NEET MDS Lessons
General Microbiology
COMPLEMENT
The complement system primarily serves to fight bacterial infections.
The complement system can be activated by at least three separate pathways.
1) alternative pathway -
- The alternative pathway of complement activation starts with the spontaneous hydroysis of an internal thioester bond in the plasma complement component C3 to result in C3(H2O).
- The smaller cleavage products C3a, C4a, C5a, sometimes called "anaphylatoxins", act as phagocytes, they cause mast cell degranulation and enhance vessel permeability, thereby facilitating access of plasma proteins and leukocytes to the site of infection
- alternative pathway provides a means of non-specific resistance against infection without the participation of antibodies and hence provides a first line of defense against a number of infectious agents.
2) Lecithin Pathway
The lectin pathway of complement activation exploits the fact that many bacterial surfaces contain mannose sugar molecules in a characteristic spacing. The oligomeric plasma protein mannan-binding lectin (MBL; lectins are proteins binding sugars) binds to such a pattern of mannose moieties, activating proteases MASP-1 and MASP-2 (MASP=MBL activated serine protease, similar in structure to C1r and C1s). These, by cleaving C4 and C2, generate a second type of C3 convertase consisting of C4b and C2b, with ensuing events identical to those of the alternative pathway.
3) classical pathway
The classical pathway usually starts with antigen-bound antibodies recruiting the C1q component, followed by binding and sequential activation of C1r and C1s serine proteases. C1s cleaves C4 and C2, with C4b and C2b forming the C3 convertase of the classical pathway. Yet, this pathway can also be activated in the absence of antibodies by the plasma protein CRP (C-reactive protein), which binds to bacterial surfaces and is able to activate C1q.
Pharmacology cross reference: humanized monoclonal antibody Eculizumab binds to complement component C5, inhibiting its cleavage and preventing activation of the lytic pathway. This is desirable when unwanted complement activation causes hemolysis, as in paroxysmal nocturnal hemoglobinuria or in some forms of hemolytic uremic syndrome. For the lytic pathway's importance in fighting meningococcal infections, Eculizumab treatment increases the risk of these infections, which may be prevented by previous vaccination.
BIOLOGICALLY ACTIVE PRODUCTS OF COMPLEMENT ACTIVATION
Activation of complement results in the production of several biologically active molecules which contribute to resistance, anaphylaxis and inflammation.
Kinin production
C2b generated during the classical pathway of C activation is a prokinin which becomes biologically active following enzymatic alteration by plasmin. Excess C2b production is prevented by limiting C2 activation by C1 inhibitor (C1-INH) also known as serpin which displaces C1rs from the C1qrs complex (Figure 10). A genetic deficiency of C1-INH results in an overproduction of C2b and is the cause of hereditary angioneurotic edema. This condition can be treated with Danazol which promotes C1-INH production or with ε-amino caproic acid which decreases plasmin activity.
Anaphylotoxins
C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which cause basophil/mast cell degranulation and smooth muscle contraction. Undesirable effects of these peptides are controlled by carboxypeptidase B (C3a-INA).
Chemotactic Factors
C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of neutrophils, basophils and macrophages and causes induction of adhesion molecules on vascular endothelial cells.
Opsonins
C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and promote phagocytosis.
Other Biologically active products of C activation
Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors and modulate their functions.
CHEMICAL AGENTS
Chlorine and iodine are most useful disinfectant Iodine as a skin disinfectant and chlorine as a water disinfectant have given consistently magnificent results. Their activity is almost exclusively bactericidal and they are effective against sporulating organisms also.
Mixtures of various surface acting agents with iodine are known as iodophores and these are used for the sterilization of dairy products.
Apart from chlorine, hypochlorite, inorganic chioramines are all good disinfectants but they act by liberating chlorine.
Hydrogen peroxide in a 3% solution is a harmless but very weak disinfectant whose primary use is in the cleansing of the wound.
Potassium permanganate is another oxidising agent which is used in the treatment of urethntzs.
Formaldehyde — is one of the least selective agent acting on proteins. It is a gas that is usually employed as its 37% solution, formalin.
When used in sufficiently high concentration it destroys the bacteria and their spores.
Classification of chemical sterilizing agents
Chemical disinfectant
Interfere with membrane functions
• Surface acting agents : Quaternary ammonium, Compounds, Soaps and fatty acids
• Phenols : Phenol, cresol, Hexylresorcinol
• Organic solvent : Chloroform, Alcohol
Denatures proteins
• Acids and alkalies : Organic acids, Hydrochloric acid , Sulphuric acid
Destroy functional groups of proteins
• Heavy metals : Copper, silver , Mercury
• Oxidizing agents: Iodine, chlorine, Hydrogen peroxide
• Dyes : Acridine orange, Acriflavine
• Alkylating agents : Formaldehyde, Ethylene oxide
Applications and in-use dilution of chemical disinfectants
Alcohols : Skin antiseptic Surface disinfectant, Dilution used 70%
Mercurials : Skin antiseptic Surface disinfectant Dilution Used 0.1 %
Silver nitrate : Antiseptic (eyes and burns) Dilution Used 1 %
Phenolic compound : Antiseptic skin washes Dilution Used .5 -5 %
Iodine : Disinfects inanimate object, Skin antiseptic Dilution used 2%
Chlorine compounds : Water treatment Disinfect inanimate objects , Dillution used 5 %
Quaternary ammonium Compounds : Skin antiseptic , Disinfects inanimate object, Dilution Used < 1 %
Glutaraldehyde: Heat sensitve instruments, Dilution used 1-2 %
Cold sterilization can be achieved by dipping the precleaned instrument in 2% solution of gluteraldehyde for 15-20 minutes. This time is sufficient to kill the vegetative form as well as spores ofthe organisms that are commonly encountered in the dentistry.
Ethylene oxide is an a agent extensively used in gaseous sterilization. It is active against all kinds of bacteria and their spores. but its greatest utility is in sterilizing those objects which are damaged by heat (e.g. heart lung machine). It is also used to sterlise fragile, heat sensitive equipment, powders as well as components of space crafts.
Evaluation of Disinfectants
Two methods which are widely employed are:
Phenol coefficient test, Kelsey -Sykes test
These tests determine the capacity of disinfectant as well as their ability to retain their activity.
DISINFECTION AND STERILIZATION
• Sterilization is the best destruction or com removal_of all forms of micro organisms.
• Disinfection is the destruction of many microorganisms but usually the b spores.
• Antisepsis is the destruction or inhibition of microorganisms in living tissues thereby limiting or preventing the harmful effect of infection.
• Astatic Agent would only inhibit the growth of microorganisms (bacteriostatic, fungistatic, sporostatic).
• Acidal agent would kill the microorganism (bactericidal. virucidal, fungicidal)
• Sterilants are the chemicals which under controlled conditions can kill sporinQ bacteria.
Neutralization Test
These are basically of two types:
• Toxin neutralization
• Virus neutralization
In toxin neutralization homologous anti-bodies prevent the biological effect of toxin as observed in vivo in experimental animals (e.g. detection of toxin of Clostridia and Corynebacterium diphthenae) or by in vitro method (e.g. Nagler’s method).
In virus neutralization test various methods are available by which identity of virus can be established as well as antibody against a virus can be estimated.
INNATE (NON-SPECIFIC) IMMUNITY
The elements of the innate (non-specific) immune system include anatomical barriers, secretory molecules and cellular components.
Among the mechanical anatomical barriers are the skin and internal epithelial layers, the movement of the intestines and the oscillation of broncho-pulmonary cilia.
Associated with these protective surfaces are chemical and biological agents.
A. Anatomical barriers to infections
1. Mechanical factors
The epithelial surfaces form a physical barrier that is very impermeable to most infectious agents. Thus, the skin acts as our first line of defense against invading organisms. The desquamation of skin epithelium also helps remove bacteria and other infectious agents that have adhered to the epithelial surfaces.
2. Chemical factors
Fatty acids in sweat inhibit the growth of bacteria. Lysozyme and phospholipase found in tears, saliva and nasal secretions can breakdown the cell wall of bacteria and destabilize bacterial membranes. The low pH of sweat and gastric secretions prevents growth of bacteria. Defensins (low molecular weight proteins) found in the lung and gastrointestinal tract have antimicrobial activity. Surfactants in the lung act as opsonins (substances that promote phagocytosis of particles by phagocytic cells).
3. Biological factors
The normal flora of the skin and in the gastrointestinal tract can prevent the colonization of pathogenic bacteria by secreting toxic substances or by competing with pathogenic bacteria for nutrients or attachment to cell surfaces.
B. Humoral barriers to infection
Humoral factors play an important role in inflammation, which is characterized by edema and the recruitment of phagocytic cells. These humoral factors are found in serum or they are formed at the site of infection.
1. Complement system – The complement system is the major humoral non-specific defense mechanism (see complement chapter). Once activated complement can lead to increased vascular permeability, recruitment of phagocytic cells, and lysis and opsonization of bacteria.
2. Coagulation system – Depending on the severity of the tissue injury, the coagulation system may or may not be activated. Some products of the coagulation system can contribute to the non-specific defenses because of their ability to increase vascular permeability and act as chemotactic agents for phagocytic cells. In addition, some of the products of the coagulation system are directly antimicrobial. For example, beta-lysin, a protein produced by platelets during coagulation can lyse many Gram positive bacteria by acting as a cationic detergent.
3. Lactoferrin and transferrin – By binding iron, an essential nutrient for bacteria, these proteins limit bacterial growth.
4. Interferons – Interferons are proteins that can limit virus replication in cells.
5. Lysozyme – Lysozyme breaks down the cell wall of bacteria.
6. Interleukin -1 – Il-1 induces fever and the production of acute phase proteins, some of which are antimicrobial because they can opsonize bacteria.
C. Cellular barriers to infection
Part of the inflammatory response is the recruitment of polymorphonuclear eosinophiles and macrophages to sites of infection. These cells are the main line of defense in the non-specific immune system.
1. Neutrophils – Polymorphonuclear cells are recruited to the site of infection where they phagocytose invading organisms and kill them intracellularly. In addition, PMNs contribute to collateral tissue damage that occurs during inflammation.
2. Macrophages – Tissue macrophages and newly recruited monocytes , which differentiate into macrophages, also function in phagocytosis and intracellular killing of microorganisms. In addition, macrophages are capable of extracellular killing of infected or altered self target cells. Furthermore, macrophages contribute to tissue repair and act as antigen-presenting cells, which are required for the induction of specific immune responses.
3. Natural killer (NK) and lymphokine activated killer (LAK) cells – NK and LAK cells can nonspecifically kill virus infected and tumor cells. These cells are not part of the inflammatory response but they are important in nonspecific immunity to viral infections and tumor surveillance.
4. Eosinophils – Eosinophils have proteins in granules that are effective in killing certain parasites.
Enzymes:
Serum lysozyme:
Provides innate & nonspecific immunity
Lysozyme is a hydrolytic enzyme capable of digesting bacterial cell walls containing peptidoglycan
• In the process of cell death, lysosomal NZs fxn mainly to aulolyse necrotic cells (NOT “mediate cell degradation”)
• Attacks bacterial cells by breaking the bond between NAG and NAM.
• Peptidoglycan – the rigid component of cell walls in most bacteria – not found in archaebacteria or eukaryotic cells
• Lysozyme is found in serum, tears, saliva, egg whites & phagocytic cells protecting the host nonspecifically from microorganisms
Superoxide dismutase: catalyzes the destruction of O2 free radicals protecting O2-metabolizing cells against harmful effects
Catalase:
- catalyzes the decomposition of H2O2 into H2O & O2
- Aerobic bacteria and facultative anaerobic w/ catalase are able to resist the effects of H2O2
- Anaerobic bacteria w/o catalase are sensitive to H2O2 (Peroxide), like Strep
- Anaerobic bacteria (obligate anaerobes) lack superoxide dismutase or catalase
- Staph makes catalase, where Strep does not have enough staff to make it
Coagulase
- Converts Fibronogen to fibrin
• Coagulase test is the prime criterion for classifying a bug as Staph aureus – from other Staph species
• Coagulase is important to the pathogenicity of S. aureus because it helps to establish the typical abscess lesion
• Coagulase also coats the surface w/ fibrin upon contact w/ blood, making it harder to phagocytize
The cell cycle
1) Labile cells (GI tract, blood cells)
- Described as parenchymal cells that are normally found in the G0 phase that can be stimulated to enter the G1
- Undergo continuous replication, and the interval between two consecutive mitoses is designated as the cell cycle
- After division, the cells enter a gap phase (G1), in which they pursue their own specialized activities
• If they continue in the cycle, after passing the restriction point (R), they are committed to a new round of division
• The G1 phase is followed by a period of nuclear DNA synthesis (S) in which all chromosomes are replicated
• The S phase is followed by a short gap phase (G2) and then by mitosis
• After each cycle, one daughter cell will become committed to differentiation, and the other will continue cycling
2) Stable cells (Hepatocytes, Kidney)
- After mitosis, the cells take up their specialized functions (G0).
- They do not re-enter the cycle unless stimulated by the loss of other cells
3) Permanent cells (neurons)
- Become terminally differentiated after mitosis and cannot re-enter the cell cycle
- Which cells do not have the ability to differentiate -> Cardiac myocytes