MICROBIAL VIRULENCE FACTORS 

Microbial virulence factors are gene products required for a microbial pathogen to establish itself in the host. These gene products are located on the bacterial chromosome, or on mobile genetic elements, such as plasmids or transposons.

Primary pathogens express virulence factors that allow them to cause disease in the normal  host.

Opportunistic pathogens are environmental organisms or normal flora that lack the means to overcome normal host defense mechanisms. They cause disease only when the normal host defenses are breached or deficient. 

Virulence factors can be divided into several categories.

Skin - Propionibacterium acnes, Staphlococcus epidermis , diptheroids; transient colonization by Staphlococcus
aureus

Oral cavity - Viridans Streptococci, Branhamella species, Prevotella melaninogenicus, Actinomyces species, Peptostreptococcus species, other anaerobes

Nasopharynx Oral organisms; transient colonization by S. pneumoniae, Haemophilus species, N. meningitidis  

Stomach Rapidly becomes sterile 

Small intestine Scant

Colon - Bacteroides species, Clostridium species, Fusobacterium species, E. coli, Proteus species, Pseudomonas aeruginosa, Enterococcus species, other bacteria and yeasts 

Vagina - Childbearing years:Lactobacillus species, yeasts, Streptococcus species 

Prepuberty / Postmenopause: colonic and skin flora 

A. Enzyme production can be of several types depending on the needs of the organism, its requirements for survival, and the local environment.
 
1. Hyaluronidase breaks down hyaluronic acid to aid in the digestion of tissue. 
2. Protease digests proteins to enhance the spread of infections. 
3. Coagulase allows coagulation of fibrinogen to clot plasma. 
4. Collagenase breaks down collagen (connective tissues). 

B. Toxins 

1. Exotoxins are heat-labile proteins with specific enzymatic activities produced by many Gram-positive and Gram-negative organisms. Exotoxins are released extracellularly and are often the sole cause of disease. 
a. Some toxins have several domains with discrete biological functions that confer maximal toxicity. An example is A-B exotoxin, where the B subunit binds to host tissue cell glycoproteins and the A subunit enzymatically attacks a susceptible target.
b. Many toxins are ADP-ribosylating toxins

2. Endotoxin is the heat-stable lipopolysaccharide moiety found in the outer membrane of Gram-negative organisms. when released by cell lysls, the lipid A portion of lipopolysaccharide can induce septic shock characterized by fever, acidosis, hypotension, complement consumption, and disseminated intravascular coagulation (DIC).  

C. Surface components 

may protect the organism from immune responses such as phagocytosis or aid in tissue invasion. For example, the polysaccharide capsules of H. influenzae type b and the acidic polysaccharide capsule of Streptococcus pneumoniae interfere with phagocytosis. Other surface proteins, such as adhesins or filamentous appendages (fimbriae, pili), are involved in adherence of invading microorganisms to cells of the host. 

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.

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.

Method of Sterilization for common items

Autoclaving :  Animal cages, Sugar tubes, Lab. Coats, Cotton , Filters, Instruments Culture media, Rubber, Gloves , Stopper, Tubing, Slides,  Syringe and Wax needles , Test tubes, Enamel metal trays ,Wire baskets, Wood, Tongue depressor, Applicator, Endodontic instruments, Orthodontic pliers , Orthodontic kits, Saliva ejector, Handpieces Cavitron heads, Steel burs, Steel tumbler, Hand instruments    

Hot air oven

Beakers, Flasks, Petri dish, Slides, Syringes, Test tubes, Glycerine, Needles ,Oil, Paper Saliva ejector, Matrix Band

Ethylene oxide

Fabric, Bedding, Blanket, Clothing, Matteresses, Pillows, Disposable instruments , Instruments, Blades, Knives, Scalpels, Scissors ,Talcum powder, Books, Cups, plates , Plastics., Flask, Petridish, Tubes, Tubing, Rubber , catheters, Drains, Gloves ,Special items - Bronchoscope, Cystoscope, Heart lung machine

Glutaraldehyde

Orthodontic kits, Orthodontic pliers , Steel burrs, 3 in 1 syringe tips ,Cystoscope ,Endoscope

Filtration

Antibiotics, Serum, Vaccines
 

Complement Fixation Test (CFT)

This test is based upon two properties of the complement viz:

a. Complent combines with all antigen-antibody complexes whether or not it is required for that reaction
b. Complement is needed in immunolytic reaction.

Test system

It contains an antigen and a serum suspected to be having antibody to that antigen. The serum is heat treated prior to the test to destroy its complement. Complement Is added in measured quantity to this system. This complement is the form of guinea pig serum which is considered a rich source of complement. The test system is incubated.

Indicator system

To test system, after incubation, is added the indicator system which consists of sheep
RBCs and antibody to sheep RBCs (haemolysin) and another incubation is allowed.
If there is specific antibody in the test system, it will bind to antigen and to this complex the complement will also get fixed. Hence, no complement will be available to combine with indicator system which though contains RBCs and their specific antibody, cannot undergo haemolysis unless complement gets attached. Absence of haemolysis shall indicated positive test or presence of specific antibody in the serum which has been added in the test system. Erythrocytes lysis is obtained in negative test.

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.