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Swedish University of Agricultural Sciences

Veterinary bacteriology: information about important bacteria
Veterinary bacteriology


There are also rapid methods for testing whether the bacteria carry enzymes that hydrolyse of inhibit specific antimicrobial substances. The enzymes that are routinely tested are the presence of beta-lactamase (penicillinase), which is an enzymes that hydrolyses the beta-lactamase ring in penicillins, which is then inactivated. Beta-lactamase-producing strains for example of Staphylococcus spp. do not always have high MIC values in the microdilution tests which means that further test is needed getting a reliable result such as detection of antibiotic-inactivating enzymes. Several methods are available; two common methods are described below.

The cloverleaf method is a simple and safe way to detect b‑-lactamase production. An agar plate is inoculated with a lawn of a bacterial strain of Staphylococcus aureus susceptible to penicillin. A paper disc impregnated with penicillin (often 10 µg) is placed in the middle of the plate. Radially out from the disc, you streak the strains you want to test for penicillinase production as well as a positive and negative control strains. After one day´s incubation, the susceptible strain can grow further towards the penicillin disc alongside strains producing b‑lactamase because the penicillin around these strains is hydrolysed and inactive. If four positive strains are tested on the same plate, zone of the inhibition will look like a four-leaf clover - hence the name of the method.

Quick method with chromogenic cephalosporins: Cephalosporins have a b‑lactam ring in their structure. Among the various cephalosporins that have been developed, there are some where hydrolysis of the b‑lactam ring causes a color change of the substances, hence he term chromogenic.

NItrocefin is a chromogenic cephalosporin that is commercially available dried on paper discs. The disc is placed directliy on the agar surface in a place with good growth of the bacteria that should be tested. The disc absorbs moisture from the medium and if b‑-lactamase is produced by the bacteria the disc will change color from yellow to red within 30 minutes. It is important that the tests are not performed on media containing blood or serum, since there are substances in serum that cause a false positive reaction. If only culture on blood agar are available, the disc can be placed on a slide and moistened with distilled water, and thereafter colony material could be applied with distilled water, and thereafter colony material could be applied on the disc with a loop. It have to be ensured that the disc does not dry during the 30 minutes that the reaction takes.

Updated: 2024-06-17.

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