How to Identify Staphylococcus aureus?
Identifying Staphylococcus aureus primarily involves specific laboratory tests that differentiate it from other Staphylococcus species, particularly its distinctive ability to produce the coagulase enzyme. This distinction is crucial for diagnosis and treatment, as S. aureus is a significant human pathogen.
Key Laboratory Methods for Staphylococcus aureus Identification
The identification of Staphylococcus aureus in a laboratory setting typically follows a sequence of tests, moving from broad differentiation to highly specific confirmation.
1. Catalase Test
The first step in differentiating gram-positive cocci is often the catalase test. This test distinguishes Staphylococcus species from Streptococcus species.
- Principle: Staphylococcus bacteria produce the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen.
- Procedure: A drop of hydrogen peroxide is added to a bacterial colony.
- Result: A rapid bubbling indicates a positive result, characteristic of Staphylococcus species, including S. aureus. Streptococcus species are catalase-negative.
2. Coagulase Test: The Definitive Method
Once a bacterium is identified as a Staphylococcus species, the coagulase test becomes the most important differentiator for S. aureus. This test distinguishes S. aureus (coagulase-positive) from coagulase-negative staphylococci (CNS).
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Tube Coagulase Test (TCT):
- This is considered the traditional and definitive method for differentiating S. aureus from coagulase-negative staphylococci (CNS) in the laboratory.
- Principle: The test detects the presence of free coagulase, an enzyme produced by S. aureus that clots rabbit plasma by activating prothrombin to form thrombin. Thrombin then converts fibrinogen to fibrin, leading to clot formation.
- Procedure: A suspension of the bacterial isolate is inoculated into a tube containing rabbit plasma.
- Result: The formation of a clot within 1 to 4 hours (or up to 24 hours for confirmation) indicates a positive result, confirming S. aureus.
- Rapid Detection: A significant advancement is the modification of this test for direct detection of S. aureus from blood cultures. By using the blood culture broth, this modified tube coagulase test can yield results in as little as 2 hours, greatly speeding up identification for critical infections.
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Slide Coagulase Test:
- Principle: This rapid test detects "clumping factor" (bound coagulase) on the bacterial cell wall, which directly agglutinates fibrinogen.
- Procedure: A bacterial colony is mixed with a drop of rabbit plasma on a slide.
- Result: Visible clumping or agglutination within 5-10 seconds indicates a positive result, suggesting S. aureus.
- Limitations: While rapid, a negative slide coagulase test should always be confirmed with a tube coagulase test, as some S. aureus strains may be slide coagulase-negative but tube coagulase-positive.
3. Other Confirmatory and Rapid Methods
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Chromogenic Agar:
- Specialized selective and differential culture media, such as chromogenic S. aureus agar, contain substrates that S. aureus metabolizes, producing specific colony colors (e.g., pink, purple, or blue-green), allowing for presumptive identification within 24 hours.
- These media simplify the screening process by making S. aureus colonies easily distinguishable from other bacteria.
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Molecular Methods:
- Techniques like Polymerase Chain Reaction (PCR) are used for highly accurate and rapid identification by detecting specific S. aureus DNA sequences (e.g., nuc gene, which codes for thermonuclease).
- These methods are particularly valuable for direct detection from clinical specimens and for resolving ambiguous cases.
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Biochemical Tests:
- Mannitol Fermentation: S. aureus typically ferments mannitol, leading to acid production and a color change (yellow) on Mannitol Salt Agar (MSA). Many CNS strains do not ferment mannitol.
- DNase Test: S. aureus produces deoxyribonuclease (DNase), an enzyme that breaks down DNA. A positive test is indicated by a clear zone around the bacterial growth on a DNase agar plate.
Summary of Key Identification Tests
| Test | Principle | S. aureus Result | Purpose |
|---|---|---|---|
| Catalase Test | Production of catalase enzyme | Positive (bubbling) | Differentiates Staphylococcus from Streptococcus. |
| Tube Coagulase Test | Production of free coagulase (clots plasma) | Positive (clot) | Definitive identification of S. aureus from CNS. |
| Slide Coagulase Test | Production of clumping factor (agglutinates plasma) | Positive (clumping) | Rapid, presumptive identification; requires confirmation if negative. |
| Chromogenic Agar | Specific enzyme activity on substrates | Characteristic color | Presumptive identification on primary culture plates. |
| Molecular Methods | Detection of specific S. aureus DNA sequences | Positive | Rapid and highly accurate confirmation, especially from samples. |
| Mannitol Fermentation | Fermentation of mannitol sugar | Positive (acid/yellow) | Aids in differentiating S. aureus on selective media. |
By utilizing a combination of these methods, clinical laboratories can accurately and efficiently identify Staphylococcus aureus, which is vital for effective patient management and infection control.
