Hippurate hydrolysis occurs in an organism that can form glycine and benzoic acid.
In the classical test (48 hours), the production of benzoic acid was detected using ferric chloride as an indicator. The gas-liquid chromatography (GLC) method is also available to detect benzoic acid.
In contrast, the rapid test (2 hours) detects glycine, the second byproduct of Hippurate hydrolysis. It uses ninhydrin as the indicator. Rapid Hippurate hydrolysis is more specific and as sensitive as the classical method.
Principle of Hippurate Hydrolysis
Hippuric acid is hydrolyzed to benzoic acid and glycine by the enzymatic action of hippuricase. The addition of a ninhydrin reagent detects glycine. Ninhydrin reacts with glycine to form a deep blue or purple color (Ruhemann’s purple). Hwang and Ederer described this rapid test for detecting glycine.
Similarly, the traditional method detects benzoic acid using ferric chloride. The ferric chloride dissolves the benzoic acid present as residue after centrifugation of hippurate solution with the test organism.
Usesof Hippurate Hydrolysis
The hippurate test identifies Campylobacter jejuni, Listeria monocytogenes, Gardnerella vaginalis, and Streptococcus agalactiae.
- The hippurate hydrolysis test is critical for separating Campylobacter jejuni (hydrolyzes hippurate) and Campylobacter coli (negative) strains.
- It also aids in differentiating β-hemolytic Streptococcus agalactiae from other β-hemolytic streptococci.
Test Procedure
Classical Method
- Firstly, take or prepare sterile sodium hippurate broth and inoculate it with the test organism.
- After that incubate it overnight at 35°C.
- Then, centrifuge the broth and remove the sediment.
- Finally, add ferric chloride reagent to the supernatant.
- If the residue remains after 10 minutes, benzoic acid is present. The test is positive for hippurate hydrolysis.
Quality Controlused in Hippurate Hydrolysis Test
Test each new lot or shipment of the reagent with known positive and negative controls, and retest at least monthly. Discard all reagents and prepare new ones if the reagents do not pass QC. Grow theATCC strains in sodium hippurate broth for quality control and look for the results.
- Streptococcus agalactiae (ATCC 4768): Moderate to heavy growth and hydrolysis of hippurate.
- Streptococcus pyogenes (ATCC 19615): Moderate to heavy growth; hippurate not hydrolyzed.
Hippurate Hydrolysis Test Positive Organisms :
The hippurate hydrolysis test is used in the presumptive identification of various bacteria. Organisms giving positive (+ve) tests are:
- Gardnerella vaginalis,
- Campylobacter jejuni,
- Listeria monocytogenes and
- Group B streptococci (Streptococcus agalactiae)
Limitations
- False-positive results can occur if incubation with ninhydrin exceeds 30 min.
- A negative test does not rule out the identification of G. vaginalis since the biotypes that cause bacterial vaginosis can be hippurate negative.
- Viridans group streptococci can be hippurate positive; performing other tests to confirm the identification of non-hemolytic colonies is essential.
- A small number of enterococci are beta-hemolytic and may hydrolyze hippurate, but they arepyrrolidinyl-β-naphthylamide (PYR) positive (S. agalactiae is PYR negative)
- Furthermore a small percentage of C. jejuniorganisms are hippurate negative and use of other methods for complete identification is necessary.
References and further readings
- Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
- Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition