DNase Test- Definition, Principle, Procedure, Result, Uses

Deoxyribonuclease (DNase) is an enzyme that can break down deoxyribonucleic acid (DNA), the genetic material of living organisms. DNase can cut DNA into smaller fragments by breaking the bonds between the nucleotides.

Some bacteria can produce DNase to help them utilize DNA as a source of nutrients or as a way to evade the immune system of the host. DNase can degrade the DNA of other organisms or cells, releasing nucleotides that can be taken up by the bacteria and used for their own growth and replication. DNase can also destroy the DNA-based traps that some immune cells use to capture and kill bacteria.

DNase test is a biochemical test that can detect the presence or absence of DNase in a bacterial culture. The test is based on the observation that DNase can change the appearance or color of a medium that contains DNA. The medium can be solid or liquid, and it can have different indicators that react with DNA or its degradation products.

DNase test is useful for identifying and differentiating some bacterial species, especially those that are pathogenic or cause infections in humans or animals. For example, DNase test can help distinguish Staphylococcus aureus, a common cause of skin and wound infections, from other staphylococci that are usually harmless. DNase test can also help identify some gram-negative bacteria, such as Serratia, Moraxella, Aeromonas, and Vibrio, that produce DNase.

In this article, we will discuss the objectives, principle, procedure, result interpretation, uses, and limitations of DNase test in detail. We will also provide some examples of microorganisms that are tested by this method and how they are differentiated by their DNase production.

The main objective of the DNase test is to determine the ability of an organism to produce the DNase enzyme. This enzyme is responsible for breaking down DNA into smaller fragments that can be utilized by the bacteria as a source of nutrients or as a virulence factor. The DNase test can also help in the identification and differentiation of some bacterial species based on their DNase production. Some of the specific objectives of the DNase test are:

• To differentiate and identify S. aureus from other Staphylococcal species. S. aureus is a common cause of skin and soft tissue infections, food poisoning, and endocarditis. It is also known to produce a heat-stable DNase enzyme that can degrade the host`s DNA and evade the immune system. The DNase test can help in confirming the presence of S. aureus in a clinical specimen or culture.
• To identify Stenotrophomonas maltophilia, a Gram-negative bacillus that is resistant to many antibiotics and can cause opportunistic infections in immunocompromised patients. S. maltophilia produces a DNase enzyme that can be detected by the DNase test.
• To identify Moraxella catarrhalis, a Gram-negative diplococcus that can cause respiratory tract infections, especially in children and elderly people. M. catarrhalis also produces a DNase enzyme that can be detected by the DNase test.
• To identify Serratia spp., a group of Gram-negative bacilli that belong to the Enterobacteriaceae family and can cause various infections, such as urinary tract infections, wound infections, and septicemia. Serratia spp. produce a DNase enzyme that can differentiate them from other Enterobacteriaceae, such as Klebsiella spp. and Enterobacter spp.
• To identify Aeromonas spp. and Vibrio cholerae, two groups of Gram-negative rods that are oxidase-positive and indole-positive and can cause gastrointestinal infections, such as diarrhea and dysentery. Aeromonas spp. and V. cholerae produce a DNase enzyme that can differentiate them from Plesiomonas shigelloides, another Gram-negative rod that is oxidase-positive and indole-positive but does not produce DNase.

The DNase test is a simple, rapid, and inexpensive method that can provide useful information for the diagnosis and treatment of bacterial infections caused by DNase-producing organisms.

DNases are enzymes that hydrolyze DNA and release free nucleotides and phosphate. The deoxyribonuclease enzyme produced by bacteria is extracellular endonucleases that break down DNA, yielding a high concentration of oligonucleotides. The media used to detect these enzymes can be made by using various indicators (toluidine blue or methyl green) or no indicators to detect the hydrolysis of DNA.

• The first method is performed with no indicator. The hydrolysis of DNA is indicated by the clearing of the agar after the addition of HCl (the oligonucleotides dissolve in acid causing a clear zone, but DNA salts are insoluble).
• When methyl green indicator is added, DNA combines with the methyl green to produce a green color. The complex is released when the DNA is hydrolyzed, and the freed methyl green is colorless at pH 7.5.
• When toluidine blue O (TBO) is added, a complex is formed with the DNA, which changes its structure when DNA is hydrolyzed, resulting in a bright pink color.

The media with dyes can inhibit some microbial growth. Using a heavy inoculum prevents this problem and makes the test more rapid, as it detects preformed enzymes.

Staphylococcus aureus possesses a heat-stable enzyme, a thermonuclease. To detect this enzyme, first, the organisms are destroyed by heat, and then the free DNase reacts with the medium.

The DNase test can be used to test various microorganisms that produce the DNase enzyme as a part of their metabolism or virulence. Some of the common microorganisms that are tested by the DNase test are:

• Gram-negative bacilli that are suggestive of Stenotrophomonas maltophilia (positive) and are colistin or polymyxin B resistant to separate from Burkholderia cepacia (negative). These bacteria are opportunistic pathogens that can cause infections in immunocompromised patients and cystic fibrosis patients. They can degrade DNA in the respiratory tract and evade the host immune system.
• Gram-negative diplococci that are presumptive for Moraxella catarrhalis (positive). This bacterium is a common cause of otitis media, sinusitis, bronchitis, and pneumonia in children and adults. It can produce DNase to break down the extracellular DNA in the biofilms formed by other bacteria and enhance its colonization and persistence.
• Gram-positive cocci that are presumptive for S. aureus (positive) and are difficult to separate from other closely related Staphylococcal species and have a questionable coagulase reaction. S. aureus is a major cause of skin and soft tissue infections, endocarditis, osteomyelitis, septicemia, and toxic shock syndrome. It can produce DNase to degrade the neutrophil extracellular traps (NETs) that are composed of DNA and antimicrobial proteins and trap the bacteria.
• Enterobacteriaceae to identify Serratia (positive) and separate them from Klebsiella spp. and Enterobacter spp. Serratia spp. are opportunistic pathogens that can cause urinary tract infections, wound infections, endocarditis, and septicemia. They can produce DNase to degrade the DNA in the pus and facilitate their spread. Serratia fonticola is the only Serratia sp. that is negative for DNase.
• Oxidase-positive, indole-positive, Gram-negative rods in order to separate Aeromonas and Vibrio cholerae (DNase positive) from Plesiomonas shigelloides (DNase negative). Aeromonas spp. and V. cholerae are waterborne pathogens that can cause gastroenteritis, diarrhea, dysentery, septicemia, and wound infections. They can produce DNase to degrade the mucosal DNA and invade the intestinal epithelium.

The DNase test can also be used to test other microorganisms that may produce the DNase enzyme under certain conditions or with certain substrates. However, these microorganisms are not routinely tested by the DNase test as they may have other more reliable tests for their identification and differentiation.

Media Used

DNase Test Agar Base is used for testing the production of the DNase enzyme. The medium can also be added with indicators like methyl green or toluidine blue O.

The composition of the DNase Test Agar Base is given below:

The final pH of the medium is adjusted to 7.4 ± 0.2 at 25°C.

Reagent Used

1 N HCl solution is used to detect the hydrolysis of DNA in the medium without an indicator.

Supplies Used

• Sterile sticks, needles, or inoculating loops
• Pasteur pipettes or drinking straws
• Boiling heat block
• Incubators at 35 and 30°C

The procedure of DNase test can vary depending on whether the medium has an indicator or not. The following steps describe the general procedure of DNase test:

A. Preparation of the media

1. In a beaker, 42 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of pure distilled or deionized water.
2. The suspension is heated with agitation to bring it to boil in order to dissolve the medium completely.
3. The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes.
4. Once the autoclaving process is complete, the beaker is taken out and cooled to a temperature of about 40-45°C.
5. The media is then poured into sterile Petri plates under sterile conditions.
6. If indicators are to be added, 0.1 gram of toluidine O or methyl green is added to the medium before sterilizing.

B. DNase Test

Without indicator

1. The agar plates are inoculated with the test organism from an 18-hour culture with a sterile inoculating loop or needle.
2. The plates are incubated at 35-37°C for 24 hours.
3. The incubated agar plates are flooded with a 1N HCl solution, and the excess acid is tipped off.
4. Some time is allowed for the reagent to be absorbed into the plates.
5. The plates are observed for a clear zone around the colonies within 5 minutes.

With indicator

1. The agar plates with an indicator are inoculated with the test organism from an 18-hour culture with a sterile inoculating loop or needle.
2. The plates are incubated for 24 hours at 35-37°C.
3. The plates are then observed for the change in color of the indicator.

The result of the DNase test depends on the presence or absence of a clear zone around the colonies after the addition of HCl or the change in color of the indicator in the medium. The following table summarizes the interpretation of the DNase test for different microorganisms:

A positive DNase test indicates that the organism produces the DNase enzyme and can hydrolyze DNA into smaller fragments. A negative DNase test indicates that the organism does not produce the DNase enzyme and cannot hydrolyze DNA.

The following images show some examples of positive and negative DNase tests:

• DNase test is used to detect the ability of an organism to produce the DNase enzyme. This enzyme can break down DNA into smaller units that can be used by the bacteria as a source of nutrients or as a virulence factor.
• DNase test can also be used to differentiate S. aureus from other Staphylococcal species. S. aureus is a common cause of skin and soft tissue infections, food poisoning, and endocarditis. It can produce a thermostable DNase that can degrade the host`s DNA and evade the immune system. Other Staphylococcal species are usually negative for DNase test.
• DNase test can also be used to identify some Gram-negative bacteria that produce DNase, such as Stenotrophomonas maltophilia, Moraxella catarrhalis, Aeromonas spp., and Vibrio cholerae. These bacteria can cause various infections in humans, such as respiratory tract infections, wound infections, gastroenteritis, and cholera. DNase test can help to distinguish them from other similar bacteria that are negative for DNase, such as Burkholderia cepacia, Plesiomonas shigelloides, and Klebsiella spp.
• DNase test can also be used to differentiate Serratia spp. from other Enterobacteriaceae. Serratia spp. are opportunistic pathogens that can cause urinary tract infections, pneumonia, septicemia, and endocarditis. They can produce a red pigment called prodigiosin and a DNase enzyme that can degrade DNA. Most other Enterobacteriaceae are negative for DNase test.

• Some microorganisms may produce DNase only under certain conditions, such as temperature, pH, or oxygen availability. Therefore, the test results may vary depending on the environmental factors.
• Some microorganisms may produce DNase that is not detected by the indicators used in the test. For example, some strains of S. aureus produce a thermonuclease that is heat-stable and requires heating to demonstrate a positive test.
• Some microorganisms may produce other enzymes that can interfere with the DNase test. For example, some strains of Pseudomonas aeruginosa produce an alkaline phosphatase that can hydrolyze the methyl green indicator and cause a false-positive result.
• Some microorganisms may not grow well on the DNase test agar base, especially if it contains dyes that can inhibit their growth. For example, some strains of Moraxella and Gram-positive cocci may require a low inoculum to avoid a false-negative result.
• The DNase test should be performed in conjunction with other biochemical and serological tests to confirm the identification of the microorganisms. A single test result may not be sufficient to differentiate between closely related species or subspecies.

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