Biochemical Test

Beta (β) Lactamase Test Principle, Procedure, Results

Many bacteria produce a group of enzymes known as beta-lactamases that are mediated through genes in plasmids or the chromosomes. Beta-lactamase can...

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This article writter by MN Editors on February 01, 2022

Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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

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

Many bacteria produce a group of enzymes known as beta-lactamases that are mediated through genes in plasmids or the chromosomes. Beta-lactamase can be a constant process or it may be triggered through contact with antimicrobials. Beta-lactamases hydrolyze (and consequently inhibit) the beta-lactam ring of many Cephalosporins as well as penicillins. Beta-Lactamase Test is a rapid test to determine the presence of beta-lactamase , an enzyme that is produced by the strains from Staphylococcus aureus Neisseria gonorrhoeae, Branhamella catarrhalis as well as Haemophilus influenzae. 

These enzymes provide resistance to a variety of penicillin antibiotics, by breaking the beta-lactam rings of cephalosporin and penicillin antibiotics and causing the inactivation of these antibiotics. This mechanism of action is the basis for the beta-Lactamase reaction.

Beta-lactamase tests that are quick can provide useful clinical data earlier than an MIC and disk-diffusion test. Numerous clinical tests have been developed to identify beta-lactamases. The tests are based on the Iodometric test, the acidometric method and Chromogenic substrates. The iodometric method is suitable to test N. the gonorrhoeae. Acidometric methods provide satisfactory results using Haemophilus Spp., N. gonorrhoeae and staphylococci. Nitrocefin offers a wide range of susceptibility as well as sensitivity to beta-lactams that are commercially available. 

Nitrocef Disks are imbued with nitrocefin, which is an chromogenic cephalosporin. Since the amide bond of the beta-lactam rings is hydrolyzed by beta-lactamase the nitrocefin’s color changes from red to yellow. Bacteria that make beta-lactamase in significant quantities produce this color change from red to yellow alteration on Nitrocef Disk. These beta-lactamases are capable of inactivating “penicillinase-labile-penicillins”, such as amoxicillin, ampicillin, penicillin, carbenicillin, mezlocillin, and piperacillin.

Procedure of Beta (β)-Lactamase Test

  1. With a single disc dispenser, drop the disc from the cartridge into a dish empty of petri dishes or onto the microscope slide.
  2. Then, moisten the disc using 1 drop of steriledistilled water.
  3. Utilizing a sterilized loop or applicator stick, remove several well-isolated similar colonies, and then smear them on the surface of a disk.
  4. Check the color of the disk.

Result Interpretation of Beta Lactamase Test

  • Positive reaction:  Yellow to red color changes in the area to which the culture is being applied.
  • Negative reaction: No colour change or color shift on the disc
OrganismsResultApproximate reaction timeInterpretation
Staphylococcus aureusPositive1 hourResistant to penicillin, ampicillin, carbenicillin. Probably susceptible to cephalothin, methicillin, oxacillin, naficillin and other penicillinaseresistant penicillins.
Enterococcus faecalisPositive5 minResistant to penicillin and ampicillin
Hameophilus influenzaePositive1 minResistant to ampicillin Susceptible to cephalosporins
Neisseria gonorrhoeae and Branhamella catarrhalisPositive1 minResistant 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.

Uses of Beta Lactamase Test

Applications that are useful include detection of:

  • N. gonorrhoeae resistance to penicillin
  • H. influenzae resistance to ampicillin
  • Staphylococcal resistance to penicillin

Limitations of Beta Lactamase Test

  • Beta-lactamase testing using the Nitrocef Disk is not meant to substitute for conventional susceptibility tests since other variables also influence the results of these tests. In some instances the intrinsic resistance to beta-lactam antibiotics hasn’t been associated with the production of beta-lactamase.
  • Don’t over-saturate the tip because it can cause the reagent to be diluted.
  • The detection of beta-lactamase activity within staphylococci could take up to 1 hour. The induction of the enzyme might also be necessary, and it can be accomplished by measuring growth along the margin of the zone around an Oxacillin disk.
  • A negative outcome does not exclude resistance due to other causes.
  • Nitrocef Disk method can’t be used to test members belonging to Enterobacteriaceae, Pseudomonas species, or any other aerobic, gram-negative Bacilli since the results may not provide an accurate prediction of susceptibility to the beta-lactams frequently used in treatment.
  • It is important to note that the Nitrocef Disk cannot be used in the case of organisms for which penicillin resistance does not occur caused by beta-lactamase, for instance Streptococcus pneumoniae or viridans streptococci.

Quality Control of Beta (β)-Lactamase Test

  • Staphylococcus aureus (ATCC 43300): Positive
  • Haemophilus influenzae (ATCC 33533): Positive
  • Branhamella catarrhalis (ATCC 25240): Negative
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