Staphylococcus aureus is a coagulase-positive staphylococcus which constitute of the S aureus species
Abstract
Staphylococcus aureus is a Gram-positive bacterium that is identified with stain purple by the Gram stain. They are cocci-shaped arranged in the form of clusters. This shape can be described as being “grape-like” when put in media containing up to 10% salt, and colonies which are yellow or golden. To identify S aureus requires an analysis of biotype. They are often tolerant of salt and hemolytic. It colonizes the axillae and nasal passage. The S aureus shows various factors that inhibit phagocytosis and surface the proteins which promote tissue colonization in the host. It has toxins that are damaging to tissues leading to symptoms of a disease. The major mechanisms for fighting S aureus infections is phagocytosis. The Antibodies produced neutralize toxins while promoting opsonization. The infections are treated using the penicillinase-resistant β-lactams. The test performed involve colonies. The staff and patients carrying these procedures should be isolated MRSA strains particularly.
Introduction
Staphylococcus aureus is a coagulase-positive staphylococcus which constitute of the S aureus species. S aureus has a variety of polysaccharides and extracellular proteins that are correlated in virulence. Virulence is the result of different effects that are expressed during the infection. Antibodies neutralize S aureus enzymes and toxins that cannot be handled by vaccines. The organisms are associated with the infections of indwelling devices. Such conditions are treated through surgical procedures. There are different tests that can be conducted to identify the S aureus bacteria infections.
- aureus has DNA – rRNA hybridization and analysis of oligonucleotide 16S rRNA that has demonstrated how Staphylococci. At the genus level, show a coherent group. The S aureus is among the 30 species recognized from biochemical analysis, particularly the hybridization of DNA-DNA. They have a diameter of 0.5 – 1.0 μm and usually occurs in short chains. This configuration distinguishes the S. aureus from Streptococci that have long chains. The culture grown in a broth should be observed because they appear in clumps.
The methods used to identify these bacteria are thermostable Deoxyribonuclease, hemolysin, and coagulase, and the test for clumping factor. The tests involve procedures of gram stain that shows the gram-positive in pairs of grape-like structures. The catalase test and the expected results to be catalase-positive. The coagulase-positive identifies S aureus in the Coagulase test. Another test used in the procedure is a DNAse test that should be positive. Application of heat-stable endonuclease tests S aureus as positive.
Materials and Methods
- Catalase Test
This test is essential to distinguish streptococci (negative to the catalase) from Staphylococci that test positive in the catalase. The test is conducted by flooding broth culture or agar slant with 3% drops of hydrogen peroxide. The positive catalase will bubble immediately. This test should no be carried on blood agar as blood usually produces bubbles. Several drops of hydrogen peroxide 3% are applied in the media. The enzyme in the catalase neutralizes the effects of hydrogen peroxide. It proceeds with a breakdown of hydrogen peroxide to water and oxygen
(2H2O2 + Catalase → 2H2O + O2).
This is evident by rapid bubbles formation from the reaction. There are different methods and application variations of the catalase test.
Slide (drop) method
Inside a petri dish, place a microscope slide, and the cover of the petri dish should be available. The petri dish is recommended to avoid viable bacterial cells. Using a wooden applicator or inoculating loop, collect a sample of organisms from an isolated colony of 18 to 24 hours isolation. Place the sample in the microscope slide. Ensure you do not pick agar in the process. Place one drop of 3% hydrogen peroxide onto the microscope slide using a dropper. Avoid mixing the substances in the slide. Cover the petri dish immediately to limit the entry of aerosol. Observe for the formation of bubbles immediately. It is clearer when observed under a dark background to enhance readability. When effervescence occurs immediately, it is evident that the results are positive. The control experiment is done using organisms that are negative and positive for the catalase.
FIG. 1. Test results for slide catalase. The top shows a positive S. aureus reaction, while the bottom shows a negative reaction for S. pyogenes.
Tube method
Add 4 drops of 3% hydrogen peroxide to a test tube. Using a wooden applicator, collect a small amount of the organisms from an isolated colony of 18 to 24 hours. Place the sample into the test tube. Do not pick any agar. Place the test tube in a dark background for clear visibility and observe for the formation of bubbles immediately. This reaction should occur at the deep end of the wooden applicator.
FIG. 2 A. the positive results for S. aureus
FIG. 2 B. the negative results produced by the S. pyogenes.
Tube slant method
Add 1.0 ml to 3% hydrogen peroxide into the inoculated pure culture grown on agar slant. Place the tube in a dark background and observe for effervescence.
FIG 3.A shows positive effects for S. aureus
FIG. 3B. Negative results for S. pyogenes.
- The test for Clumping Factor A
The S. aureus shows a clumping factor A (ClfA) by the protein surface binding to fibrinogen through molecular interactions. The pathogen S. aureus binds to the matrix of extracellular host using different proteins anchored to the cell. The clumping factor A is an important factor used by the S. aureus in infections. The ClfA enhances the attachment of bacteria to the fibrinogen that allows the bacteria to form a biofilm after colonizing. The vaccination using ClfA to protect against infections is included in the trials of a vaccine against S. aureus. ClfA is an essential factor as it enhances the adhesion of blood proteins during stress.
To investigate the presence of Clumping factor A and fibrinogen proteins, the use of S. aureus SH1000 clfA clfB fnbA-fnbB were used. The strain transformed, expressing the clfA gene in the test. The fibrinogen was immobilized on the substrates contain solid by using N-hydroxy succinimide surface. The images observed in the microscope confirmed ClfA positive cells in large amounts on the surface coated with fibrinogen substrates. However, no adhesion was noted on the ClfA negative cells. This result indicates that the ClfA is represented and expressed in fibrinogen-binding proteins found on the surface of strains.
Another test is using the single-cell force spectroscopy. This analyzed the Clumping factor A and fibrinogen binding forces at the cell level. Single bacteria are attached to the colloidal cantilevers. The force between the fibrinogen substrates and the cell probes is measured. The figure 1.a below shows the lengths of rapture and adhesion forces obtained from the S. aureus representatives.
F3. large
The adhesion forces spread across a large range. This suggests that various molecular bonds in the process were probed. The forces of adhesion measured were larger than those measured for other species of Staphylococcus. This is clear that the fibrinogen forces are very strong. The large forces were removed by the negative ClfA cells. This reflects that the interactions were between specific ClfA and fibrinogen interactions.
Discussion
The ClfA is a factor of S. aureus that binds to the fibrinogen coated substrates. The adhesion factor favors the adhesive bacterial under extreme physical stress. The ClfA is a sensitive molecular force that activates the adhesion of S, aureus, and fibrinogen. The results emphasize the importance of mechanobiology in regulating the adhesion of S. aureus that contributes to strategic anti-adhesion using inhibitors. We refer to the theory that “unbinding forces between ligands and receptors increases with the rate at which the force is applied.” This theory helps us understand the strength of the force induced in the bond between the ClfA and fibrinogen. The strength and weakness of bonds usually differ by the magnitude of the intermediate force. Strong bonds are not the result of the rupture of various multiple weak bonds.
- Gram stain test
This procedure uses the blood culture bottles. The identification of S. aureus from blood cultures that test positive gives essential therapeutic and clinical information. The criteria of using characteristics of Gram stain to distinguish S. aureus and other species of Staphylococci is done by isolating in Bac T/ALERT blood culture bottles. This has an overall of 98% specificity and 89% sensitivity. The identification of positive blood cultures is becoming simpler from the monitored systems. Once a culture is detected positive, it relies on Gram stain broth. The Gram-positive cocci usually appear in clusters suggesting the presence of S. aureus. However, the differentiation of the S. aureus from negative staphylococci is carried in 18 to 24 hours after the organism is cultured. This process of distinction is essential for virulence as high coagulant frequencies that test negative are usually isolated as being contaminants. There are morphological characteristics that distinguish the S. aureus from the negative staphylococci seen in Gram stains in cultured blood bottles from the Bac/ALERT tests. The features are the size of cells, number of cells in a cluster, and the type of blood culture, whether aerobic or non-aerobic.
Some criteria were used to distinguish S. aureus from the negative staphylococci in Gram stain from the BacT/ALERT blood cultures. The first bottle contained an anaerobic culture. The S. aureus was selected for organisms below 1 μm. This was characterized by irregular clusters in large numbers of between 200 and 300 cells. In the same blood culture bottle, they used the negative coagulase staphylococci above 1 μm. The results were a small or tetrads of small clusters of up to 16 cells. Another bottle type tested consisted of aerobic cultures. The S. aureus tested were above 1 μm. This resulted in tight clusters where the individuals could not be differentiated. The negative coagulase staphylococci were exposed to similar conditions. The cells used were below 1 μm in size. The small tetrads clusters up to about 16 cells.
Discussion
The ability to identify S. aureus is affected by the change of appropriate antibiotic regimes. This test showed that the bacteria S. aureus growing in a blood culture BacT/ALERT is identified through direct characteristics and degree of accuracy. The S. aureus is identified in anaerobic cultures compared to aerobic. This may be caused by charcoal in the aerobic bottles, which made the identification of Gram-positive difficult. However, this method is highly effective compared to the immunological tests, thermostable endonuclease detection, and the two-hour coagulase tube test. This method is also not as reactive as the polymerase chain. This method is more effective because it’s cheap, and the detection of results is immediate. The criteria for identifying S. aureus using the BacT/ ALERT blood cultures can not be applied for BACTEC blood cultures. When the Gram-positive bacteria resembling S. aureus are identified during the test BacT/ALERT bottles, the person under observation likely has S. aureus bacteremia.
- Deoxyribonuclease test (DNase)
The DNase test is applied in determining the organism’s ability to hydrolyze DNA as it utilizes the energy and carbon sources for growth. The S. aureus can produce enzymes that breakdown the DNA. This test is used to differentiate the S. aureus that produces the deoxyribonuclease enzyme. The S. aureus possesses heat enzyme, thermonuclear. The organisms are heated and destroyed, and the free DNase reacts with the set medium to detect the enzyme. The DNase agar hydrolysis DNA that is observed through clearing agar by adding HCL. The oligonucleotides dissolve in HCL acid, but the DNA is insoluble. The medium use is pale green because of the DNA indicator that is methyl green.
If the organism grows in medium, they produce Deoxyribonuclease, which break their own DNA into fragments. After the DNA is broken down, it binds to methyl green, and the color fades. The colony now is surrounded by the colorless zone. The expected results are the DNA is hydrolyzed, methyl green released, and turns the medium colorless in the test organism. The test is negative when the degradation of the medium is green throughout.
This test is used to differentiate S. aureus when the DNase turns positive from the other species of staphylococci, which don’t produce similar enzymes. The test is useful when the plasma is not available to perform the coagulase test. However, this test is faced by various limitations where the MRSA strains usually do not produce positive test results for DNase. 1N HCL of bactericidal for the staphylococci organisms is applied and should be observed within 5 minutes, otherwise it cannot continue through re-incubation.
Overall, the antibiotics used for treatment f staphylococci infections include nafcilin, cephalospirin, or vancomycin. The major test used is the coagulase reaction that divides the Staphylococcus into coagulase-positive or negative species. The bacteria is spread by having contact with infected persons, sharing contaminated objects, and inhaling droplets from sneezing or coughing. The skin infections are also common that can be spread to infect the skin and bloodstream in distant organs. They are found on the nose, groin, armpit, the skin, and other areas. Diagnosis is based on the skin appearance or bacterial identification in infected material samples. Ensure you wash hands thoroughly to prevent infection spread.