Staphylococcus Aureus

Staphylococcus aureus is a widely spread microorganism on the body surfaces (especially on the skin and the mucous membranes such as nose) of human beings and warm blooded animals. Scientifically, this organism is classified as follows kingdom, bacteria phylum, firm cutes class, bacilli order, bacillales family, staphylococcaceae genus, staphylococcus and species, staphylococci. The organism has the ability to produce toxins and cause food poisoning as well as making direct infections that causes diseases such as septicemia and boils in humans. Staphylococcal food poisoning results after ingesting enterotoxins together with food in case of improper storage though direct contamination (for instance in milk) may be responsible. Besides, the microorganism can infect animals and cause inflammation of the udder. This leads to mastitis disease in dairy animals. The cells of Staphylococcus aureus are round in shape and appear as grape-like clusters under microscopic observation. Its colonies have golden-yellow color.

Staphylococcus aureus is a facultative anaerobe, gram-positive, catalase positive, and coagulase positive microorganism. Besides, it has a rigid cell wall made of peptidoglycan molecules (at least 90 thick) which gives shape to the cells besides enhancing their protection. The microorganism is usually distinguished from other staphylococci species because of its ability to produce an enzymatic compound called coagulase. This attribute makes the organism to be highly virulent and forms the basis of Staphylococcus aureus classification. Although it is coagulase-positive, some strains do not produce coagulase. Hence, catalase test is done to identify Staphylococcus aureus as it is capable of producing catalase enzyme which hydrolyzes hydrogen peroxide into water and oxygen. Staphylococcus aureus thrives well in the presence of oxygen to produce enterotoxins though it also survives in anaerobic environments. However, its growth is inhibited in environments with 80 CO2. The microorganism requires an optimum temperature of 370c but it can also tolerate up to 440 c in case of high NaCL concentrations. The optimum PH for supporting its growth is at least 7.3 but maximum PH may rise up to 9.3. On the other hand, growth may be inhibited by addition 0.1 acetic acid which modifies PH value to 5.1.

Staphylococcus aureus is in particular resistant to dry conditions and is capable of growing and producing enterotoxins at low water activity values for instance 0.85. This significantly enables it to tolerate adverse conditions thus making the microorganism a good competitor. In addition, it is also capable of tolerating high concentrations NaCl for instance 25 although the levels may be as low as 10. For Staphylococcus aureus to produce toxins, it requires a PH range between 5.3 and 7.0. The minimum PH for toxin production is 4.8 and can also produce toxins at PH 9. In addition, optimum temperatures for toxin production fall between 35 0c and 400c. Besides, the least temperature for toxin production is 10oc whereas the highest temperature value goes up to 450c for toxin production. Staphylococcus aureus requires optimum water activity value of 0.9 to produce toxins. The minimum water activity value for toxin production is 0.86. High toxin levels are produced in aerobic environments. This microorganism has improved heat resistance especially in dry foods with high fat and salt content. Staphylococcus aureus is also capable of surviving frozen conditions and produce highly heat resistant toxins. Extremely high and low PH conditions are known to destroy vegetative cells of Staphylococcus aureus effectively although this may depend on the type of acid. During preservation, reduced PH, water activity, modified osmotic balance, high CO2 concentration, and use of preservatives such as benzoate and sorbate salts can be used to inhibit Staphylococcus aureus (Ministry of Health, 2000).

Microbiological Tests
Thyioglycollate broth is a media used to culture and isolate both obligate and facultative anaerobic microorganisms as well as testing sterility. Thyioglycollate and cystine in the broth, acts as reducing agents necessary for creating anaerobiosis for fastidious anaerobes. The broth has high viscosity that prohibits fast oxygen uptake. Culture medium is inoculated at the bottom of the broth and incubated for several days at 35-37oc. Gram staining is a test used to differentiate between gram-positive and gram-negative bacteria. The most distinct attribute tested in this process is the composition of bacterial cell wall. Gram positive microorganisms have thick and compact cell wall that forms the outermost layer containing about 90 peptidoglycan molecule. Mannitol salt agar is normally used as either selective or differential media for gram-positive cocci. This media selects salt tolerant and fermentative microorganisms especially Staphylococcus aureus. Lactose and sucrose tests are performed to determine whether they are fermented by microorganisms in the media. This is done using Durham tubes and phenol red reagent. Deviation of color from red and production of gas bubbles are the attributes of interest.

Starch utilization test is done by inoculating the culture on a starch agar and using iodine to establish whether microorganisms present are capable of hydrolyzing the starch present by observing color change. Glucose fermentation test is done by using iron agar slant. In case fermentation takes place, fissures are formed on the surface of the media. The Indole test is usually used to differentiate between gram-positive and gram-negative organisms. The gram-negative bacteria hydrolyze tryptophan in the SIM medium. The test is done by adding three to four drops of Kovacs reagent into the culture and then observing for appearance of a red ring.

Methyl Red test is used to differentiate between those organisms which are able to reduce the PH of the inoculating media by overcoming the phosphate buffer present. After inoculation for about five to six days, methyl red is added and color observation is done. Vogues-Proskauer test tests for microorganisms which are capable of fermenting and converting acidic products to acetone concurrently. During the test, Vogues-Proskauer reagent -KOH and -Napththylamine- converts acetone into di-acetyl. It then reacts with guanidine nuclei to give a red coloration/

The Gelatin hydrolysis is done to test whether microorganisms in a cultural medium are capable of producing gelatinases from gelatin nutrient broth. Gelatinases are enzymes of protein nature. After incubation at 35-37oc, test tubes containing gelatin are put on ice. The gelatin is observed either for solidification or liquefaction.

Deoxyribonuclease (DNAse) enzyme activity test is used to differentiate between Staphylococcus aureus and Staphylococcus epidermis microorganisms. The culture is inoculated on DNase test agar plate which contains methyl green. Production of the enzyme is then established by looking for a pink halo around the colonies. Biochemical reactions include tests such as Coagulase and Catalase. Coagulase test identifies the Staphylococcus aureus.

This microorganism produces coagulase enzyme which renders blood plasma to clot within 24 hours of incubation at 35 oc to 37 oc. As earlier mentioned, the catalase test is used to establish the production of catalase enzyme. It properly differentiates Staphylococcus aureus from among other gram positive bacteria because of the microorganisms ability to produce catalase enzyme. Hydrogen peroxide is added to the culture medium and bubbles are observed.

Results and Conclusions
Thioglycollate Broth specifically allows only the growth of anaerobic bacteria at the lower part of the media. Aerobic microorganisms are eliminated. Staphylococcus aureus colonies demonstrate good growth and are observable due to their golden color. This differentiates them from Staphylococcus epidermitis which appear as white colonies.

Gram staining identifies Staphylococcus aureus as a gram-positive microorganism. Because there a number of gram-positive cocci such as Staphylococcus epidermitis and Staphylococcus saprophyticus, they are well differentiated using other tests as later explained.

Staphylococcus aureus ferments Mannitol Salt Agar, which contains 7.5 NaCl, into acid. This is detected by measuring the PH using an indicator and change of color from red to yellow indicates positive results. This test distinguishes Staphylococcus aureus from other gram-positive cocci for instance Staphylococcus epidermidis. The fermentation of sucrose and lactose gives positive results if the phenol-red changes to yellow color. However, this may be confused with other gram-negative which also gives positive color results such as Citrobacter fruendii. Furthermore, fermentation is evidenced by the presence of a gas bubble on the Durham tube. In both fermentations, Staphylococcus aureus do not produce any gas. In the starch test, if an organism is able to hydrolyze starch in the media, a yellow halo on the culture indicates positive results for hydrolysis. Staphylococcus aureus gives negative results in this test.

In the Iron agar test, which tests for glucose and lactose fermentation, positive results are displayed by the presence of cracks on the medium. This establishes gas production due to fermentation. Microbial fermentation changes the color from red to yellow. Further, reduction of thiosulfate in the media results in formation of a black precipitate. Usually, Staphylococcus aureus is able to give positive color results without successfully breaking down thiosulfate in the medium and producing hydrogen peroxide gas.

The Indole test gives positive results upon tryptophan hydrolysis. This is evident if a red ring appears on the culture after adding a few drops of Kovacs reagent. It is usually done to differentiate gram-negative from gram positive microorganisms.

Methyl red test reveals positive results for gram negative bacteria by observing a red color after adding methyl red reagent into the medium. Negative results are noted if yellow andor orange color appears. This test eliminates Staphylococcus aureus by giving positive results for the presence of enteric microorganisms.

Positive Vogues-Proskauer test results are evidenced by observing a red coloration on the medium whereas no color change indicates negative results. Fermentative organisms produce acetone which is further converted into diacetyl and reacts with guanidine to give positive results. Mainly, this test is done to differentiate gram negative organisms.

Gelatin test gives positive results by displaying agar liquefaction. This proofs that gelatinase enzyme was produced by the bacteria during inoculation. Negative results are evidenced by gelatin solidification. Staphylococcus aureus usually gives negative results for this test. DNAse test has positive results if a pink colored halo is produced on a clear growth on the medium. This test eliminates gram-negative bacteria to differentiate Staphylococcus aureus and Staphylococcus epidermis. Gram-positive cocci organisms are capable of producing DNAse enzyme. The coagulase test shows positive results if the microorganism capable of coagulating or solidifying plasma sample. It separates Staphylococcus aureus from coagulase negative microorganisms such as Staphylococcus epidermitis and Staphylococcus saprophyticus.

The Catalase test gives positive results by producing gas which is evidenced by bubble production. Absence of bubble production gives negative results. This test differentiates Staphylococcus aureus from aerobic gram-positive microorganisms. It also eliminates catalase negative microorganisms for instance enterococci and streptococci. In summary, identifying and differentiating a particular microorganism cannot be done by only a single test. However, specific attributes are significantly outstanding for instance in the case of Staphylococcus aureus. Gram staining test acts as the initial step to differentiate between gram-positive and gram-negative bacteria.  Afterwards, specific tests for instance, coagulase, starch, and Catalase further differentiate between Staphylococcus aureus and other gram positive cocci such as Staphylococcus epidermitis and Staphylococcus saprophyticus which are the most common gram-positive cocci. Coagulase test specifically differentiates Staphylococcus aureus from the others as it is the only coagulase positive microorganism.


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