Test for Production of Beta Lactamase

In this test for production of beta lactamase post we have briefly explained beta lactamase producing bacteria, beta lactamase test principle, objectives, requirements, beta lactamase producing bacteria test procedure, uses and limitations.

Beta Lactamase Producing Bacteria

In Beta Lactamase Test, Beta-lactamases are a type of enzyme produced by a variety of bacteria and are mediated by genes on plasmids or chromosomes. Beta lactamase producing bacteria can be either constitutive or triggered by antibiotic exposure.

The beta-lactam rings of a variety of susceptible penicillins and cephalosporins are hydrolyzed by beta-lactamases. Beta-lactamases are categorised according to their preferred antibacterial substrate and how inhibitors affect them.

Some antibiotics, like cefazolin and cloxacillin, are resistant to this type of hydrolysis. Many forms of penicillin are resistant to beta-lactamase generating strains of the following bacteria: Staphylococcus species, Hemophilus influenzae, Neisseria gonorrhoeae, Bacteroides species, Enterococcus species, and Moraxella catarrhalis.

When isolated from important specimens such as blood or spinal fluid, the organisms listed above should always be screened for beta-lactamase production. When a beta-lactamase inhibitor is added to a beta-lactam, the antimicrobials’ action is restored.

Principle

Nitrocefin, a chromogenic cephalosporin, is imbedded in the Cefinase disc. When a beta-lactamase hydrolyses the amide link in the beta lactam ring, the colour changes from yellow to red fairly quickly.

When a bacterium produces large amounts of this enzyme, the yellow-colored disc in the area where the isolate is smeared turns red.

While other penicillin’s and Cephalosporins can be employed as enzyme substrates, Nitrocefin has the broadest range of susceptibility and sensitivity of the commercially available beta lactams.

Other microbial enzymes are not known to react with it. Each disc is used to check for the presence of betalactamase in one bacterial strain.

Procedure

1. Dispense the disc from the cartridge into an empty petri dish or onto a microscope slide using a single disc dispenser. 1 drop of sterile distilled water should be used to moisten the disc.

2. Remove several well-isolated comparable colonies with a sterilised loop or applicator stick and smear onto a disc surface. Keep an eye on the disc for any colour changes.

Results

Beta Lactamase Test

Beta Lactamase Producing Bacteria: A positive reaction will show a yellow to red change on the area where culture was applied. A negative result will show no color change on disc.

Quality Control

Haemophilus influenzae: Resistant to ampicillin for a positive reaction.

Neisseria gonorrhoeae: Resistant to penicillin for a positive reaction.

Moraxella catarrhalis: Resistant to penicillin for a positive reaction.

Enterococcus faecalis: Resistant to penicillin and ampicillin for a positive reaction.

Further Readings

Reference