Decarboxylase and Dihydrolase Tests

Many bacteria have the ability to use amino acids as energy and carbon sources. Decarboxylase tests determine whether the bacterial species possess enzymes capable of decarboxylating (removing the carboxyl group, COOH)specific amino acids in the test medium.

Two amino acids commonly used to test for decarboxylase activity are lysine and ornithine. The products of decarboxylation are amine or diamine molecules and CO2,with resulting alkalinity. Degradation of the amino acids and their specific end products are shown in the following reaction: Degradation of amino acids and their specific end products

Lysine (amino acid) — Lysine decarboxylase — Cadaverine

(amine) + CO2

Ornithine — Ornithine decarboxylase — Putrescine

Arginine — Arginine dihydrolase — Citrulline —

Ornithine — Putrescine

Lysine is decarboxylated by the enzyme lysine decarboxylase to cadaverine, a diamine, and CO2, Ornithine is cleaved by ornithine decarboxylase to putrescine, a diamine, and carbon dioxide. Both cadaverine and putrescine are stable in anaerobic conditions.

Arginine can be decarboxylated in a two-step process. In the first step, arginine undergoes decarboxylation by arginine decarboxylase to form agmatine and CO2, Then agmatine is further metabolized to putrescine and urea. If the bacteria also produce urease, the urea will be degraded to ammonia (NHJ and CO2,Arginine can also be degraded by arginine dihydrolase forming citrulline, ammonia, and inorganic phosphate. In the next step, citrulline undergoes phosphorolytic cleavage to yield ornithine. If the bacteria also possess ornithine decarboxylase, ornithine will be converted to putrescine.

The test to detect decarboxylation uses Moeller decarboxylase base medium. This base is a broth containing glucose; peptones; two pH indicators, bromcresol purple and cresol red; and the specific amino acid at a concentration of 1%.The medium has an initial Ph of 6.0. Having glucose in the medium is important because decarboxylases are inducible enzymes produced in an acid pH. The uninoculated medium is purple; metabolism of the small amount of glucose drops the pH to about 5.5, turning the medium yellow. For decarboxylation to take place, two conditions must be met: an acid pH and an anaerobic environment. A control tube containing only the base medium without the amino acid is tested along with the test organism to determine the viability of the organism. The control tube also determines whether sufficient acid is produced. Both tubes are inoculated with the test organism; are overlayed with a layer of sterile mineral oil, which creates anaerobic conditions; and then are incubated at 35°C.

During the first few hours of incubation, organisms attack the glucose first, changing the pH to acid. If the organism produces the specific decarboxylase and the amino acid in the medium is attacked, release of the amine products causes an alkaline pH shift. This results in a purple (positive result) color in the medium. If the organism does not possess the specific decarboxylase, the medium remains yellow (negative result). The control tube remains yellow. Results can usually be recorded in 24 hours; however, bacteria with weak decarboxylase activity may take up to 4 days to be positive. Modifications of the decarboxylase test to detect other biochemical reactions are also routinely used. Examples of these include the motility-indole-ornithine (MIO) and lysine iron agar (LIA)tests (Figure 9-6).


Deaminase Test

Amino acids can be metabolized by deaminases that remove an amine (NH2) group. The phenylalanine deaminase (PAD)test determines whether an organism possesses the enzyme that deaminates phenylalanine to phenylpyruvic acid. The test medium is an agar slant that contains a 0.2% concentration of phenylalanine.

The surface of the slant is inoculated with a bacterial colony. Addition of a 10% ferric chloride (FeCI;) reagent after incubation results in a green color if phenylpyruvic acid is present. This test is helpful in initial differentiation of Proteus, Morganella, and Providencia organisms, which are positive, from the rest of the Enterobacteriaceae (Figure 9-7.

figure 9-6

Deaminationof Phenylalanine Phenylalanine-+ Phenylalanine deaminase -+ Phenylpyruvic acid + (FeCIJ Green

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