2. Add the test organism to make an opaque, milky suspension.
3. Add 2 drops of the starch solution and mix. Let it sit at room temperature (approximately 25°C) for 30 to 60 minutes.
4. Add 1 drop of the iodine reagent. Shake or stir the mixture for 1 minute.
5. Observe for color change. Decolorization (to white) in less than 10 minutes indicates a positive reaction.

Results and Interpretation:

• Positive: Fading of blue to colorless.
• Negative: Blue or purple color.

Applications:

• Similar results to the Nitrocefin disk test, making it useful in detecting Staphylococcal β-lactamase when chromogenic methods are not available.
• More sensitive and accurate compared to the acidimetric method for the detection of beta-lactamase production by Staphylococcus.
• Most reliable method for testing beta-lactamase production by N. gonorrhoeae.

Limitations:

• Specific to the β-lactamase test but not a chromogenic method for detecting enzymes from cephalosporinase activity.
• Acquisition of freshly prepared starch and iodine solution may hinder routine use.