Beta Lactamase Test is a crucial diagnostic tool that determines if bacteria have beta-lactamase enzymes. Certain bacteria manufacture these enzymes to provide resistance to antibiotics that are beta-lactam, such as cephalosporins, carbapenems, monobactams, and penicillins. For the purpose of treating bacterial infections, especially those brought on by multi-drug resistant organisms, and directing efficient antimicrobial therapy, the ability to quickly and correctly detect the development of beta-lactamases in bacterial isolates is essential.
Table of Contents
Principle of Beta Lactamase Test
- The chromogenic cephalosporinase test is one of the most helpful assays in clinical laboratories for β-lactamase identification.
- The substrate for this test, a chromogenic cephalosporin, is included on the test disk.
- When β-lactamase-containing organisms are added to the disk, they work by hydrolyzing the substrate’s β-lactam ring.
- The outcome of this hydrolysis process is the production of an immediately identifiable colored product.
- In particular, the substrate on the test disk is nitrocefin. When a bacteria generates substantial amounts of β-lactamase, the yellow-colored disk in the region where the isolate is spread turns red.
Types of Beta Lactamase Test
1. Nitrocefin (Chromogenic Cephalosporin) Test
Principle: When hydrolyzed by beta-lactamase, nitrocefin, a chromogenic cephalosporin substrate, changes color.
Method: The chromogenic substrate is impregnated into a nitrocefin disk or strip. After applying the bacterial isolate to the disk, a positive outcome is indicated by the emergence of a red hue.
Findings: A red hue denotes the presence of beta-lactamase, whereas a colorless result means the test was unsuccessful.
2. Acidimetric Method
Principle: The hydrolysis of the beta-lactam ring by beta-lactamase activity results in the generation of acid, which lowers pH.
Procedure: A beta-lactam antibiotic is taken in combination with a pH indicator (such as phenol red). The antibiotic and pH indicator are incubated with bacterial isolates. Enzyme activity is shown by a color change brought on by a pH shift.
Results: Acid production and a positive test are shown by a shift from red to yellow; a negative test is indicated by no color change.
3. Iodometric Method
Principle: When Beta Lactamase Test hydrolyzes penicillin, it releases penicilloic acid, which subsequently reduces iodine. This process results in the presence of free iodine, which is detected by this approach.
Method: An iodine-starch combination is used. Penicillin is administered to bacterial isolates, and they are then incubated with iodine. The beta-lactamase activity is shown by the starch-iodine complex’s blue-black hue disappearing.
Findings: A reduction in the blue-black hue denotes a favorable outcome, whereas its preservation signifies an unfavorable outcome.
4. Cefinase Test
Principle: A chromogenic cephalosporin substrate is used, just like in the nitrocefin test.
Method: Wet the paper disk and inoculate it with the bacterial isolate once it has been impregnated with the substrate. It is seen that the hue changes.
Outcomes: A favorable outcome is indicated by a red color; a poor outcome is shown by no color change.
Procedure Beta Lactamase Test
- Preparing the Sample: Take the bacterial colony off of the culture plate.
- Exposure to Substrates: The bacterial isolate can be applied to a solution containing the Beta Lactamase Test substrate or a disk that has been impregnated with substrate.
- Incubation: Give the enzyme-substrate reaction enough time to happen; this might take a few minutes to many hours, depending on the technique.
- Observation: Using the technique employed, look for any discernible alterations, including a specific hue shift.
Results Interpretation of Beta Lactamase Test
Positive Result: The presence of beta-lactamase is shown by a noticeable color shift (red in nitrocefin, yellow in acidimetric, and loss of blue-black in iodometric), which suggests that the bacterial isolate is probably resistant to beta-lactam antibiotics.
Negative Outcome: The lack of beta-lactamase, which indicates resistance to beta-lactam antibiotics, is implied by neither color change nor the predicted indication.
Frequently Asked Question
1. What is a Beta Lactamase Test?
The eta (β) Lactamase Test is employed to identify the beta-lactamase enzymes generated by various bacterial species. The enzyme beta-lactamase, which is either chromosomally or plasmid-encoded, hydrolyzes the beta-lactam ring of antibiotics in the beta-lactam class, rendering the medications inactive.
2. What bacteria are beta-lactamase positive?
Genes from various genera, including Enterobacter sp., Salmonella sp., Proteus sp., Serratia marcescens, Shigella dysenteriae, Pseudomonas aeruginosa, and gram-negative bacteria like Klebsiella pneumoniae and Escherichia coli, produce extended-spectrum beta-lactamases (ESBL) (24).
3. What color is positive for beta-lactamase?
When beta-lactamase hydrolyzes nitrocefin, a chromogenic cephalosporin substrate, it becomes red instead of yellow, indicating the presence of beta-lactamase enzymatic activity. Disk-diffusion can be used for extended spectrum beta lactamase (ESBL) screening.
4. What is the meaning of lactamase?
any of a number of bacterial enzymes that hydrolyze beta-lactam antibiotics (like cephalosporin and penicillin) to render them ineffective. A number of pathogenic bacteria will produce the enzyme beta-lactamase in response to an antibiotic.
Related Article