Hippurate Hydrolysis Test- Principle, Procedure, Result, Uses

Hippurate hydrolysis test is a biochemical test that differentiates microorganisms on the basis of their ability to hydrolyze hippurate into glycine and benzoic acid by the enzymatic action of hippuricase. Hippurate is a salt or ester of hippuric acid, which is a compound formed by the conjugation of benzoic acid and glycine in the liver and excreted in urine. Some bacteria can break down hippurate by producing an enzyme called hippuricase, which cleaves the bond between benzoic acid and glycine. The resulting products are then detected by different indicators, such as ferric chloride or ninhydrin.

The hippurate hydrolysis test has two main objectives:

• To detect the production of the enzyme hippuricase that hydrolyzes the substrate hippurate into glycine and benzoic acid.
• To differentiate bacteria on the basis of their ability to hydrolyze hippurate.

The test is useful for the presumptive identification of some bacteria that can hydrolyze hippurate, such as:

• Streptococcus agalactiae (Group B streptococci), which can be distinguished from other beta-hemolytic streptococci that cannot hydrolyze hippurate.
• Campylobacter jejuni, which can be separated from Campylobacter coli that is hippurate negative.
• Listeria monocytogenes, which can be identified by its ability to hydrolyze hippurate along with other characteristics such as catalase positivity, motility at 25°C, beta-hemolysis, and CAMP positivity.
• Gardnerella vaginalis, which can be detected by its hippurate positivity along with other features such as catalase negativity, gram-variable morphology, and hemolysis on human blood agar.

The hippurate hydrolysis test is used to test the ability of certain microorganisms to produce the enzyme hippuricase, which can break down the substrate hippurate into glycine and benzoic acid. The test is mainly used for the presumptive identification of four groups of bacteria:

• Streptococcus agalactiae: These are Gram-positive cocci that are catalase-negative and beta-hemolytic. They are also known as group B streptococci (GBS) and can cause infections in newborns, pregnant women, and immunocompromised individuals.
• Campylobacter jejuni: These are curved Gram-negative rods that are oxidase-positive and catalase-positive. They are one of the most common causes of bacterial gastroenteritis worldwide and can also cause Guillain-Barré syndrome and reactive arthritis.
• Listeria monocytogenes: These are small Gram-positive rods that are catalase-positive and motile at 25°C. They can cause listeriosis, a serious infection that can affect pregnant women, newborns, elderly people, and immunocompromised individuals. Listeriosis can manifest as meningitis, septicemia, or abortion.
• Gardnerella vaginalis: These are tiny Gram-variable rods that are catalase-negative and hemolytic on human blood agar. They are associated with bacterial vaginosis, a condition characterized by vaginal discharge, itching, and odor.

The hippurate hydrolysis test can help differentiate these bacteria from other closely related or similar-looking bacteria that do not hydrolyze hippurate. However, the test is not confirmatory and should be supplemented by other biochemical, immunological, molecular, or mass spectrometry tests for accurate identification.

The hippurate hydrolysis test is based on the ability of some bacteria to produce an enzyme called hippuricase, which can break down the substrate hippurate into glycine and benzoic acid. Hippurate is a compound formed by the conjugation of benzoic acid and glycine in the liver of mammals. It is excreted in urine and can be used as a source of nitrogen by some bacteria.

The test can be performed in two ways: the classical method and the rapid method. The classical method uses ferric chloride as an indicator to detect the presence of benzoic acid in the reaction mixture. The rapid method uses ninhydrin as an indicator to detect the presence of glycine in the reaction mixture. Both methods require a hippurate solution as the test medium, which should not contain any other protein source that could interfere with the indicator reaction.

The classical method involves inoculating a tube containing 1% hippurate solution with a pure culture of the test organism and incubating it for 24 to 48 hours at 35°C. Then, a few drops of ferric chloride solution are added to the tube and mixed well. A positive result is indicated by the formation of a red-brown precipitate of ferric benzoate within 10 minutes. A negative result is indicated by no color change or a slight greenish color.

The rapid method involves inoculating a tube containing 0.2 ml of hippurate solution with a heavy suspension of the test organism and incubating it for 2 hours at 35°C. Then, two drops of ninhydrin solution are added to the tube and mixed well. The tube is then reincubated for another 30 minutes at 35°C. A positive result is indicated by the appearance of a deep blue color (about the color of crystal violet) within 30 minutes. A negative result is indicated by no color change or a faint purple color.

The principle behind both methods is the same: the hydrolysis of hippurate by hippuricase releases benzoic acid and glycine, which can be detected by different indicators. The difference lies in the sensitivity and specificity of the indicators and the time required for the test. The rapid method using ninhydrin is more sensitive and specific than the classical method using ferric chloride, and it also reduces the time required for the test from days to hours.

The hippurate hydrolysis test is mainly used to differentiate between different species of Streptococcus, especially Group B streptococci (S. agalactiae) from other beta-hemolytic streptococci (Groups A, C, F, and G). It can also be used to identify other bacteria that can hydrolyze hippurate, such as Listeria monocytogenes, Campylobacter jejuni, and Gardnerella vaginalis.

Media Used

The hippurate hydrolysis test does not require any specific culture medium, as it is performed on bacterial colonies grown on standard media such as blood agar or chocolate agar. However, the test medium used must only have hippurate as a protein source since ninhydrin reacts with any free amino acids present.

Reagent Used

The main reagent used for the hippurate hydrolysis test is ninhydrin, which is a chemical indicator that reacts with glycine, the product of hippurate hydrolysis. Ninhydrin can be found commercially as a liquid solution or as a reagent-impregnated disk, tablet, or strip. Alternatively, it can be prepared in the laboratory by dissolving 3.5 grams of ninhydrin in 50 ml of acetone and 50 ml of 1-butanol.

Supplies Used

The supplies needed for the hippurate hydrolysis test are:

• Sterile wooden sticks or inoculating loops
• Test tubes
• Distilled water
• Incubator at 35°C to 37°C
• Timer

The procedure of the hippurate hydrolysis test can be divided into two steps: preparation of the hippurate solution and hydrolysis test.

A. Preparation of hippurate solution

• Depending on the source of the hippurate reagent, different amounts of distilled water are added to it. For commercially bought dehydrated hippurate tubes, 0.2 ml (3 or 4 drops) of distilled water at a pH of 6.8 to 7.2 is added to the test reagent. For disk or tablet tests, 2 drops of distilled water are added to an empty tube. For laboratory prepared hippurate solution, 0.4 ml of the reagent is added to a tube per test.
• The hippurate solution should be clear and colorless. If it is cloudy or discolored, it should be discarded and replaced with a fresh one.

B. Hydrolysis Test

• A heavy suspension (equivalent to no. 3 McFarland standard) of the test organism is prepared from an 18- to 24-hr culture in the tube containing the hippurate solution. The colony should be picked up carefully as not to pick up agar, which contains protein that can interfere with the test.
• The tube is then incubated for 2 h at 35°C to 37°C.
• After the 2-h incubation period, 2 drops of the ninhydrin solution are added to the hippurate reagent-organism mixture. An additional 2 drops are to be added if the test has 0.4 ml or more of the hippurate.
• The tubes are then reincubated at 35 to 37°C for 30 min.
• The observation of the tubes is made at 10-min intervals for the appearance of deep blue color. The color change will usually appear within 10 to 15 min after the ninhydrin indicator solution has been added.

The result of the hippurate hydrolysis test is based on the color change of the test medium after the addition of ninhydrin reagent. The color change indicates the presence or absence of glycine, which is produced by the hydrolysis of hippurate by the enzyme hippuricase.

• A positive hippurate hydrolysis reaction is indicated by the appearance of a deep blue color (about the color of crystal violet) within 30 minutes. This means that the organism has hippuricase enzyme and can hydrolyze hippurate to glycine and benzoic acid. Examples of hippurate positive organisms are Streptococcus agalactiae, Listeria monocytogenes, Campylobacter jejuni, and Gardnerella vaginalis.
• A negative hippurate hydrolysis reaction is indicated by the appearance of a faint purple color or no color change. This means that the organism does not have hippuricase enzyme and cannot hydrolyze hippurate. Examples of hippurate negative organisms are Streptococcus pyogenes, Enterococcus faecalis, and some strains of Campylobacter jejuni.

The result should be interpreted along with other biochemical, immunological, molecular, or mass spectrometry tests to confirm the identification of an organism. Some factors that can affect the accuracy of the test are:

• The quality and quantity of the inoculum
• The pH and freshness of the hippurate reagent
• The incubation time and temperature
• The amount and timing of the ninhydrin reagent

To ensure the accuracy and reliability of the hippurate hydrolysis test, it is important to use control organisms along with the test isolates. Control organisms are bacteria that are known to be either positive or negative for hippurate hydrolysis, and they serve as a reference for comparison with the test results. Control organisms should be inoculated and incubated with each run of test isolates.

Some examples of control organisms for the hippurate hydrolysis test are:

• Streptococcus agalactiae: hippurate positive. This organism is a Gram-positive coccus that belongs to Lancefield group B and causes infections in newborns, pregnant women, and immunocompromised individuals. It can be identified by its beta-hemolysis on blood agar and its positive hippurate hydrolysis test.
• Streptococcus pyogenes: hippurate negative. This organism is a Gram-positive coccus that belongs to Lancefield group A and causes pharyngitis, impetigo, rheumatic fever, and necrotizing fasciitis. It can be identified by its beta-hemolysis on blood agar and its negative hippurate hydrolysis test.
• Campylobacter jejuni: hippurate positive. This organism is a curved Gram-negative rod that causes gastroenteritis, enterocolitis, and Guillain-Barré syndrome. It can be identified by its oxidase and catalase positivity, its microaerophilic growth at 42°C, and its positive hippurate hydrolysis test.
• Campylobacter coli: hippurate negative. This organism is a curved Gram-negative rod that causes gastroenteritis and enterocolitis. It can be identified by its oxidase and catalase positivity, its microaerophilic growth at 42°C, and its negative hippurate hydrolysis test.
• Listeria monocytogenes: hippurate positive. This organism is a small Gram-positive rod that causes listeriosis, a serious infection that can affect pregnant women, newborns, elderly people, and immunocompromised individuals. It can be identified by its catalase positivity, its motility at 25°C, its beta-hemolysis on blood agar, its CAMP positivity, and its positive hippurate hydrolysis test.
• Gardnerella vaginalis: hippurate positive. This organism is a tiny Gram-variable rod that causes bacterial vaginosis, a condition characterized by vaginal discharge, itching, and odor. It can be identified by its catalase negativity, its hemolysis on human blood agar, and its positive hippurate hydrolysis test.

The results of the hippurate hydrolysis test should be reported based on the appearance of color change after adding the ninhydrin reagent. The following are some examples of how to report the results for different organisms:

• L. monocytogenes organisms are tiny Gram-positive rods that are catalase-positive, motile at 25°C, beta-hemolytic, CAMP positive, and hippurate positive. A positive hippurate test is indicated by a deep blue color within 30 minutes.
• S. agalactiae organisms are catalase-negative, Gram-positive cocci that are identified by having a characteristic narrow zone of beta-hemolysis and are hippurate positive. A positive hippurate test is indicated by a deep blue color within 30 minutes.
• C. jejuni organisms are curved, Gram-negative rods that are identified by having a positive oxidase and catalase reaction, having no growth aerobically at 35°C, and being hippurate positive. A positive hippurate test is indicated by a deep blue color within 30 minutes.
• G. vaginalis organisms are catalase-negative, Gram-variable rods that are hemolytic on human blood agar and are hippurate positive. A positive hippurate test is indicated by a deep blue color within 30 minutes.

A negative hippurate test is indicated by a faint purple color or no color change within 30 minutes. This result can be seen in other streptococci, enterococci, C. coli, and other bacteria that do not hydrolyze hippurate.

The results of the hippurate hydrolysis test should be interpreted along with other biochemical, immunological, molecular, or mass spectrometry tests to confirm the identification of an organism. The hippurate hydrolysis test is not a definitive test and may have some limitations and sources of error.

The hippurate hydrolysis test is used for the identification and differentiation of various bacterial species based on their ability to hydrolyze hippurate. Some of the main uses of the test are:

• To differentiate Group B streptococci (S. agalactiae) from other beta-hemolytic streptococci (Groups A, C, F, and G) that cannot hydrolyze hippurate. Group B streptococci are important pathogens that cause neonatal sepsis, meningitis, pneumonia, and other infections in humans and animals. The test is also useful for distinguishing bovine Group B streptococci from human Group B streptococci.
• To detect the production of hippuricase enzyme by certain bacteria that can hydrolyze hippurate. Hippuricase is a rare enzyme that is found in only a few bacterial genera, such as Campylobacter, Listeria, Gardnerella, and some Streptococcus species. The presence of hippuricase indicates the metabolic capability of these bacteria to utilize hippurate as a nitrogen source.
• To presumptively identify Listeria monocytogenes, a Gram-positive rod that causes listeriosis, a serious foodborne infection that can affect pregnant women, newborns, immunocompromised individuals, and elderly people. L. monocytogenes is one of the few bacteria that can grow at refrigeration temperatures and can contaminate dairy products, meat products, vegetables, and ready-to-eat foods. L. monocytogenes is positive for hippurate hydrolysis, catalase, motility at 25°C, beta-hemolysis, and CAMP test.
• To presumptively identify Campylobacter jejuni, a curved Gram-negative rod that causes campylobacteriosis, a common diarrheal disease in humans and animals. C. jejuni is transmitted through contaminated food (especially poultry), water, or contact with infected animals. C. jejuni is positive for hippurate hydrolysis, oxidase, catalase, and motility at 42°C. It does not grow aerobically at 35°C.
• To presumptively identify Gardnerella vaginalis, a Gram-variable rod that is associated with bacterial vaginosis (BV), a condition characterized by vaginal discharge, itching, burning, and odor. BV is caused by an imbalance of the normal vaginal flora and can increase the risk of sexually transmitted infections and adverse pregnancy outcomes. G. vaginalis is positive for hippurate hydrolysis and beta-hemolysis on human blood agar. It is negative for catalase and oxidase.

The hippurate hydrolysis test is a simple and rapid method that can provide useful information for the identification of some clinically relevant bacteria. However, it should not be used as a sole criterion for diagnosis and should be confirmed by other tests such as immunological, molecular, or mass spectrometry methods.

• Not all S. agalactiae organisms are beta-hemolytic, and some viridans group streptococci can be hippurate positive; thus, another test must be done on nonhemolytic colonies to confirm the identification.
• A small number of enterococci are beta-hemolytic and may hydrolyze hippurate, but they are pyrrolidonyl-β-naphthylamide (PYR) positive.
• A small percentage of C. jejuni organisms are hippurate negative and must be identified by other methods.
• A negative test does not rule out the identification of G. vaginalis, since the biotypes that cause bacterial vaginosis can be hippurate negative.
• False-positive results can occur if incubation with ninhydrin exceeds 30 min.
• The hippurate reagent deteriorates in about 7 days when kept at 4°C.
• The inoculation of low inoculum might result in false-negative results.
• The test should not be taken as a confirmatory test, and other immunological, molecular, or mass spectrometry testing must be performed to identify an organism accurately.

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