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.
 

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. 

NON-SPECIFIC KILLER CELLS

Several different cells including NK and LAK cells, K cells, activated macrophages and eosinophils are capable of killing foreign and altered self target cells in a non-specific manner. These cells play an important role in the innate immune system.

A. NK and LAK cells

Natural killer (NK) cells are also known as large granular lymphocytes (LGL) because they resemble lymphocytes in their morphology, except that they are slightly larger and have numerous granules.

NK cells can be identified by the presence of CD56 and CD16 and a lack of CD3 cell surface markers.

NK cells are capable of killing virus-infected and malignant target cells but they are relatively inefficient in doing so.

However, upon exposure to IL-2 and IFN-gamma, NK cells become lymphokine-activated killer (LAK) cells, which are capable of killing malignant cells.

Continued exposure to IL-2 and IFN-gamma enables the LAK cells to kill transformed as well as malignant cells. LAK cell therapy is one approach for the treatment of malignancies.

NK and LAK cells have two kinds of receptors on their surface – a killer activating receptor (KAR) and a killer inhibiting receptor (KIR). 

When the KAR encounters its ligand, a killer activating ligand (KAL) on the target cell the NK or LAK cells are capable of killing the target. However, if the KIR also binds to its ligand then killing is inhibited even if KAR binds to KAL. 

The ligands for KIR are MHC-class I molecules. Thus, if a target cell expresses class I MHC molecules it will not be killed by NK or LAK cells even if the target also has a KAL which could bind to KAR. 

Normal cells constitutively express MHC class I molecules on their surface, however, virus infected and malignant cells down regulate expression of class I MHC. Thus, NK and LAK cells selectively kill virus-infected and malignant cells while sparing normal cells.

B. K cells 

Killer (K) cells are not a morphologically distinct type of cell. Rather a K cell is any cell that mediates antibody-dependent cellular cytotoxicity (ADCC). 

In ADCC antibody acts as a link to bring the K cell and the target cell together to allow killing to occur. K cells have on their surface an Fc receptor for antibody and thus they can recognize, bind and kill target cells coated with antibody. 

Killer cells which have Fc receptors include NK, LAK, and macrophages which have an Fc receptor for IgG antibodies and eosinophils which have an Fc receptor for IgE antibodies.

BACTERIAL GROWTH

The conversion of a parental cell into two daughters constitutes the bacterial life cycle and the time taken to complete cell cycle is known as generation_time. This is around 15 minutes in vegetative bacteria except mycobacteria.

Bacterial Growth Curve

In the presence of fresh growth medium a bacterium shows following four phases;

The Lag phase -> The Log phase -> The Stationary phase  -> The Decline phase

The Lag Phase : short duration , bacteria adapt themselves to new environment 

The Log Phase (Exponential Phase) : Regular growth of bacteria occurs The morphology of bacteria is best developed in this phase and organisms manifest typical biochemical characters. 

- Most of the cidal Abx work best in this phase
•    i.e. Ampicillin
- Best phase for staining bacterial cultures

Chemostat and turbidostat are examples of technique by which this phase can be prolonged.

Stationary Phase : balanced growth and cell division cannot be sustained. The total cell Count remains static till lysis supervenes, but the viable cell count quickly declines.

Decline Phase: death phase. Dyeing bacteria exceed the dividing bacterias.
 

NUTRITION OF BACTERIA

Nutrients

Chemoheterotrophs: nutrient source is organic material
Bacteria also requires a source of  minerals.

Oxygen

On this basis bacteria have been divided into four groups.

Obligate Anaerobes: These grow only under conditions of high reducing intensity. These bacteria catalase peroxidase, superoxide dismutase and cytochrome systems
Clostridium and Bacteroides are important examples.

Facultalive Anaerobes. These can grow under both aerobic and anaerobic conditions and include members of family enterobacteriaceae and many other bacteria.

Obligatory Aerobes. These cannot grow unless oxygen is present in the medium. Pseudomonas belong to this group.

Microaerophillic. These organisms can grow under conditions with low oxygen tension. Clostridium tetani is an important example.
The strict anaerobes are unable to grow unless Eh is as low as 0.2 volt

Temperature

•    On the basis of temperature requirements, three groups of bacteria are recognised.

•    Psychrophilic : Growth in  the range of —5 to 30°C with an optimum of 10-20 

•    Mesophillic : bacteria grow best at 20-40°C with a range of 10-45°C. 

•    Medically important bacteria belong to this group

•    Myco. leprae is one such important example and it can grow only at reduced temperature such as footpad of mouse

•    Thermophillic organisms prefer high temperature (25-80°C) for growth and yield maximum growth at 50-60°C

pH :  Most pathogenic bacteria require a pH of  7.2-7.6 for their own optimal growth.
 

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.