Staphylococcus aureus is a bacterium known for its ability to cause a wide range of illnesses, from superficial skin infections to life-threatening conditions like sepsis and endocarditis. Its increasing resistance to antibiotics makes rapid and accurate laboratory identification necessary for effective clinical management. Quick identification allows healthcare providers to initiate appropriate treatment, which directly impacts patient outcomes. Identification involves a systematic pipeline of techniques, beginning with growth on specialized media and progressing through specific biochemical and advanced molecular testing. This approach ensures S. aureus is correctly distinguished from other staphylococcal species.
Culturing on Selective and Differential Media
Initial isolation and presumptive identification of S. aureus begins with culturing the specimen on specialized media. These media are selective, allowing only certain organisms to grow, and differential, enabling visual distinction between species. The primary medium used is Mannitol Salt Agar (MSA).
MSA is selective due to its high concentration of sodium chloride, typically 7.5%. This salt level inhibits most bacteria, including Gram-negative organisms, but allows halotolerant species, such as those belonging to the genus Staphylococcus, to flourish.
The differential component is the sugar alcohol mannitol, combined with the pH indicator phenol red. S. aureus ferments mannitol, producing acidic byproducts. Phenol red detects this change, turning the surrounding medium and the S. aureus colonies from pinkish-red to yellow.
In contrast, Coagulase-Negative Staphylococci (CoNS) grow well in the high-salt environment but cannot ferment mannitol. These organisms produce small, typically red or pink colonies with no color change in the surrounding medium. The visual distinction between a yellow colony and a red colony on MSA provides a strong, presumptive indication of S. aureus presence, leading to confirmatory tests.
The Coagulase Test: The Definitive Biochemical Marker
The coagulase test is the definitive method for confirming S. aureus identity, differentiating it from Coagulase-Negative Staphylococci. This test detects the coagulase enzyme, a protein that causes the clotting of blood plasma. The unique ability of S. aureus to produce this enzyme makes the test a gold standard in the clinical laboratory.
S. aureus produces two forms of coagulase: bound and free. Bound coagulase, also known as clumping factor, is a cell wall protein that binds directly to fibrinogen in the plasma. This binding causes bacterial cells to clump into visible aggregates, detected by the rapid slide coagulase test. The slide test is a screening method because not all S. aureus strains produce detectable bound coagulase.
Free coagulase is an extracellular enzyme that reacts with a plasma component called Coagulase Reacting Factor (CRF), forming a complex that acts like thrombin. This complex converts soluble fibrinogen into an insoluble fibrin clot, resulting in visible gelation. This reaction is detected by the tube coagulase test, which involves mixing the suspected organism with plasma and incubating it at 37°C.
A positive tube test result is the formation of a solid clot, observed within one to four hours of incubation. Since the tube test detects the free enzyme, produced by nearly all pathogenic S. aureus strains, it is considered the definitive confirmatory test. Any negative slide test result must be confirmed with the tube test to prevent misidentification.
Supplementary Enzymatic and Resistance Testing
Supplementary tests are used to further characterize S. aureus and distinguish it from other Gram-positive cocci. The Catalase test quickly distinguishes the genus Staphylococcus (catalase-positive) from Streptococcus (catalase-negative). This test confirms the catalase enzyme, which breaks down hydrogen peroxide into water and oxygen, producing immediate bubbles.
The DNase test checks for the organism’s ability to produce deoxyribonuclease, an enzyme that breaks down DNA. S. aureus is one of the few staphylococci that produces thermostable DNase, which correlates strongly with its pathogenic potential. A positive DNase test, indicated by a clear zone around the bacterial growth on a DNase agar plate, provides additional evidence for identification.
Screening for methicillin resistance defines Methicillin-Resistant S. aureus (MRSA). Traditional screening involves culturing the isolate on specialized media, such as chromogenic agar or Mueller-Hinton agar supplemented with NaCl and oxacillin or cefoxitin. The resistance gene mecA or its variant mecC allows the organism to produce an altered penicillin-binding protein (PBP2a), which is the biochemical mechanism of resistance. These phenotypic tests determine the clinical relevance of the isolated organism.
Modern and Automated Identification Systems
Modern clinical laboratories rely on rapid, high-throughput systems to reduce identification time. Automated systems, such as the Vitek or Phoenix platforms, use pre-filled cards containing numerous biochemical substrates to perform multiple tests simultaneously. These instruments read colorimetric or turbidity changes, compare results to a database, and provide definitive species identification and antimicrobial susceptibility profiles.
For the fastest results, molecular methods based on Nucleic Acid Amplification Tests (NAATs), particularly Polymerase Chain Reaction (PCR), are standard practice. PCR assays directly detect species-specific genes, such as nuc or sa442, which are exclusive to S. aureus. This technique offers high sensitivity and specificity, often identifying the organism directly from a patient sample in one to three hours.
PCR is also used to detect resistance genes, such as mecA, confirming the presence of MRSA. The ability to simultaneously identify the species and its resistance profile is a major advantage of molecular methods. This allows clinicians to begin targeted therapy sooner than with traditional culture-based methods, significantly improving the overall turnaround time for diagnosis.