To understand the biochemical process of hydrogen peroxide detoxification by aerobic bacteria through the production of the enzyme catalase.
To describe how catalase production can be determined.
To perform the test.
Some bacteria contain flavoproteins that reduce oxygen (O2), resulting in the production of hydrogen peroxide (H2O2) and, in some cases, an extremely toxic superoxide (O2–). Accumulation of these substances will result in death of the organism as they are powerful oxidizing agents and destroy cellular constituents very rapidly unless they can be enzymatically degraded. These substances are produced when aerobes, facultative anaerobes, and microaerophiles use the aerobic respiratory pathway, in which oxygen is the final electron acceptor, during degradation of carbohydrates for energy production.
A bacterium must be able to protect itself against such O2 products or it will be killed. Many bacteria possess enzymes that afford protection against toxic O2 products. Facultative anaerobes and Obligate aerobes usually contain the enzymes superoxide dismutase, which has the ability to catalyze the destruction of superoxide, and either catalase or peroxidase, which catalyze the destruction of hydrogen peroxide as follows:
The inability of strict anaerobes to synthesize catalase, peroxidase, or superoxide dismutase may explain why oxygen is poisonous to these microorganisms. In the absence of these enzymes, the toxic concentration of H2O2 cannot be degraded when these organisms are cultivated in the presence of oxygen.
Organisms capable of producing catalase rapidly degrade hydrogen peroxide which is a tetramer containing four polypeptide chains, which are usually 500 amino acids long. It also contains four porphyrin heme groups(ie., iron groups) that will allow the enzyme to react with the hydrogen peroxide.
The enzyme catalase is present in most cytochrome-containing aerobic and facultative anaerobic bacteria. Catalase is the enzyme which has one of the highest turnover numbers compared to all other enzymes; one molecule of catalase has the ability to convert millions of molecules of hydrogen peroxide to water and oxygen in each second.
Catalase production and activity can be detected by adding the substrate H2O2 to an appropriately incubated (18- to 24-hour) tryptic soy agar slant culture. Organisms which produce the enzyme break down the hydrogen peroxide, and the resulting O2 production produces bubbles in the reagent drop, indicating a positive test. Organisms lacking the cytochrome system also lack the catalase enzyme and are unable to break down hydrogen peroxide, into O2 and water and are catalase negative.
Catalase activity is very useful in differentiating between groups of bacteria. For example, the morphologically similar Enterococcus (catalase negative) and Staphylococcus (catalase positive) can be differentiated using the catalase test
II) Coagulase Test
To understand the biochemistry of the enzyme coagulase.
To explain how coagulase confer a survival advantage to bacteria that produce this enzyme.
To describe how pathogenic species of Staphylococci can be differentiated from nonpathogenic species.
To perform coagulase test.
Coagulases are enzymes that clot blood plasma by a mechanism that is similar to normal clotting. The coagulase test identifies whether an organism produces this exoenzyme. This enzyme clots the plasma component of blood. The only significant disease causing bacteria of humans that produce coagulase enzyme are Staphylococcus aureus. Thus this enzyme is a good indicator of the pathogenic potential of S. aureus.
In human host, the action of coagulase enzyme produces clotting of the plasma by converting fibrinogen to fibrin in the immediate vicinity of the bacterium as a means of protection by itself. The fibrin meshwork that is formed by this conversion surrounds the bacterial cells or infected tissues, protecting the organism from non-specific host resistance mechanisms such as phagocytosis and the anti staphylococcal activity of normal serum. This enables the bacterium to persist in the presence of a host immune response, which can lead to the establishment of infection. Thus, coagulase is described as a virulence factor( disease- causing factor) of Staphylococcus aureus. Citrate and EDTA are usually added to act as anticoagulants and prevent false-positive results.
Most strains of S.aureus produce one or two types of coagulase; free coagulase and bound coagulase. Free coagulase is an extracellular enzyme which reacts with prothrombin and its derivatives. Bound coagulase is localized on the surface of the cell wall and reacts with α- and β-chains of the plasma fibrinogens to form a coagulate. Free coagulase is an enzyme that is secreted extracellularly and bound coagulase is a cell wall associated protein. Free coagulase can be detected in tube coagulase test and bound coagulase can be detected in slide coagulase test.
Slide coagulase test may be used to screen isolates of S.aureus and tube coagulase may be used for further confirmation. There are seven antigenic types of free coagulase, but only one antigenic type of bound coagulase exists. Free coagulase is always heat labile while bound coagulase is heat stable.
In the test, the sample is added to rabbit plasma and held at 37° C for a specified period of time. Clot formation occurs within 4 hours is interpreted as a positive result and indicative of a virulent Staphylococcus aureus strain. The absence of coagulation after 24 hours of incubation is a negative result, indicative of an avirulent strain.
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