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

Objective of Beta (β)-Lactamase Test

  • To detect the enzyme beta-lactamase, which confers penicillin resistance to various bacterial organisms.

Principle of Beta (β)-Lactamase Test

Various bacteria produce a class of enzymes called beta-lactamases, which may be mediated by genes on plasmids or chromosomes. Production of beta-lactamase may be constitutive or induced by exposure to antimicrobials. Beta-lactamases hydrolyze (and thereby inactivate) the beta-lactam rings of a variety of susceptible penicillins and cephalosporins. Beta-Lactamase Test rapidly detects the presence of beta-lactamase enzyme produced by strains of Staphylococcus aureus, Neisseria gonorrhoeae, Branhamella catarrhalis, and Haemophilus influenzae. These enzymes confer resistance to a number of penicillin antibiotics by cleaving the beta-lactam ring of penicillin and cephalosporin antibiotics, resulting in the inactivation of these drugs. This mode of action forms the basis of the beta-Lactamase test reaction.

Rapid beta-lactamase tests can yield clinically relevant information earlier than a MIC or disk diffusion test. Several clinical tests have been devised to detect beta-lactamases. These tests include the iodometric method, the acidometric method, and chromogenic substrates. Iodometric methods are suitable for testing N. gonorrhoeae. Acidometric methods produce acceptable results with Haemophilus spp., N. gonorrhoeae, and staphylococci. Nitrocefin has a wide spectrum of susceptibility and sensitivity to the commercially available beta-lactams. Nitrocef Disks are impregnated with nitrocefin, a chromogenic cephalosporin. As the amide bond in a beta-lactam ring is hydrolyzed by a beta-lactamase, nitrocefin changes color from yellow to red. Bacteria that produce beta-lactamase in significant amounts produce this yellow to red color change on the 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

  • Using a single disk dispenser, dispense the disk from the cartridge into an empty petri dish or onto a microscope slide.
  • Moisten disc with 1 drop of sterile distilled water.
  • With a sterilized loop or applicator stick remove several well-isolated similar colonies and smear onto a disk surface.
  • Observe disk for color change.

Result Interpretation of Beta (β)-Lactamase Test

Result Interpretation of Beta (β)-Lactamase Test

Positive reaction:  yellow to red color change on the area where the culture is applied.

Note: For most bacterial strains a positive result will develop within 5 minutes. However, positive reactions for some staphylococci may take up to 1 hour to develop, and color change does not usually develop over the entire disk.

Negative reaction:  no color change 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

Limitations of Beta (β)-Lactamase Test

  • Beta-lactamase detection with the Nitrocef Disk should not entirely replace conventional susceptibility test methods, as other factors also influence the results of such tests, and on occasion, intrinsic resistance to beta-lactam antimicrobials has not been correlated with the production of beta-lactamase.
  • Do not over-saturate the tip as it could dilute the reagent.
  • Detection of beta-lactamase activity in staphylococci may take up to one hour. Induction of the enzyme may also be required, this can be done by testing growth from the zone margin around an oxacillin disk.
  • A negative result does not rule out resistance due to other mechanisms.
  • Nitrocef Disk method cannot be used to test members of EnterobacteriaceaePseudomonas species, or other aerobic, gram-negative bacilli because the results may not be predictive of susceptibility to the beta-lactams most often used for therapy.
  • The Nitrocef Disk cannot be used for organisms where penicillin resistance is not due to beta-lactamase production, such as Streptococcus pneumoniae and viridans streptococci.

Quality Control of Beta (β)-Lactamase Test

Staphylococcus aureus (ATCC 43300): Positive

Haemophilus influenzae (ATCC 33533): Positive

Branhamella catarrhalis (ATCC 25240): Negative



About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar, Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for more than four years, he joined the Central Department of Microbiology, Tribhuvan University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is interested in research on actinobacteria, myxobacteria, and natural products. He has published more than 15 research articles and book chapters in international journals and well-renowned publishers.

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