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.
NITRIC OXIDE-DEPENDENT KILLING
Binding of bacteria to macrophages, particularly binding via Toll-like receptors, results in the production of TNF-alpha, which acts in an autocrine manner to induce the expression of the inducible nitric oxide synthetase gene (i-nos ) resulting in the production of nitric oxide (NO) . If the cell is also exposed to interferon gamma (IFN-gamma) additional nitric oxide will be produced (figure 12). Nitric oxide released by the cell is toxic and can kill microorganism in the vicinity of the macrophage.
Bacteria
A bacterial cell has a nuclear apparatus which is a loose arrangement of DNA This is surrounded cytoplasm which contains ribosomes, mesosomes and inclusion granules. The cytoplasm is enclosed within a cytoplasmic membrane. Bacterium has a rigid cell wall Fimbriae and flagella are the surface adherents. Some bacteria may have a capsule (or loose slime) around the cell wall.
Shape and Size of Bacteria
The bacteria can be spheroidal (coccus), rod or cylindrical (bacillus) and spirillar (spirochaete). Very short bacilli are called as coccobacilli Some of the bacilli may be curved or comma shaped (Vibrio cholerae).
Arrangement of Bacterial Cells
Streptococci are present in chains; staphylococci in grape-like clusters Cocci in pairs (diplococci) are suggestive of pneumococci, gonococci or menigococci.
Bacilli do not exhibit typical arrangement pattern except the Chinese letter arrangement shown by Corynebacterium diphtheriae
Surface Adherents and Appendages
CAPSULE The gels formed by the capsule adhere to the cell Capsule can be detected by negative staining ,with specific antiserum and observing the capsular swelling phenomenon called as Quellung reaction
Usually weakly antigenic Capsule production is better in vivo as compared to in vitro environment.
Eg. Capsules seen in Pneumococci, Klebsiella, Escherichia coli, Haemophilus influenzae
Flagella : provide motility to the bacterium.
Motile organisms: vibrios, pseudomonas, Esch.coli, salmonellae, spirochaetes and spirilla.
Pathogenic cocci are nomotile.
Flagella measure in length from 3 to 20 µm and in diameter from 0.01 to 0.0 13 µm.
Arrangement
Bacteria with one polar flagellum are known as monotrichous;
Tuft of several polar flagellae is known as lophotrichous
Presence of Flagellae at both the ends of organism is amphitrichous
Flagellae distributed all over the surface of the bacterium, it is called peritrichous.
• Filament is composed of a protein-flagellin. The flagellar antigen is called as H (Hauch) antigen in contrast to somatic antigen which is called as O (Ohne haunch)
PILI (fimbriae) : hair like structures help in attachment also called sex pilli, transfers genetic material through conjugation , Present in Certain Gram negative bacteria. Only Composed of protein pilin
Gram positive bacterium that has pili is Cornebacterium renale
The Cell Wall
The cell wall of bacteria is multilayered structure. The external surface of cell wall is smooth in Gram positive bacteria Gram negative bacteria have convoluted cell surfaces. The average thickness of cell wall is 0.15 to 0.50 .µm. Chemically composed of mucopeptide scaffolding formed by N acetyl glucosamine and N acetyl muramic acid
The cell wall is a three layered structure in Gram negative bacteria: outer membrane middle layer and plasma membrane. The outer membrane consists of lipoprotein and 1ipoppolysaccaride component
Functions of bacterial cell wall
Provides shape , Gives rigidity , Protection, Surface has receptor sites for phages, Site of antibody action, Provides attachment to complement, Contains components toxic to host
Cytoplasmic Structures
The Plasma Membrane: This delicate membrane separates rigid cell wall from cytoplasm. It accounts for 30% of total cell weight. Chemically, it is 60% protein, 20-30% lipids and remaining carbohydrates.
Mesosomes:
Principal sites of respiratory enzyme , Seen well in Gram positive bacteria as compared to Gram negative batcteria. Attachement of mesosomes to both DNA chromatin and membrane have been noticed thus help in cell division
Ribosomes:
sites of protein synthesis. These are composed of RNA and proteins and constitute upto 4 of total cell protein and 90% of total cellular RNA.
Cytoplasmic Granules: Glycogen : Enteric bacteria
Poly-beta & hydroxy Butyrate : Bacillus & Pseudomonas
Babes-Ernst :Corynebacterium & Yersinia pestis
Nuclear Apparatus
Bacterial DNA represents 2-3% of the cell weight and 10% of the volume of bacterium. Nucleous can be demonstrated by staining it with DNA specific Fuelgen stain .Consists of a single molecule of double stranded DNA arranged in a circular form. Bacterial chromosome is haploid and replicates by binary fission, the bacteria may have plasmid an extrachromosomal genetic material.
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.
Test for Antigen - Antibody Reactions
Antigens are those substance that stimulates the production of antibodies which, when enter into the body it reacts specifically in a manner that are clearly visible.
Some antigens may not induce antibody production, but instead creates immunological tolerance.
An antigen introduced into the body produces only specific antibodies and will react with only those specific antigens.
These antibodies appear in the serum and tissue fluids. All antibodies are considered as immunoglobulin. They are mainly of five classes; IgG, IgA, IgM, IgD and IgE.
Antigen- antibody reactions are known as serological reactions and are used as serological diagnostic tests for the identification of infectious diseases.
The reaction occurs mainly in three stages;
1. The initial interaction between the antigen and antibody, which produces no visible effects. It is a reversible and rapid reaction.
2. The secondary stage leads to the demonstration proceedings, such as precipitation, agglutination, etc.
3. The tertiary reaction follows the neutralization or destruction of injurious antigens. These results in clinical allergy and other immunological diseases.
There are certain characteristics for antigen-antibody reactions;
1. Reaction is specific.
2. The whole molecules participate in the reaction, and not just a part of it.
3. No denaturation of antigen or antibody occurs during the reaction.
4. The combination usually occurs at the surface.
5. The combination is firm, but reversible
6. Agglutinins formed after agglutination usually are formed by both antigen and antibody together.
7. They can combine in varying proportions.
Measurement of antigen and antibody are made in terms of mass or as units or titre.
Serological reactions include;
1. Precipitation reaction
a soluble antigen combining with the specific antibody in the presence of electrolytes at a suitable temperature and pH forming insoluble precipitins. Commonly used tests are ring test, slide test, tube test, immunodiffusion, etc.
Radial Immunodiffusion
In radial immunodiffusion antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed .
This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples.
Immunoelectrophoresis
In immunoelectrophoresis, a complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs .
This tests is used for the qualitative analysis of complex mixtures of antigens
This test can also be used to evaluate purity of isolated serum proteins.
Countercurrent electrophoresis
In this test the antigen and antibody are placed in wells punched out of an agar gel and the antigen and antibody are electrophoresed into each other where they form a precipitation line.
2. Agglutination reaction
when a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. When the antigen is an erythrocyte the term hemagglutination is used.
Applications of agglutination tests
i. Determination of blood types or antibodies to blood group antigens.
ii. To assess bacterial infections
e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.
Passive hemagglutination
The agglutination test only works with particulate antigens. However, it is possible to coat erythrocytes with a soluble antigen (e.g. viral antigen, a polysaccharide or a hapten) and use the coated red blood cells in an agglutination test for antibody to the soluble antigen . This is called passive hemagglutination.
The test is performed just like the agglutination test.
Applications include detection of antibodies to soluble antigens and detection of antibodies to viral antigens.
Coomb's Test (Antiglobulin Test)
DIRECT ANTIGLOBULIN TEST (DAT)
The DAT is used to detect IgG or C3 bound to the surface of the red cell. In patients with hemolysis, the DAT is useful in determining whether there is an immune etiology.
A positive DAT can occur without hemolysis
Immune causes of hemolysis including autoimmune hemolytic anemias, drug induced hemolysis, and delayed or acute hemolytic transfusion reactions are characterized by a positive DAT.
INDIRECT ANTIGLOBULIN TEST (IAT)
The IAT (antibody screen) is performed by incubating patient serum with reagent screening red cells for approximately 20 minutes and then observing for agglutination. If the antibody screen is positive, additional testing is required to determine the specificity of the antibody.
The IAT is used to detect red cell antibodies in patient serum. Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both.
3. Complement fixation test (CFT)
the ability of antigen antibody complexes to fix complement is made use in this test. Complement is something which takes part in any immunological reaction and absorbed during the combining of antigen with its specific antibody.
The best example of CFT is the Wassermann reaction done for the detection of Syphilis.
4. Neutralization test
different types of these are available. Virus neutralization, toxin neutralization, etc. are some of its kind.
5. Opsonization
this makes use of the determination of opsonic index, which is the ratio of the phagocytic activity of patient’s blood to the phagocytic activity of the normal patient’s for a given bacterium.
6. Immunfluorescence
the method of labeling the antibodies with fluorescent dyes and using them for the detection of antigens in tissues.
7. Radioimmunoassay (RIA)
is a competitive binding radioisotopes and enzymes are used as labels to conjugate with antigens or antibodies.
8. Enzyme Immuno Assay (EIA)
the assays based on the measurement of enzyme labeled antigen or antibody. The most common example is ELISA used to detect HIV.
9. Immunoelectroblot
it uses the sensitivity of Enzyme immunoassay with a greater specificity. Example is Western blot done for the serodiagnosis of HIV infection.
CELLS ORGANELLES
Cell parts:
Mitochondrion – double MB structure responsible for cellular metabolism – powerhouse of the cell
Nucleus – controls synthetic activities and stores genetic information
Ribosome – site of mRNA attachment and amino acid assembly, protein synthesis
Endoplasmic reticulum – functions in intracellular transportation
Gogli apparatus/complex – composed of membranous sacs – involved in production of large CHO molecules & lysosomes
Lysosome – organelle contains hydrolytic enzymes necessary for intracellular digestion
Membrane bag containing digestive enzymes
Cellular food digestion – lysosome MB fuses w/ MB of food vacuole & squirts the enzymes inside. Digested food diffuses through the vacuole MB to enter the cell to be used for energy or growth. Lysosome MB keeps the cell iself from being digested
-Involved mostly in cells that like to phagocytose
-Involved in autolytic and digestive processes
-Formed when the Golgi complex packages up an especially large vesicle of digestive enzyme proteins
Phagosome
– vesicle that forms around a particle (bacterial or other) w/in the phagocyte that engulfed it
- Then separates from the cell membrane bag & fuses w/ lysozome to receive contents
- This coupling forms phagolysosomes in which digestion of the engulfed particle occurs
Microbodies:
- Contain catalase
- Bounded by a single membrane bag
- Compartments specialized for specific metabolic pathways
- Similar in function to lysosomes, but are smaller & isolate metabolic reactions involving H2O2
- Two general families:
· Peroxisomes: transfer H2 to O2, producing H2O2 – generally not found in plants
· Glyoxysomes: common in fat-storing tissues of the germinating seeds of plants
¨ Contain enzymes that convert fats to sugar to make the energy stored in the oils of the seed available
Inclusions
– transitory, non-living metabolic byproducts found in the cytoplasm of the cell
- May appear as fat droplets, CHO accumulations, or engulfed foreign matter.
Classification:
Neutrophiles (pH = 7.0)
- P. aeruginosaqo
- Clostridium sporogenes
- Proteus species
Acidophiles (pH < 7.0)
- Thiobacillus thiooxidans
- Sulfollobus acidocaldaarius
- Bacillus acidocaldarius
Alkaliphiles (pH > 7.0)
- Nitrobacter species
- Streptococcus pneumoniae