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.
PHAGOCYTOSIS AND INTRACELLULAR KILLING
A. Phagocytic cells
1. Neutrophiles/Polymorphonuclear cells
PMNs are motile phagocytic cells that have lobed nuclei. They can be identified by their characteristic nucleus or by an antigen present on the cell surface called CD66. They contain two kinds of granules the contents of which are involved in the antimicrobial properties of these cells.
The second type of granule found in more mature PMNs is the secondary or specific granule. These contain lysozyme, NADPH oxidase components, which are involved in the generation of toxic oxygen products, and characteristically lactoferrin, an iron chelating protein and B12-binding protein.
2. Monocytes/Macrophages
Macrophages are phagocytic cells . They can be identified morphologically or by the presence of the CD14 cell surface marker.
B. Response of phagocytes to infection
Circulating PMNs and monocytes respond to danger (SOS) signals generated at the site of an infection. SOS signals include N-formyl-methionine containing peptides released by bacteria, clotting system peptides, complement products and cytokines released from tissue macrophages that have encountered bacteria in tissue.
Some of the SOS signals stimulate endothelial cells near the site of the infection to express cell adhesion molecules such as ICAM-1 and selectins which bind to components on the surface of phagocytic cells and cause the phagocytes to adhere to the endothelium.
Vasodilators produced at the site of infection cause the junctions between endothelial cells to loosen and the phagocytes then cross the endothelial barrier by “squeezing” between the endothelial cells in a process called diapedesis.
Once in the tissue spaces some of the SOS signals attract phagocytes to the infection site by chemotaxis (movement toward an increasing chemical gradient). The SOS signals also activate the phagocytes, which results in increased phagocytosis and intracellular killing of the invading organisms.
C. Initiation of Phagocytosis
Phagocytic cells have a variety of receptors on their cell membranes through which infectious agents bind to the cells. These include:
1. Fc receptors – Bacteria with IgG antibody on their surface have the Fc region exposed and this part of the Ig molecule can bind to the receptor on phagocytes. Binding to the Fc receptor requires prior interaction of the antibody with an antigen. Binding of IgG-coated bacteria to Fc receptors results in enhanced phagocytosis and activation of the metabolic activity of phagocytes (respiratory burst).
2. Complement receptors – Phagocytic cells have a receptor for the 3rd component of complement, C3b. Binding of C3b-coated bacteria to this receptor also results in enhanced phagocytosis and stimulation of the respiratory burst.
3. Scavenger receptors – Scavenger receptors bind a wide variety of polyanions on bacterial surfaces resulting in phagocytosis of bacteria.
4. Toll-like receptors – Phagocytes have a variety of Toll-like receptors (Pattern Recognition Receptors or PRRs) which recognize broad molecular patterns called PAMPs (pathogen associated molecular patterns) on infectious agents. Binding of infectious agents via Toll-like receptors results in phagocytosis and the release of inflammatory cytokines (IL-1, TNF-alpha and IL-6) by the phagocytes.
D. Phagocytosis
The pseudopods eventually surround the bacterium and engulf it, and the bacterium is enclosed in a phagosome. During phagocytosis the granules or lysosomes of the phagocyte fuse with the phagosome and empty their contents. The result is a bacterium engulfed in a phagolysosome which contains the contents of the granules or lysosomes.
E. Respiratory burst and intracellular killing
During phagocytosis there is an increase in glucose and oxygen consumption which is referred to as the respiratory burst. The consequence of the respiratory burst is that a number of oxygen-containing compounds are produced which kill the bacteria being phagocytosed. This is referred to as oxygen-dependent intracellular killing. In addition, bacteria can be killed by pre-formed substances released from granules or lysosomes when they fuse with the phagosome. This is referred to as oxygen-independent intracellular killing.
1. Oxygen-dependent myeloperoxidase-independent intracellular killing
During phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is formed. Cytochrome B which was part of the specific granule combines with the plasma membrane NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the NADPH. The result is the production of superoxide anion. Some of the superoxide anion is converted to H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion can react with H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The result of all of these reactions is the production of the toxic oxygen compounds superoxide anion (O2-), H2O2, singlet oxygen (1O2) and hydroxyl radical (OH•).
2. Oxygen-dependent myeloperoxidase-dependent intracellular killing
As the azurophilic granules fuse with the phagosome, myeloperoxidase is released into the phagolysosome. Myeloperoxidase utilizes H2O2 and halide ions (usually Cl-) to produce hypochlorite, a highly toxic substance. Some of the hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions is the production of toxic hypochlorite (OCl-) and singlet oxygen (1O2).
3. Detoxification reactions
PMNs and macrophages have means to protect themselves from the toxic oxygen intermediates. These reactions involve the dismutation of superoxide anion to hydrogen peroxide by superoxide dismutase and the conversion of hydrogen peroxide to water by catalase.
4. Oxygen-independent intracellular killing
In addition to the oxygen-dependent mechanisms of killing there are also oxygen–independent killing mechanisms in phagocytes: cationic proteins (cathepsin) released into the phagolysosome can damage bacterial membranes; lysozyme breaks down bacterial cell walls; lactoferrin chelates iron, which deprives bacteria of this required nutrient; hydrolytic enzymes break down bacterial proteins. Thus, even patients who have defects in the oxygen-dependent killing pathways are able to kill bacteria. However, since the oxygen-dependent mechanisms are much more efficient in killing, patients with defects in these pathways are more susceptible and get more serious infections.
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.
Autoantibodies
Anti-nuclear antibodies (ANA) Systemic Lupus
Anti-dsDNA, anti-Smith Specific for Systemic Lupus
Anti-histone Drug-induced Lupus
Anti-IgG Rheumatoid arthritis
Anti-neutrophil Vasculitis
Anti-centromere Scleroderma (CREST)
Anti-Scl-70 Sclerderma (diffuse)
Anti-mitochondria 1oary biliary cirrhosis
Anti-gliadin Celiac disease
Anti-basement membrane Goodpasture’s syndrome
Anti-epithelial cell Pemphigus vulgaris
Anti-microsomal Hashimoto’s thryoiditis
Immunology:
The branch of life science which deals with immune reaction is known as immunology.
Components of Immune System:
The immune system consists of a network of diverse organs and tissue which vary structurally as well as functionally from each other. These organs remain spreaded throughout the body. Basically, immune system is a complex network of lymphoid organs, tissues and cells.
These lymphoid organs can be categorized under three types depending upon their functional aspects:
i. Primary lymphoid organ.
ii. Secondary lymphoid organ.
iii.Tertiary lymphoid organ.
White blood cells or leukocytes are the basic cell types which help to give rise to different types of cells which participate in the development of immune response . WBC are classified into granulocytes and agranulocytes depending on the presence or absence of granules in the cytoplasm.
Agranular leukocytes are of two types, viz., lymphocytes and monocytes. Lymphocytes play pivotal role in producing defensive molecules of immune system. Out of all leukocytes, only lymphocytes possess the quality of diversity, specificity, memory and self-non self recognition as various important aspects of immune response.
Other cell types remain as accessory one; help to activate lymphocytes, to generate various immune effector cells, to increase the rate of antigen clearance
All cells of the immune system have their origin in the bone marrow
myeloid (neutrophils, basophils, eosinpophils, macrophages and dendritic cells)
lymphoid (B lymphocyte, T lymphocyte and Natural Killer) cells .
The myeloid progenitor (stem) cell in the bone marrow gives rise to erythrocytes, platelets, neutrophils, monocytes/macrophages and dendritic cells whereas the lymphoid progenitor (stem) cell gives rise to the NK, T cells and B cells.
For T cell development the precursor T cells must migrate to the thymus where they undergo differentiation into two distinct types of T cells, the CD4+ T helper cell and the CD8+ pre-cytotoxic T cell.
Two types of T helper cells are produced in the thymus the TH1 cells, which help the CD8+ pre-cytotoxic cells to differentiate into cytotoxic T cells, and TH2 cells, which help B cells, differentiate into plasma cells, which secrete antibodies.
Function of the immune system is self/non-self discrimination.
This ability to distinguish between self and non-self is necessary to protect the organism from invading pathogens and to eliminate modified or altered cells (e.g. malignant cells).
Since pathogens may replicate intracellularly (viruses and some bacteria and parasites) or extracellularly (most bacteria, fungi and parasites), different components of the immune system have evolved to protect against these different types of pathogens.
ANTIGEN-ANTIBODY REACTIONS
I. NATURE OF ANTIGEN-ANTIBODY REACTIONS
A. Lock and Key Concept
The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. Antigen-antibody reactions is one of a key (i.e. the antigen) which fits into a lock (i.e. the antibody).
B. Non-covalent Bonds
The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds.
C. Reversibility
Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible.
II. AFFINITY AND AVIDITY
A. Affinity
Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody .
B. Avidity
Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities.
III. SPECIFICITY AND CROSS REACTIVITY
A. Specificity
Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. In general, there is a high degree of specificity in antigen-antibody reactions.
B. Cross reactivity
Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen.
PHYSICAL AGENTS
Heat occupies the most important place as a physical agent.
Moist Heat : This is heating in the presence of water and can be employed in the following ways:
Temperature below 100°C: This includes holder method of Pasteurization where 60°C for 30 minutes is employed for sterilization and in its flash modification where in objects are subjected to a temperature of 71.1°C for 15 seconds. This method does not destroy spores.
Temperatures Around 100°C : Tyndallization is an example of this methodology in which steaming of the object is done for 30 minutes on each of three consecutive days. Spores which survive the heating process would germinate before the next thermal exposure and would then be killed.
Temperatures Above 100°C : Dry saturated steam acts as an excellent agent for sterilization. Autoclaves have been designed on the principles of moist heat.
Time-temperature relationship in heat sterilization
Moist heat (autoclaving)
121°C 15 minutes
126°C 10 minutes
134 C 3 minutes
Dry heat
>160°C >120 minutes
>170°C >60minutes
>180°C >30 minutes
Mechanism of microbial inactivation
The autoclaving is in use for the sterilization of many ophthalmic and parentral products. surgical dressings, rubber gloves, bacteriological media as well a of lab and hospital reusable goods.
Dry Heat: Less efficient, bacterial spores are most resistant. Spores may require a temperature of 140° C for three hours to get killed.
Dry heat sterilization is usually carried out by flaming as is done in microbiology laboratory to sterilize the inoculating loop and in hot air ovens in which a number of time-temperature combinations can be used. It is essential that hot air should circulate between the objects to be sterilized. Microbial inactivation by dry heat is primarily an oxidation process.
Dry heat is employed for sterilization of glassware glass syringes, oils and oily injections as well as metal instruments. -
Indicators of Sterilization:
These determine the efficacy of heat sterilization and can be in the form of spores of Bacillus stearothermophilus (killed at 121C in 12 minutes) or in the form of chemical indicators, autoclave tapes and thermocouples.
Ionizing Radiations
Ionizing radiations include X-rays, gamma rays and beta rays, and these induce defects in the microbial DNA synthesis is inhibited resulting in cell death. Spores are more resistant to ionizing radiations than nonsporulating bacteria.
The ionizing radiations are used for the sterilization of single use disposable medical items.
Mechanism of microbial inactivation by moist heat
Bacterial spores
• Denaturation of spore_epzymes
• Impairment of germination
• Damage to cell membrane
• Increased sensitivity to inhibitory agents
• Structural damage
• Damage to chromosome
Nonsporulating bacteria
• Damage to cytoplasmic membrane
• Breakdown of RNA
• Coagulation of proteins
• Damage to bacterial chromosome
Ultraviolet Radiations :
wave length 240-280 nm have been found to be most efficient in sterilizing. Bacterial spores are more resistant to U.V. rays than the vegetative forms. Even viruses are sometimes more resistant than vegetative bacteria.
Mechanism of Action :
Exposure to UV rays results in the formation of purine and pyrimidine diamers between adjacent molecules in the same strand of DNA. This results into noncoding lesions in DNA and bacterial death.
Used to disinfect drinking water, obtaining pyrogen free water, air disinfection (especially in safety laboratories, hospitals, operation theatres) and in places where dangerous microorganisms are being handled.
Filteration
Type of Filters
Various types of filters that are available are /
Unglazed ceramic filter (Chamberland and Doulton filters)
Asbestos filters (Seitz, Carlson and Sterimat filters)
Sintered glass filters
Membrane filters
Membrane filters are widely used now a days. Made up of cellulose ester and are most suitable for preparing_sterile solutions. The range of pore size in which these are available is 0.05-12 µm whereas the required pore size for sterlization is in range of 0.2-0.22 p.m.