Hippurate hydrolysis test - Introduction, History, Principle, Procedure, Result

Introduction to Hippurate hydrolysis test

Hippurate hydrolase is an enzyme that reversibly catalyzes Hippurate, a bacterial product of phenylalanine metabolism, into sodium benzoate and glycine in the presence of water.

History of Hippurate hydrolysis test

Ayers and Rupp were the first to discover that the Hippurate hydrolysis test can differentiate bovine β-hemolytic Group B streptococci, from human β-hemolytic Group B Streptococcus species.

The Hippurate hydrolysis test was later modified by Facklam et al for presumptive identification of Group A, B, and D streptococci.

Hippurate hydrolysis test

The hippurate hydrolysis test aims to detect hippurate hydrolyzing bacteria, mainly Streptococcus species.

The hippurate hydrolysis test can now be used to distinguish β-hemolytic Group B streptococci from β-hemolytic Group A streptococci as well as non-enterococcal Group D streptococci.

Principle of Hippurate hydrolysis test

Group B streptococci including Streptococcus agalactiae and some species of enterococci are able to hydrolyze 1% aqueous sodium hippurate to produce glycine and sodium benzoate. When the oxidizing agent ninhydrin deaminates glycine, it gets reduced to produce purple coloration.

Hippuric acid == ^{(hippuricase)} ==> Glycine + benzoic acid ==^(Ninhydrin)==> Purple colour (positive)

Procedure of Hippurate hydrolysis test

The procedure of Hippurate hydrolysis test is as follows:

1. A rapid hippurate disc is placed aseptically in the test tube containing Brain Heart Infusion Broth inoculated with β-hemolytic Streptococcus.

2. The broth is then incubated at 35-37°C for 2 hours.

3. In the test tube, 0.2 mL ninhydrin reagent

4. The mixture is further reincubated for another 15 to 30 minutes.

5. Observe if the solution's color changes to purple

Result of Hippurate hydrolysis test

The result of Hippurate hydrolysis test is interpreted as:

Positive

Deep purple/blue color within 30 minutes (eg: Streptococcus agalactiae)

Negative

Colorless or slightly yellow-pink (eg: Streptococcus pyogenes)

Uses of Hippurate hydrolysis test

Since, groups A, C, F, and G streptococci cannot hydrolyze sodium hippurate, group B streptococci are easily identified by Hippurate hydrolysis test.

Hippurate hydrolysis test is also used in the identification of Gardnerella vaginalis, Streptococcus agalactiae, Campylobacter jejuni, and Listeria monocytogenes.

This test also separates Campylobacter jejuni from other Campylobacter spp.

Limitation of Hippurate hydrolysis test

The setback of Hippurate hydrolysis test is that the test medium must only contain hippurate as ninhydrin readily reacts with any free amino acids, giving false positive results.