bacteriology

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bacteriology

مشاركةبواسطة دكتور كمال سيد » الأربعاء إبريل 24, 2013 4:46 pm

introduction

Bacteriology is the study of bacteria.
This subdivision of microbiology involves the identification, classification, and characterization of bacterial species
A person who studies bacteriology is a bacteriologist.

Bacteriology and microbiology

Because of the similarity of thinking and working with microorganisms other than bacteria, such as protozoa, fungi, and viruses, there has been a tendency for the field of bacteriology to extend as microbiology. The terms were formerly often used interchangeably. However, bacteriology can be classified as a distinct science.
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مشاركةبواسطة دكتور كمال سيد » الثلاثاء أغسطس 26, 2014 4:04 pm

Sterilization & Disinfection

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Sterilization
Sterilization is the killing or removal of all microorganisms, including bacterial spores which are highly resistant. Sterilization is an absolute term, i.e. the article must be sterile meaning the absence of all microorganisms.

Disinfection is the killing of many, but not all microorganisms. It is a process of reduction of number of contaminating organisms to a level that cannot cause infection, i.e. pathogens must be killed. Some organisms and bacterial spores may survive

Disinfectants are chemicals that are used for disinfection. Disinfectants should be used only on inanimate objects

Antiseptics are mild forms of disinfectants that are used externally on living tissues to kill microorganisms, e.g. on the surface of skin and mucous membranes


Uses of Sterilization
1. Sterilization for Surgical Procedures : Gloves, aprons, surgical instruments, syringes etc. are to be sterilized

2. Sterilization in Microbiological works like preparation of culture media, reagents and equipments where a sterile condition is to be maintained


CLASSIFICATION OF METHODS
: Sterilization and disinfection are done by
(A). Physical Agents
1. Heat
2. Radiation
3. Filtration

(B). Chemical Agents
In practice, certain methods are placed under sterilization which in fact do not fulfill the definition of sterilization such as boiling for 1/2 hr and pasteurization which will not kill spores

STERILIZATION BY HEAT
Heat is most effective and a rapid method of sterilization and disinfection. Excessive heat acts by coagulation of cell proteins. Less heat interferes metabolic reactions. Sterilization occurs by heating above 100°C which ensure lolling of bacterial spores. Sterilization by hot air in hot air oven and sterilization by autoclaving are the two most common method used in the laboratory.

: Types of Heat
A. Sterilization by moist heat
B. Sterilization by dry heat

A. Sterilization by Moist Heat
Moist heat acts by denaturation and coagulation of protein, breakage of DNA strands, and loss of functional integrity of cell membrane

(I. Sterilization at 100°C
1. Boiling: Boiling at 100°C for 30 minutes is done in a water bath. Syringes, rubber goods and surgical instruments may be sterilized by this method. All bacteria and certain spores are killed. It leads to disinfection

2. Steaming: Steam (100°C) is more effective than dry heat at the same temperature as: (a) Bacteria are more susceptible to moist heat, (b) Steam has more penetrating power, and (c) Steam has more sterilizing power as more heat is given up during condensation

Steam Sterilizer: It works at 100°C under normal atmospheric pressure i.e. without extra pressure. It is ideally suitable for sterilizing media which may be damaged at a temperature higher than 100°C
It is a metallic vessel having 2 perforated diaphragms (Shelves), one above boiling water, and the other about 4" above the floor. Water is boiled by electricity, gas or stove. Steam passes up. There is a small opening on the roof of the instrument for the escape of steam

: Sterilization is done by two methods

(a) Single Exposure for 11/2 hours. It leads to disinfection

(b) Tyndallization (Fractional Sterilization) : Heat labile media like those containing sugar, milk, gelatin can be sterilized by this method. Steaming at 100°C is done in steam sterilizer for 20 minutes followed by incubation at 37°C overnight
This procedure is repeated for another 2 successive days. That is 'steaming' is done for 3 successive days. Spores, if any, germinate to vegetative bacteria during incubation and are destroyed during steaming on second and third day. It leads to sterilization

II. Sterilization above 100°C: Autoclaving
Autoclaving is one of the most common methods of sterilization. Principle: In this method sterilization is done by steam under pressure. Steaming at temperature higher than 100°C is used in autoclaving. The temperature of boiling depends on the surrounding atmospheric pressure

A higher temperature of steaming is obtained by employing a higher pressure. When the autoclave is closed and made air-tight, and water starts boiling, the inside pressures increases and now the water boils above 100°C. At 15 ib per sq. inch pressure, 121°C temperatures is obtained. This is kept for 15 minutes for sterilization to kill spores. It works like a pressure cooker

'Sterilization holding time' is the time for which the entire load in the autoclave requires to be exposed

: Autoclave is a metallic cylindrical vessel. On the lid, there are
1A gauge for indicating the pressure, (2) A safety valve, which can be set to blow off at any desired pressure, and (3) A stopcock to release the pressure. It is provided with a perforated diaphragm

Water is placed below the diaphragm and heated from below by electricity, gas or stove

Working of Autoclave : (a) Place materials inside, (b) Close the lid. Leave stopcock open, (c) Set the safety valve at the desired pressure, (d) Heat the autoclave. Air is forced out and eventually steam ensures out through the tap, (e) close the tap

The inside pressure now rises until it reaches the set level (i.e. 15 Win), when the safety valve opens and the excess steam escapes, (f) Keep it for 15 minutes (holding time), (g) Stop heating, (h) Cool the autoclave below 100°C, (i) Open the stopcock slowly to allow air to enter the autoclave.

Checking of Autoclave for Efficiency. Methods
(i) Spores of Bacillus stearothermophilus are used. Spores withstand 121°C heat for up to 12 min. Strips containing this bacteria are included with the material being autoclaved. Strips are cultured between 50°C and 60°C for surviving spores. If the spores are killed the autoclave is functioning properly.
(ii)Automatic Monitoring System


III. Sterilization below 100°C
1. Pasteurization : Pasteurization is heating of milk to such temperature and for such a period of time so as to kill pathogenic bacteria that may be present in milk without changing colour, flavour and nutritive value of the milk. Mycobacterium bovis, Salmonella species, Escherichia coli and Brucella species may be present in milk. It does not sterilize the milk as many living organisms including spores are not destroyed..

Methods of Pasteurization
(i) Flash Method: It is "high temperature- short time method". Heating is done at 72°C for 15 seconds.
(ii) Holding Method: Heating is done between 63°C and 66°C for 30 minutes.

2. Inspissation : Inspissation is done between 75°C to 80°C. Inspissation means stiffening of protein without coagulation as the temperature is below coagulation temperature. Media containing serum or egg is sterilized by heating for 3 successive days. It is done in 'Serum Inspissator'.


B. Sterilization by Dry Heat
Mechanisms. (1) Protein denaturation, (2) Oxidative damage, (3) Toxic effect of elevated electrolyte (in absence of water).

Dry heat at 160°C (holding temperature for one hour is required to kill the most resistant spores). The articles remain dry. It is unsuitable for clothing which may be spoiled.

1. Red Heat. Wire loops used in microbiology laboratory are sterilized by heating to 'red' in bunsen burner or spirit lamp flame. Temperature is above 100°C. It leads to sterilization

2. Flaming. The article is passed through flame without allowing it to become red hot, e.g. scalpel. Temperature is not high to cause sterilization

3. Sterilization by Hot Air

Hot Air Oven (Sterilizer). It Is one of the most common method used for sterilization. Glass wares, swab sticks, all-glass syringes, powder and oily substances are sterilized in hot air oven. For sterilization, a temperature of 160°C is maintained (holding) for one hour. Spores are killed at this temperature. It leads to sterilization.

Hot Air Oven is an apparatus with double metallic walls and a door. There is an air space between these walls. The apparatus is heated by electricity or gas at the bottom. On heating, the air at the bottom becomes hot and passes between the two walls from below upwards, and then passes in the inner chamber through the holes on Me top of the apparatus. A thermostat is fitted to maintain a constant temperature of 160°C
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Re: bacteriology

مشاركةبواسطة دكتور كمال سيد » الثلاثاء أغسطس 26, 2014 4:24 pm

SHAPE, SIZE AND ARRANGEMENT OF BACTERIA

:Bacteria are classified into coccus and bacillus
Coccus (Pleural- Cocci): A coccus is a round or oval shaped bacterium. In most cocci, one axis of the cell is almost equal to any other axis. But a coccus may be slightly oval, pointed at the ends; lancet, bean or kidney shaped (Gonococcus). Typical cocci are about 0.5-1.0 pm in diameter. During multiplication, cocci may arrange in irregular groups, chains, pairs or singles.

(a) Cocci in irregular groups
(b) Cocci in chains
(c) Cocci in pairs are called diplococcus

Bacillus (Pleural- Bacilli) or Rod. Bacillus is rod shaped. Bacterium having one axis of the cell longer than the other. Size : 1-5 Iim x 0.5-1.0 pm. Rods are mostly arranged singly. When the size of a rod is short and approximates to coccal form, it is called coccobacillus. Vibrio is comma-shaped single curved rod. Actinomyces and Nocardia are mycelial, filamentous or rods

Corynebacterium is club-shaped rod. Spirochaetes are long slender flexible spiral rods with several curves. Most are 5-20 pm x 0.1-0.2 gm. Fusobacterium is spindle shaped rod. Spirillum is spiral rod with several curves. Mycoplasma lack cell wall. Chlamydiae and Rickettsiae are strict intracellular bacteria.


On Gram-staining, cocci and bacilli (except Mycobacterium) are classified into Gram-positive bacteria and Gram-negative bacteria. This difference in the staining depends on the composition of bacterial cell wall. It is used to study the shape, size and arrangement of bacteria beside the staining character.

Pleomorphism indicates that there is considerable variation in the size and/or shape of a strain

Involution form indicates that there is morphologically different forms of a strain. Most commonly occurs in the culture of Corynebacterium

CELL STRUCTURE OF BACTERIA
(Prokaryotic Cell Structure)

1. Cell Envelope: Layers that surround prokaryotic cell
(a) Cytoplasmic or cell membrane. It is the innermost layer present in all bacteria which surrounds the cytoplasm.
(b) Cell wall. The layer between cytoplasmic membrane and capsule. It is present in all bacteria except mycoplasma, i.e. it is absent in mycoplasma.
(c) Capsule and Glycocalyx : Outermost layer. Some bacteria may have capsule and glycocalyx
(d) S-layers

2. Genetic Elements
(a) Nucleoid. Nucleoid is present in all bacteria.
(b) Plasmid. Plasmid is present in certain bacteria.
(c) Transposons are present in certain bacteria.

3. Cytoplasm. It is present in all bacteria.

4. Flagella. May or may not be present.

5. Pili (Fimbriae). May or may not be present.

6. Spores. Endospores are formed by certain bacteria.


CELL ENVELOPE
The layers that surround the bacterial cells are referred collectively as the cell envelope. Cell envelope consists of cytoplasmic membrane, cell wall, and for some bacteria capsule and glycocalyx from inner to outer surface. Many bacteria may have an S-layer as the outermost component of the cell envelope. Cell envelope differs in Gram-positive and Gram-negative bacteria as follows:

1. Gram-Positive Cell Envelope. Cell envelope consists of two to three layers: (a) Cytoplasmic membrane, (b) Cell wall-thick peptidoglycan layer: May be 40 sheets, and (c) Capsule or an S-layer in some bacteria.

2. Gram-Negative Cell Envelope. Highly complex, multila­yered envelope: (a) Cytoplasmic membrane or inner membrane, (b) Cell wall- peptidoglycan- only one or two sheets, (c) Outer membrane, (d) Capsule or S-layer may also be present, (e) Periplasmic space. The space between the inner and outer membrane is called periplasmic space.

Cytoplasmic Membrane

Structure : Cytoplasmic membrane is the innermost layer of cell envelope that externally limits cytoplasm. It is composed of phospholipids and proteins.

Mesosomes are convoluted invaginations of the cytoplasmic membrane and visible with electron microscope. There are two types: septa) mesosomes and lateral mesosomes. Septa) mesosome function in the formation of cross wall during cell division.

Functions : (1) Selective permeability and transport of solutes, (2) bearing the receptors and other proteins of the chemotactic system, (3) bearing the enzymes and carrier molecules that function in the biosynthesis of DNA, membrane lipids, and cell wall polymers, (4) electron transport and oxidative phosphorylation in aerobic species, and (5) secretion of hydrolytic enzymes and toxins.

Bacterial chromosome (DNA) is attached to a septa) mesosome during cell division.


Cell Wall
Layers of the cell envelope lying between the cytoplasmic membrane and capsule is the 'cell wall'. It is external to cytoplasmic membrane. A bacterial cell wall is prokaryotic. Mycoplasmas have no cell wall. Functions: The cell wall is mostly rigid but also exhibit tensile strength. The strength depends on peptidoglycan, murein or mucopeptide which is the main component. The cell wall maintains the bacterial shape and protects the cell from lysis

The cell wall plays an important part in cell division. During cell division, a transverse partition is formed from the cell wall by its inward growth. Many antibacterial agents inhibit cell wall synthesis. Differential Gram-staining character leading to classification into Gram- positive and Gram-negative bacteria reside in the cell-wall. Peptidoglycan layer is common component, others are special components.


1. Cell-walls of Gram- positive bacteria
(a) Peptidoglycan Layer. Peptidoglycan comprise up to 50% of the cell wall material and may be 40 sheets. It is composed of N-acetyl muramic acid, N-acetyl glucosamine and peptides. Lysozyme acts on it,
(b) Teichoic acids. Teichoic acids lie on the outside surface of the peptidoglycan layer in most Gram positive bacteria. Teichoic acids are polymers containing ribitol or glycerol. There are two types of teichoic acid-wall teichoic acid linked to peptidoglycan and membrane teichoic acid linked to membrane.
(c) Polysaccharides.


2. Cell walls of Gram-negative bacteria
(a) Peptidoglycan Layer. Peptidoglycan comprises 5 to 10% of the wall material. Only one or two sheets.
(b) Lipoprotein. Lipoprotein is the most abundant protein of Gram negative cells. It probably stabilizes the outer membrane.
(c) Outer membrane. Lipid in nature. Serves to protect the cell of enteric bacteria from bile salts.
(d) Lipopolysaccharide (LPS). LPS molecule is toxic and is referred to as endotoxin of Gram negative bacteria. LPS is split into lipid A and polysaccharide. Lipid A is the toxin, and polysaccharide is the major surface antigen of the bacterial cell- called 0 antigen. Lipooligosaccharides (LOS) are important virulence factors and are antigenic.
(e) Periplasmic space. The space between the inner and outer membrane. It contains gel-like solutions of proteins and murein layer. Periplasmic proteins include (a) binding proteins for specific substrates like vitamins, sugar, amino acids, ions, and (b) enzymes like P-lactamase, alkaline phosphatase.


Capsule And Glycocalyx
Many bacteria synthesize extracellular polymers forming variable outer layer when growing in their natural environment. (1) Capsule. Capsule forms a well-defined layer closely surrounding the cell. Functions: Capsule plays role in the virulence of pathogenic bacteria by contributing to invasiveness and preventing phagocytosis. Typing of certain bacteria are done on antigenic character of the capsule. Capsulated bacteria form smooth or mucoid colonies, and if they become noncapsulated then form rough colonies, e.g. Streptococcus pneumoniae, Klebsiella pneumoniae

The extracellular material is polysaccharide except the capsule of Bacillus anthraces which is poly D-glutamic acid, a polypeptide. Capsule is demonstrated by special stain. (2) Glycocalyx. When the polymer forms a loose meshwork of fibrils, it is called glycocalyx. Glycocalyx plays role in adherence of bacteria on surface, e.g. Streptococcus mutans adheres to tooth enamel. (3) If the polymer is detached from the cells then it is called "slime layer". Bacteria may be trapped in slime layer
Capsule Stain. The bacterial film on the slide is stained with hot crystal violet solution followed by rinsing with copper
sulphate solution. The cell and background appear dark blue and the capsule a much paler blue.

: Examples
Capsulated Bacteria: Streptococcus pneumoniae, Kleb-iella pneumoniae (large capsule), Bacillus anthraces. Staphylococcus (some strains), Streptococcus pyogenes (some strains), Bordetella pertussis.

Non-capsulated Bacteria : Neisseria, Corynebacterium, Salmonella, Shigella, Vibrio, Proteus, Pseudomonas
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Re: bacteriology

مشاركةبواسطة دكتور كمال سيد » الثلاثاء أغسطس 26, 2014 4:34 pm

GENETIC ELEMENTS

Nucleoid- Bacterial Chromosome

Bacterial nucleoid consists of a single circular molecule. It is a single, haploid chromosome of about 1 mm long in unfolded state. Bacterial chromosome (DNA) is attached to a septa) mesosome. Nucleoids consist of DNA, smaller amounts of RNA, RNA polymerase and probably other proteins. The nucleoid replicates by fission. It has no nucleolus, nuclear membrane, spindle or non-identical chromosomes. Genes of the nucleus determines the amino acid sequences and hence the structure of a protein and thereby all the properties of the organism. These account for heredity. Recently it has been found that some prokaryotes have a linear chromosome. Staining of Nucleus. Nuclei are stained with Feulgan stain which is specific for DNA.


Plasmid
Certain bacteria contain plasmid. These are extra-chromosomal genetic material (DNA), ie. genetic determinant, and are mostly circular. Certain properties are determined by plasmid:

1.Virulence factors, eg. In enteropathogenic E. coli strains, both enterotoxins and colonization antigens (pill). Other exotoxin productions are frequently controlled by plasmids.

2. (Drug resistance by R factors (Resistance plasmids

3.Metabolic activities may be determined. Examples: Sucrose uptake and metabolism, and citrate uptake by Escherichia coli.

4.Production of colicins (Bacteriocins) by many Gram-negative bacteria is controlled by plasmids

5. (Transfer of genetic material by sex factors (See Chap. 40


Transposon
Transposons are genetic elements with DNA sequences


CYTOPLASMIC STRUCTURES
Mitochondria are absent in prokaryotic cells.

1. Ribosomes: They are present as minute granules in large number. RNA is the principal component of ribosomes

2. : Nutrient granules: These are not essential or permanent structures These may be

(a) Metachromatic, Volutin or Babes-Ernst granules. These may be present in Corynebacterium diphtheriae and may be used for identification. These act as reserve of energy. These have affinity for basic dyes. Albert staining is done to demonstrate metachromatic granules.
(b)Lipid granules. These are P- hyd roxybu rate.
(c)Polysaccharide granules, eg. glycogen, starch.
(d)Other granules, eg. sulphur.

3. Carotenoids- Photosynthetic pigments are present in photosynthetic bacteria

FLAGELLA
A large number of bacteria possess flagella. Flagellum is the organ of locomotion. Flagellated bacteria are motile. Nonflagellated bacteria are non-motile.

: Examples
Motile Bacteria: Escherichia, Salmonella, Proteus, Pseudomonas, Vibrio, Aeromonas, Plesiomonas, Campylo­bacter, Helicobacter, Clostridium tetani.

Nonmotile Bacteria: All Cocci, Shigella, Klebsiella, Corynebacterium diphtheriae, Clostridium perfringens, Bacillus anthraces, Haemophilus, Bordetella, Brucella, Yersinia, Pasteurella, Francisella.

Structure . Flagella are long, slender thread-like (12-30 nm in diameter) organs of locomotion. A flagellum is attached to the bacterial cell body by a complex structure consisting of filament, hook and basal body. The basal body has one pair of rings in Gram-positive bacteria and two pairs in Gram-negative bacteria. A bacterial flagellum is composed entirely of protein. A flagellum is made up of several thousand molecules of a protein subunit called flagellin. The flagellum is formed by the aggregation of subunits to form a helical structure.

A flagellated bacterium may become non-flagellated but not the reverse. The arrangement of flagella for a particular bacterium is constant and is one of the four types.

1. Peritrichous. Flagella are distributed over the entire cell, e.g. Escherichia coli, Salmonella genus.

2. Amphitrichous. There is a single flagellum at each pole.

3. Lophotrichous. There is a bunch of flagella at one or both poles, e.g. Spirillum minus.

4. Monotrichous. There is a single polar flagellum at one pole, e.g. Vibrio cholerae.

They have no function in the virulence of the bacteria. Antigenic structure of the flagella (H antigens) may be used for identification and classification of bacteria.

Sensory transduction is the behavior of the flagellated bacteria in response to the environment. This may be: (1) Chemotaxis is the movement of the bacterium towards a chemical attractant, e.g. sugar, amino-acid, (2) Aerotaxis­movement towards the optimal oxygen concentration, (3) Electron acceptor taxis- movement towards alternative electron receptors, (4) Phototaxis- movement towards light of photosynthetic bacteria.

Motility Test. Hanging-drop preparation is done by placing a drop of bacterial suspension between a slide and cover slip separated by a circular layer of vaseline on the surface of the slide.

Flagella Staining. They are demonstrated by special staining. Flagella are not visible under light microscope. Bacteria are first treated with tannic acid salt which precipitates on flagella and cell walls and increases the diameter of the flagella. They are then visible under light microscope.

PILI (FIMBRIAE)
Pill or fimbriae are very thin, hair-like short rigid surface appendages. Many Gram-negative bacteria possess pili or fimbriae. There may be 100-500 pili in one cell. They are shorter and finer than flagella and composed of protein subunits termed pilins. Minor proteins, located at the tips of pili, are responsible for the attachment properties. They are seen by electron microscope. Pellicle formation in the fluid media by bacteria is due to fimbriae. Pill of different bacteria are antigenically distinct. Some bacteria, e.g. N. gonorrhoeae are able to make pili of different antigenic types (antigenic variation) and this can still adhere to cells by a type in presence of antibodies to other types. Fimbriae of group A streptococci are the site of the surface antigen, M protein. Lipoteichoic acid, associated with these fimbriae is responsible for adherence of these streptococci to epithelial cells of the hosts. There are two types of pili on function:

1. Ordinary pill. These play roles in adherence of bacteria to host cell surfaces. 'Colonization antigens' are ordinary pili that provide the cells with adherent properties, genetically determined by plasmids.

2. Sex pill. These give the 'maleness'. They are few in number and very long, e.g. in enterobacteria. During bacterial conjugation the male (donor) cell adhere with its sex pili and transfer DNA to non-male (recipient) cell. The pili act as conjugation tubes. In this way the genetic material which determines antibiotic resistance may pass from one bacterium to another. Sex pili possess receptors to which bacteriophage can become attached.
BACTERIAL SPORES (ENDOSPORES)
Bacterial endospores are formed when conditions for vegetative growth are not favourable.


Spore-Forming Bacteria
1.Bacillus and Clostridium genera form spores.
2. Sporosarcina (Gram-positive coccus) and possibly Coxiella burnetii can produce spores.


Demonstration of Spore:
1.In Gram stain, the spore appears as unstained colourless areas in cells.
2.Spores are stained with malachite green, or carbol fuchsin (by modified Ziehl-Neelsen stain).

Nature. The spore is a resting cell. Spores are in a subdued metabolic state and non-reproducing. Each cell forms a single internal spore that is liberated when the mother cell undergoes autolysis. The spore is highly resistant to heat, chemical agents and desiccation. In sporulation, each vegetative cell forms only one spore, and in subsequent germination each spore gives rise to a single vegetative cell.
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مشاركةبواسطة دكتور كمال سيد » الثلاثاء أغسطس 26, 2014 4:39 pm

BACTERIAL GROWTH

Growth of Bacteria is the orderly increase of all the chemical constituents of the bacteria. Multiplication is the consequence of growth. Death of bacteria is the irreversible loss of ability to reproduce.

Bacteria are composed of proteins, carbohydrates, lipids, water and trace elements.


Factors Required for Bacterial Growth
: The requirements for bacterial growth are
A) Environmental factors affecting growth, and)
B) Sources of metabolic energy)

A. Environmental Factors affecting Growth
1. Nutrients. Nutrients in growth media must contain all the elements necessary for the synthesis of new organisms. In general the following must be provided : (a) Hydrogen donors and acceptors, (b) Carbon source, (c) Nitrogen source, (d) Minerals : sulphur and phosphorus, (e) Growth factors: amino acids, purines, pyrimidines; vitamins, (f) Trace elements: Mg, Fe, Mn.

Growth Factors: A growth factor is an organic compound which a cell must contain in order to grow but which it is unable to synthesize. These substances are essential for the organism and are to be supplied as nutrients. Thiamine, nicotinic acid, folic acid and para-aminobenzoic acid are examples of growth factors.

Essential Metabolites: These metabolites are essential for growth of bacterium. These must be synthesized by the bacterium, or be provided in the medium. Mg, Fe and Mn are essential trace elements.

Autotrophs live only on inorganic substances, i.e. do not require organic nutrients for growth. They are not of medical importance.

Heterotrophs require organic materials for growth, e.g. proteins, carbohydrates, lipids as source of energy. All bacteria of medical importance belong to heterotrophs. Parasites may depend on the host for certain foods. Saprophytes grow, on dead organic matter.

2. pH of the medium. Most pathogenic bacteria grow best in pH 7.2-7.4. Vibno cholerae can grow in pH 8.2-9.0.

3. Gaseous Requirement
(a) Role of Oxygen. Bacteria may be classified into four groups on oxygen requirement :
(i) Aerobes. They cannot grow without oxygen, e.g. Mycobacterium tuberculosis.
(ii) Facultative anaerobes. These grow under both aerobic and anaerobic conditions. Most bacteria are facultative anaerobes, e.g. Enterobacteriaceae.
(iii)Anaerobes. They only grow in absence of free oxygen, e.g. Clostridium, Bacteroides.
(iv) Microaerophils grow best in oxygen less than that present in the air, e.g. Campylobacter.

Aerobes and facultative anaerobes have the metabolic systems: (1) cytochrome systems for the metabolism of oxygen, (2) Superoxide dismutase, (3) catalase.

Anaerobic bacteria do not grow in the presence of oxygen. They do not use oxygen for growth and metabolism but obtain their energy from fermentation reactions. Anaerobic bacteria are killed by oxygen or toxic oxygen radicals. Multiple mechanisms play role for oxygen toxicity : (1) They do not have cytochrome systems for oxygen metabolism, (2) They may have low levels of superoxide dismutase, and (3) They may or may not have catalase.

(b) Carbon dioxide. All bacteria require CO2 for their growth. Most bacteria produce CO2. N. gonorrhoeae and N. meningitides and Br abortus grow better in presence of 5 per cent CO2.

4. Temperature. Most bacteria are mesophilic. Mesophilic bacteria grow best at 30-37°C. Optimum temperature for growth of common pathogenic bacteria is 37°C. Bacteria of a species will not grow but may remain alive at a maximum and a minimum temperature.

5. Ionic strength and osmotic pressure.

6. Light. Optimum condition for growth is darkness.


B.Sources of Metabolic Energy
Mainly three mechanisms generate metabolic energy. These are fermentation, respiration and photosynthesis. An organism to grow, at least one of these mechanisms must be used.

REPRODUCTION
Bacteria reproduce by binary fission. Multiplication takes place in geometric progression. The nucleus (chromosome) undergoes duplication prior to cell division. When the cell grows about twice its size, the nuclear material divides, and a transverse septum originates from plasma membrane and cell wall and divides the cell into two parts. The two daughter cells receive an identical set of chromosomes. The daughter cells separate and may be arranged singly, in pairs, clumps, or chains.

GROWTH CURVE
The growth curve indicates multiplication and death of bacteria. When a bacterium is inoculated in a medium, it passes through four growth phases which will be evident in a growth curve drawn by plotting the logarithm of the number of bacteria against time. Number of bacteria in the culture at different periods may be : (1) Total count. It includes both living and dead bacteria, or (2) Viable count. It includes only the living bacteria. Microbial concentration can be measured in terms of cell concentration, i.e. the number of viable cells per unit volume of culture, or of biomass concentration, i.e. dry weight of cells per unit volume of culture.

Bacterial count is of use in the examination of water and milk.

Growth Phases
1. Lag Phase. In this phase there is increase in cell size but not multiplication. Time is required for adaptation (synthesis of new enzymes) to new environment. During this phase vigorous metabolic activity occurs but cells do not divide. Enzymes and intermediates are formed and accumulate until they are present in concentration that permits growth to start. Antibiotics have little effect at this stage.

2. Exponential Phase or Logarithmic (Log) Phase. The cells multiply at the maximum rate in this exponential phase, i.e. there is linear relationship between time and logarithm of the number of cells. Mass increases in an exponential manner
This continues until one of two things happens: either one or more nutrients in the medium become exhausted, or toxic metabolic products, accumulate and inhibit growth. Nutrient oxygen becomes limited for aerobic organisms. In exponential phase, the biomass increases exponentially with respect to time, i.e. the biomass doubles with each doubling time
The average time required for the population, or the biomass, to double is known as the generation time or doubling time. Linear plots of exponential growth can be produced by plotting the logarithm of biomass concentration as a function of time. Importance : Antibiotics act better at this phase.

3. Maximal Stationary Phase. Due to exhaustion of nutrients or accumulation of toxic products death of bacteria starts and the growth cease completely. The count remains stationary due to balance between multiplication and death rate. Importance: Production of exotoxins, antibiotics, metachromatic granules, and spore formation takes place in this phase.

4. Decline phase or death phase. In this phase there is progressive death of cells. However, some living bacteria use the breakdown products of dead bacteria as nutrient and remain as persister.

Recently, some authors are dividing the growth curve into six phases by the letters A to F as follows
(a) Lag phase- Growth rate is zero.
(b) Acceleration phase- Increasing growth rate. C Exponential phase — Constant growth rate.
(c) Retardation phase- Growth rate is decreasing.
(e) Maximum stationary phase- Growth rate is zero. F Decline phase- Growth rate is negative (death).
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BACTERIAL GENETICS

Genetic.
Genetic is the science that deals with heredity. Thus, it is the study of the mechanisms by which different characteristics are passed on from parents to offsprings (progeny).

Gene. Genes are genetic determinants and thus control heredity and determine properties of the organism. The genes are functional units of the chromosomes. Synthesis of protein components and enzymes of a cell is regulated by genes. DNA is responsible for both gene function and replication. By replication heredity or stability of a type is maintained. An organism containing a normal gene is known as 'wild type'. Genes may rarely mutate (change) resulting in heritable variations called mutations (See chapter 8). The changed organism is called a mutant.

Genotype. It is the genetic determinant of a cell.

Phenotype. This is the structural and physiological manifestations of the organism due to a particular genotype.

Genome. Genome is the entire set of genes and thus is the totality of genetic information in an organism.

BACTERIAL GENOME (PROKARYOTIC GENOME)
Genes are carried on :
1. Bacterial chromosome. Chromosomes carry most prokaryotic genes. Genes essential for bacterial growth are carried on chromosome. It is present in all bacteria. Only one chromo-some is present in a bacterial nucleoid. Bacterial chromosome is a single circle containing about 4000 kbp (kilobase pairs) of DNA. DNA is a double stranded helical structure. A molecule of DNA consists of many mononucleotides. A mononucleotide consists of a molecule of sugar, a molecule of phosphate and a molecule of base. In each strand of DNA, molecules of phosphate and sugar alternate, and one of the four bases present in DNA is attached with the sugar. The four bases are adenine, thymine, guanine and cytosine. A base of a strand joins with its complementary base of the other strand. Adenine is always complementary to thymine, and guanine to cytosine. During cell division, duplication of chromosome occurs so that each daughter cell receives an identical set.

2.Plasmid. Certain bacteria contain plasmids. Plasmids carry genes associated with specialized function. These are extra-chromosomal genetic elements. Plasmid is a circular double stranded DNA having several to 100 kbp. Plasmids replicate autonomously and they code for functions which are normally not indispensable. In some cases, plasmids may be transferred from one cell to another and thus may carry sets of specialized genetic information through a population, e.g. Drug resistance plasmids (R factors) may render diverse bacteria resistant to antimicrobial drugs.

3.Transposon. Transposons are genetic elements with DNA sequences of several kbp. They can migrate from one genetic

locus to another. Transfer of transposons can occur between one plasmid to another, or between plasmid and chromosome within a bacterial cell. The process is called transposition. Transposons carry genes for specialized functions, e.g. antibiotic resistance. Transposons do not contain genetic information for their replication.

NOTE: Bacteria infected with bacterial viruses (bacteriopha­ges) contain the genes of the phage. Lysogenic bacteria are the bacteria which contain nonlytic prophage state of temperate phages.

Phenotypic changes that can occur in bacteria due to Mutation:
1. Loss of capsule formation by capsulated bacteria.
2. Change in colony characters.
3. Change in fermentation activity.
4. Loss of sensitivity to antibiotics.
5. Loss of sensitivity to bacteriophage (Bacterial virus).

TRANSFER OF GENE
Transfer of DNA is widespread among bacterial cells. In intercellular transfer, the genetic material passes from the donor cell to the recipient cell. It leads to genetic diversity of bacteria. Intercellular transfer of DNA between bacterial strains can occur by conjugation, transduction and transformation.

1. Conjugation
In conjugation, a suitable donor cell (male) comes near a recipient cell (female), establishes direct cell-to-cell contact and transfers genetic material. Plasmids are most frequently transferred by conjugation. Plasmids can also mobilize portions of chromosome for transfer. This is mediated by a fertility or F factor which is carried on a plasmid. Sex pilus is responsible for the attachment of donor (F*) cell and recipient cell (F) in bacterial conjugation (mating) process. Cell with sex pilus is the male and without sex pilus is the female bacteria.

Example: Transfer of antibiotic resistance can occur by conjugation. Transfer of drug resistant plasmid is called R-factor (Resistance plasmid). This occurs via sex pilus from the male (donor) cell to the female (recipient) cell.

2.Transduction
Transduction is phage-mediated. In transduction, genetic material of donor bacterial cell is enclosed in a bacterial virus (phage) and transferred to the recipient cell.

3.Transformation
In transformation, the recipient cell directly takes up naked DNA released from the donor cell altering its genotype. It can occur in the medium. Natural transformation also can occur.
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TYPES OF CULTURE MEDIA

: Media are of different types on consistency and chemical composition

A. On Consistency
1. Solid Media. Advantages of solid media: (a) Bacteria may be identified by studying the colony character, (b) Mixed bacteria can be separated. Solid media is used for the isolation of bacteria as pure culture. 'Agar' is most commonly used to prepare solid media. Agar is polysaccharide extract obtained from seaweed. Agar is an ideal solidifying agent as it is : (a) Bacteriologically inert, i.e. no influence on bacterial growth, (b) It remains solid at 37°C, and (c) It is transparent.
2. Liquid Media. It is used for profuse growth, e.g. blood culture in liquid media. Mixed organisms cannot be separated.

: B. On Chemical Composition
1. Routine Laboratory Media
2. Synthetic Media. These are chemically defined media prepared from pure chemical substances. It is used in research work

ROUTINE LABORATORY MEDIA
These are classified into six types: (1) Basal media, (2) Enriched media, (3) Selective media, (4) Indicator media, (5) Transport media, and (6) Storage media.

1. BASAL MEDIA. Basal media are those that may be used for growth (culture) of bacteria that do not need enrichment of the media. Examples: Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in these media

2. ENRICHED MEDIA. The media are enriched usually by adding blood, serum or egg. Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar media

3. SELECTIVE MEDIA. These media favour the growth of a particular bacterium by inhibiting the growth of undesired bacteria and allowing growth of desirable bacteria. Examples: MacConkey agar, Lowenstein-Jensen media, tellurite media (Tellurite inhibits the growth of most of the throat organisms except diphtheria bacilli). Antibiotic may be added to a medium for inhibition

4. INDICATOR (DIFFERENTIAL) MEDIA. An indicator is included in the medium. A particular organism causes change in the indicator, e.g. blood, neutral red, tellurite. Examples: Blood agar and MacConkey agar are indicator media

5. TRANSPORT MEDIA. These media are used when specie-men cannot be cultured soon after collection. Examples: Cary-Blair medium, Amies medium, Stuart medium

6. STORAGE MEDIA. Media used for storing the bacteria for a long period of time. Examples: Egg saline medium, chalk cooked meat broth

COMMON MEDIA IN ROUTINE USE
Nutrient Broth. 500 g meat, e.g. ox heart is minced and mixed with 1 litre water. 10 g peptone and 5 g sodium chloride are added, pH is adjusted to 7.3. Uses: (1) As a basal media for the preparation of other media, (2) To study soluble products of bacteria.

Nutrient Agar. It is solid at 37°C. 2.5% agar is added in nutrient broth. It is heated at 100°C to melt the agar and then cooled.

Peptone Water. Peptone 1% and sodium chloride 0.5%. It is used as base for sugar media and to test indole formation.

Blood Agar. Most commonly used medium. 5-10% defibrinated sheep or horse blood is added to melted agar at 45-50°C. Blood acts as an enrichment material and also as an indicator. Certain bacteria when grown in blood agar produce haemolysis around their colonies. Certain bacteria produce no haemolysis

Types of changes : (a) beta (p) haemolysis. The colony is surrounded by a clear zone of complete haemolysis, e.g. Streptococcus pyogenes is a beta haemolytic streptococci, (b) Alpha (a) haemolysis. The colony is surrounded by a zone of greenish discolouration due to formation of biliverdin, e.g. Viridans streptococci, (c) Gamma (y) haemolysis, or, No haemolysis. There is no change in the medium surrounding the colony,

Chocolate Agar or Heated Blood agar. Prepared by heating blood agar. It is used for culture of pneumococcus, gonococcus, meningococcus and Haemophilus. Heating the blood inactivates inhibitor of growths.

MacConkey Agar. Most commonly used for enterobac­teriaceae. It contains agar, peptone, sodium chloride, bile salt, lactose and neutral red. It is a selective and indicator medium

(1) Selective as bile salt does not inhibit the growth of enterobactericeae but inhibits growth of many other bacteria

(2) Indicator medium as the colonies of bacteria that ferment lactose take a pink colour due to production of acid. Acid turns the indicator neutral red to pink. These bacteria are called 'lactose fermenter', e.g. Escherichia coll. Colourless colony indicates that lactose is not fermented, i.e. the bacterium is non-lactose fermenter, e.g. Salmonella. Shigella, Vibrio.

Mueller Hinton Agar. Disc diffusion sensitivity tests for antimicrobial drugs should be carried out on this media as per WHO recommendation to promote reproducibility and comparability of results.

Hiss's Serum Water Medium. This medium is used to study the fermentation reactions of bacteria which can not grow in peptone water sugar media, e.g. pneumococcus, Neisseria, Corynebacterium.

Lowenstein-Jensen Medium. It is used to culture tubercle bacilli. It contains egg, malachite green and glycerol. (1) Egg is an enrichment material which stimulates the growth of tubercle bacilli, (2) Malachite green inhibits growth of organisms other than mycobacteria, (3) Glycerol promotes the growth of Mycobacterium tuberculosis but not Mycobacterium bovis.

Dubos Medium. This liquid medium is used for tubercle bacilli. In this medium drug sensitivity of tubercle bacilli can be carried out. It contains 'tween 80', bovine serum albumin, casein hydrolysate, asparagin and salts. Tween 80 causes dispersed growth and bovine albumin causes rapid growth.

Loeffler Serum. Serum is used for enrichment. Diphtheria bacilli grow in this medium in 6 hours when the secondary bacteria do not grow. It is used for rapid diagnosis of diphtheria and to demonstrate volutin granules. It contains sheep, ox or horse serum.

Tellurite Blood Agar. It is used as a selective medium for isolation of Cotynebacterium diphtheriae. Tellurite inhibits the growth of most secondary bacteria without an inhibitory effect on diphtheria bacilli. It is also an indicator medium as the diphtheria bacilli produce black colonies. Tellurite metabolized to tellbrism, which has black colour.

EMB (Eosin-methylene blue) Agar. A selective and differential medium for enteric Gram-negative rods. Lactose-fermenting colonies are coloured and nonlactose-fermenting colonies are nonpigmented. Selects against gram positive bacteria.

XLD (Xylose Lysine Deoxychoiate). It is used to isolate Salmonella and Shigella species from stool specimens. This is a selective media.

SS (Salmonella-Shigella) Agar. It is a selective medium used to isolate Salmonella and Shigella species. SS Agar with additional bile salt is used if Yersinia enterocolitica is suspected.

DCA (Desoxycholate Citrate Agar). It is used for isolation of Salmonella and Shigella. The other enteric bacteria are mostly inhibited (a selective medium). It is also a differential (indicator) medium due to presence of lactose and neutral red.

Tetrathionate Broth. This medium is used for isolating Salmonella from stool. It acts as a selective medium. It inhibits normal intestinal bacteria and permits multiplication of Salmonella.

Selenite F Broth. Uses and functions are same as that of tetrathionate broth.

Thiosulphate-Citrate-Bile-Sucrose (TCBS) Agar. TCBS agar is a selective medium used to isolate Vibrio cholerae and other Vibrio species from stool.

Charcoal-yeast agar. Used for Legionella pneumophila. Increased concentration of iron and cysteine allows growth.

Tellurite-Gelatin Agar Medium (TGAM). It may be used as transport, selective and indicator medium.

(Alkaline peptone water. See under Vibrio. (Chapter 51

Campylobacter Medium. This selective medium is used to isolate Campylobacter jejuni and Campylobacter coli from stool.

Cary-Blair Medium. It is used as a transport medium for faeces that may contain Salmonella, Shigella, Vibrio or Cam­pylobacter species.

Amies medium is used for gonococci and other pathogens.
Peptone Water Sugar Media. These indicator media are used to study 'Sugar fermentation'. 1 % solution of a sugar (lactose, glucose, mannitol etc) is added to peptone water containing Andrade's indicator in a test tube. A small inverted Durham tube is placed in the medium. The media are colourless. After culture, change of a medium to red colour indicates acid production. Gas, if produced collects in Durham tube.

Motility Indole Urea (MIU) Medium. This is used to differentiate enterobacteria species by their motility, urease, and indole reactions.

TSI (Triple sugar iron) Agar- See chapter 53.

KIA (Kligler Iron Agar). This is a differential slope medium used in the identification of enteric bacteria. The reactions are based on the fermentation of lactose and glucose and the production of hydrogen sulphide (chapter 53).

Christensen's Urea Medium. This is used to identify urea splitting organisms, e.g. Proteus. A purple pink colour indicates urea splitting (See chapter 53).

Bordet-Gengou Medium. This medium is used for culture of Bordetella pertussis. Increased concentration of blood allows growth. It contains agar, potato, sodium chloride, glycerol, peptone and 50% horse blood. Penicillin may be added to it.
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Host-parasite Interactions

Infectious Diseases. These are caused by infectious agents which are bacteria, viruses, fungi, protozoa and helminths.

Parasite is a living organism (bacteria, viruses, fungi, protozoa, helminths) that lives in another organism, and receives shelter and nourishment. However, in medical science parasitology traditionally deals with animal parasites- protozoa and helminths.

Strict or obligate parasite is an organism that cannot live without a host. That is they have no free-living existence. Examples: Treponema pallidum, viruses, malarial parasite

Facultative parasite is an organism that has both a free-living and a parasitic existence, e.g. Clostridium species, Pseudomonas species

Pathogen. Pathogen is an organism which can cause disease

Nonpathogen. An organism that does not cause disease. It may be a member of normal flora

Opportunistic pathogen is an organism (nonpathogen, commensal or saprophyte) that can cause disease only in immunocompromised individuals that is having impaired
resistance. Example : Cytomegalovirus, Pneumocystis carinii, Atypical mycobacteria, opportunist fungi

Saprophyte is an organism that lives on dead organic matter


(NORMAL FLORA (Commensals
Microorganisms that is present on the skin and mucous membrane of normal (healthy) persons. One particular microorganism of the normal flora may be a nonpathogen, or opportunistic pathogen. Normal flora is of two types: (1) Resident flora. These are microorganisms regularly present in the region at a given age, e.g. Viridans streptococci in mouth and throat, Escherichia coli in intestine, (2) Transient flora.

The microorganism is present only for hours to weeks, e.g. Streptococcus pyogenes in throat

Microorganisms of transient flora play no role when the normal resident flora remains intact. But if the resident flora is disturbed than the microorganisms of transient flora may colonize and produce disease. See chapter 62

Beneficial Functions of Normal Flora. Examples: (1) In mouth and lower bowel an invading pathogen may fail to compete for nutrients and receptor sites with normal flora, (2) Some bacteria of the bowel produce antimicrobial substances, (3) In new born, bacteria acts as a powerful stimulus for the development of immune system, (4) Bacteria of the gut can produce
vitamin K.

: Harmful Effects of Normal Flora. Clinical diseases by opportunist pathogen of normal flora arise under

(1) when the organism leaves the normal site and localizes to another site, e.g. Escherichia coli in the urinary tract from gut, (2) competitive advantage due to antibiotic therapy, e.g. colitis by Clostridium difficile, (3) In immunocompromised individuals.

HOST
: Host is the harbouring organism of a parasite. They fall into three groups or classes

1. Definitive host. In this host the adult stage of animal parasites lives and sexual reproduction takes place. Man is the definitive host for all animal parasites except malarial parasites and hydatid tapeworm.

2. Intermediate host. In this host the asexual reproduction takes place, or the larval stages of animal parasites develops. When development of larval stage takes place in two different hosts they are called 'first' and 'second' intermediate hosts. Man is the intermediate host for malarial parasites and hydatid tapeworm. Man is both definitive and intermediate host for Taenia solium and Trichinella spiralis.

3. Paratenic host. A carrier or transport host.

Note : The term infestation is used when the parasite lives on the outer surface. They are arthropodes- mites, ticks and insects.

INFECTION
Infection is the multiplication of an infectious agent within the body. Multiplication of pathogenic bacteria (e.g. Salmonella typhi) even if the person is asymptomatic is taken as an infection. Multiplication of bacteria of normal flora at its normal site is not an infection. However, if they multiply and cause disease it is an infection, e.g. Escherichia coli when causes diarrhoea.

An infection involves the following : (1) Source or reservoir of infectious agent, (2) Transmission of the infectious agent from the source to the host (3) Susceptible host- Portal or route of entry of the agent in the host, its localization, multiplication and finally host-parasite interactions which result in either (a) Destruction of the agent or, (b) Infectious disease.

: (Source (Reservoir
1. Human Source
a) Exogenous source : Patient or carrier)

b) Endogenous source : The individual himself)

Carrier. A person with asymptomatic infection which can be transmitted to another susceptible person. An individual may be a carrier : (a) in the incubation period (Incubatory carrier), e.g. in measles, (b) during convalescence (convalescent carrier), e.g. typhoid fever, or (c) for a prolonged period, e.g. typhoid fever

2. Foods and Drinks
(a) Food :Any contaminated food, (b) Water contaminated with bacteria of typhoid fever, cholera, diarrhoea and dysentery,
(c) Milk contaminated with salmonella, M. bovis.

3. Animals : Zoonoses are diseases which are transmitted from infected animals to humans.
(a) Cow : Bovine tuberculosis, brucellosis by Brucella abortus, Salmonella food poisoning, anthrax, Taenia saginata, (b) Fowl. Salmonella food poisoning by eggs and meat, (c) Dog. Rabies, hydatid disease, Weil's disease, (d) Horse. Tetanus and glanders, (e) Goat. Anthrax and brucellosis (by B. melitensis ), (f) Sheep. Anthrax and tetanus, (g) Cat. Cat scratch disease, (h) Rat. Plaque, Weil's disease, (i) Parrot and pigeons. Psittacosis.

4. Soil. Tetanus, gas gangrene.

Modes of Transmission
1. Water-borne, e.g. cholera and other diarrhoea) diseases, enteric fever from contaminated water.

2. Food-borne (contaminated food) e.g. enteric fever, salmo­nella food poisoning (e.g. eggs of duck, foul). Milk and milk products- enteric fever, bovine tuberculosis.

3. Air-borne e.g. Droplets. During coughing, sneezing and talking: Diphtheria, tuberculosis, measles, chickenpox, mumps, influenza,

4. Dust-borne. e.g. tuberculosis,

5. Soil- Tetanus, gas gangrene.

6. Contaminated fomites like beddings, clothings, utensils: Diphtheria, enteric fever, food poisoning.

7. Direct contact- STD like gonorrhoea, syphilis, AIDS.

8. Transplacental: Rubella, toxoplasma, congenital syphilis.

9. Insect borne. (a) Actively, e.g. malaria, filaria, kala azar, dengue, plague, yellow fever, (b) Passively, e.g. diarrhoea) diseases, enteric fever, salmonella food poisoning.


Portal or Route of Entry
(1) Alimentary system by ingestion, (2) Respiratory system by inhalation, (3) Genitourinary system, (4) Skin due to trauma, bite of insects, (5) Placenta.

Self-infection and cross-infection
Self-infection is the infection that occurs from the patient's own flora. Examples : (i) infection of an wound of a patient by staphylococci carried by the individual in his nose, (2) coliforms and anaerobes released from his bowel during surgery.

Cross infection is the infection derived from other patients or healthy carriers by direct spread as droplets during talking, coughing, sneezing, by air-born dust, food, fluids etc. Cross-infection is common in hospitals

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BACTERIAL TOXINS


Bacterial toxins are exotoxins and endotoxins. Characteristics and differences of exotoxins and endotoxin are listed in down the page

EXOTOXINS
Exotoxin producing bacteria

1. Corynebacterium diphtheriae. Strains that carry a temperate bacteriophage are toxigenic causes diphtheria. Exotoxin inhibits protein synthesis and causes cell death. Causes diphtheria.

2. Clostridium tetani. Exotoxin blocks action of inhibitory neurones of spinal cord. Causes tetanus.

3. Clostridium perfringens. (a) Causes gas gangrene. Exotoxin (alpha toxin) has lacithinase activity and thereby causes cell death, (b) Enterotoxin causes hyper secretion of water and electrolytes in diarrhoea.

4. Clostridium botulinum. Exotoxin causes paralysis of deglutination and respiratory muscles. It blocks release of acetylcholine of synapses and neuromuscular junctions. Causes botulism.

5. Vibrio cholerae 01 and 0139. Enterotoxin (Exotoxin) causes hyper secretion of water and electrolytes within gut in diarrhoea.

6. Enterotoxigenic E. coll. Produce enterotoxin (LT- heat labile exotoxin) causes hypersecretion of water and electrolytes within gut.

7. Shigella dysenteriae type 1 (Shiga bacillus). Exotoxin causes acute inflammation.

8. Staphylococcus aureus- some strains : (a) Toxic shock syndrome toxin-1. Causes toxic shock syndrome. (b) Staphylococcal enterotoxin causes toxin type food poisoning and stimulates vomiting centre of brain.

9. Streptococcus pyogenes. Pyrogenic (Erythrogenic) exotoxin causes scarlet fever and toxic shock syndrome.

NOTE : Enterotoxins are exotoxins that are associated with diarrhoeal diseases and food poisoning. Bacteria producing enterotoxins are V. cholerae, enterotoxigenic E. coli (ETEC), some strains of S. aureus, V. parahaemolyticus, Y. enterocolitica, Aeromonas species, and C. perfringens

: Toxoid. Toxoid is modified exotoxin. An exotoxin has two main properties
(1) toxicity, and
(2) antigenicity. In toxoid, the toxicity of the toxin is destroyed but its antigenicity is preserved. As such toxoids e.g. diphtheria toxoid, tetanus toxoid can be safely used for vaccines. Toxins can be converted to toxoid by different methods e.g. formalin treatment.

ENDOTOXINS
Endotoxins are the integral part of the cell walls of Gram-negative bacteria, and are liberated when bacteria are disintegrated (lysed). Cell wall of Gram negative bacteria contain lipopolysaccharides (LPS, endotoxin) which consists of : (1) Lipid A. This is the endotoxin and is the core, and (2) Polysaccharide form coat. This is the 0 antigen which can induce specific immunity
Physiological, pathological and clinical effects of endotoxins of different Gram negative bacteria are similar. These are

1. Fever. The endotoxin acts on mononuclear phagocytes (monocytes/macrophages), with liberation of interleukin-1 (endogenous pyrogen). Interleukin-1 acts on thermoregulatory centre. Chill is due to widespread arteriolar and venular constriction.
2. Leucopenia occurs early with onset of fever. It may be followed by leucocytosis.

3. Hypoglycaemia. LPS enhances glycolysis in many cell types and can lead to hypoglycaemia.

4. Hypotension occurs early in Gram-negative bacteraemia.

5. Shock. 'Endotoxic' or 'septic' shock may develop in severe Gram-negative bacteraemia (See chapter 4).

6. Activation of complement. Endotoxin activates complement system by alternative pathway.

7. Disseminated Intravascular Coagulation (DIC). DIC may occur in Gram negative bacteraemia. It is initiated on activation of factor XII (Hageman factor) of coagulation cascade by endotoxin which finally leads to conversion of fibrinogen to fibrin. Endotoxin leads platelets to adhere on vascular endothelium. Endotoxin can activate plasminogen to plasmin which acts on fibrin producing fibrin-split products. Shwartzman phenomenon is taken as a specialized form of DIC.

8. Death may occur due to shock and/or DIC.

NOTE: Peptidoglycan of Gram-positive Bacteria: Peptidoglycan of Gram-positive bacteria released during infection may produce similar activities as LPS of Gram-negative bacteria. However, peptidoglycan is much less potent than LPS.


Characteristics and Differences of Exotoxins and Endotoxins
: Endotoxin
1. Integral part of the cell wall of Gram-negative bacteria. Released on bacterial death and in part during growth. Release is not required for biologic activity.
2. Formed only by Gram-negative bacteria
3. Lipopolysaccharides. Lipid A portion is responsible for toxicity.
4. No specific receptor.
5. Moderately toxic. Fatal to animals in large doses.
6. Relatively heat stable. Toxicity is not destroyed above 60°C for hours.
7. Weakly antigenic. Antibodies are protective.
8. Not converted to toxoid.
9. Synthesis directed by chromosomal genes.
10. Usually produce fever in the host by release of interleukin-1 and other mediators

: Exotoxins
1.Excreted by living cells
2. Produced by Gram-positive and Gram-negative bacteria
3. Polypeptides
4. Usually bind to specific receptors on cells
5. Highly toxic. Fatal to animals in very small doses
6. Relatively heat labile. Toxicity destroyed over 60°C
7. Highly antigenic. Stimulate formation of antitoxin. Antitoxin neutralizes the toxin
8. Converted to toxoid by formalin. Toxoid is nontoxic but antigenic. Toxoids are used to immunize, e.g. tetanus toxoid
9. Usually controlled by extra-chromosomal genes, e.g. plasmids, phage gene
10. Usually do not produce fever in the host

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MECHANISMS OF ACTION OF ANTIMICROBIAL DRUGS


Selective Toxicity : Selective toxicity is the property of an ideal antimicrobial agent. Selective toxicity means that the drug is harmful to a parasite but not to the host. Usually the effect is relative rather absolute, i.e. a drug in a concentration tolerated by the host may damage an infecting microorganism

Selective toxicity may be a function of a specific receptor required for drug attachment, or it may depend on inhibition of biochemical events essential to the organism but not to the host. The mechanisms are discussed under four headings: (1) Inhibition of cell wall synthesis, (2) Inhibition of cell membrane function, (3) Inhibition of protein synthesis, and (4) Inhibition of nucleic acid synthesis

: Classification on mode of Action
1. Inhibition of cell wall synthesis. Penicillins, cephalo­sporin, vancomycin, bacitracin, cycloserin are examples

All (3-Iactarn drugs like penicillins, cephalosporins are selective inhibitors of bacterial cell wall synthesis and therefore active against growing bacteria. Mechanism of action: Initial step is the binding of the drug to specific drug receptor PBPs (Penicillin- binding proteins) on bacteria. There are 3 to 6 PBPs having different effects. At least some of which are enzymes involved in transpeptidation (cross-linking) reactions
After attachment, peptidoglycan synthesis is inhibited as final transpeptidation is blocked. Then there occurs inactivation of an inhibitor of autolytic enzyme in the cell wall. This activates the autolytic enzymes in the cell wall that results in lysis resulting in bacterial death. Organisms with defective autolysin function are inhibited but not killed by l3dactam drugs, and they are said to be "tolerant

The Gram-positive and Gram-negative bacteria differ in susceptibility to penicillins or cephalosporins on structural differences in their cell walls that decide binding, penetration, and activity of the drugs. The differences in cell wall are in the amount of peptidoglycan, presence of receptors and lipids, nature of cross-linking and activity of autolytic enzymes. Peptidoglycan layer of the cell wall is much thicker in Gram-positive than in Gram-negative bacteria.

Lack of toxicity of p-lactam drugs to animal cells is attributed to the absence of bacterial type cell wall with its peptidoglycan in animal cells.

Extended-spectrum P lactum (ESBLs) are enzymes that mediate resistance to extended-spectrum (third generation) cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone) and monobactams (e.g., aztreonam) but do not affect cephamycins (e.g., cefoxitin and cefotetan) or carbapenems (e.g., meropenem or imipenem). Production of ESBLs can be screened by broth microdilution and disk diffusion screening tests using selected antimicrobial agents.

2. Alteration of cell membrane function. Polymyxin, amph­otericin B, imidazole are examples.
These drugs act by disruption of the functional integrity of the cytoplasmic membrane. Macromolecules and ions escape from the cell causing cell damage or death. Selective chemotherapy is possible as the cytoplasmic membrane of bacteria and fungi has a structure different from that of animal cells, and can be more readily disrupted by certain chernothe rape utics.

3. Inhibition of protein synthesis. These drugs inhibit protein synthesis in bacteria. Bacteria have 70S ribosomes, whereas mammalian cells have 80S ribosomes. Subunits of each type of ribosome, their chemical composition, and functional specifities differ. Thus a drug can inhibit protein synthesis in bacterial ribosomes but not in mammalian ribosomes.

(a) Action on 50S ribosomal subunit. Chloramphenicol, erythromycin and chindamycin are examples.
(b) Action on 30S ribosomal subunit. Tetracycline and aminoglycosides are examples.

4. Inhibition of nucleic acid synthesis. These drugs inhibit nucleic acid synthesis by different mechanisms. (a) Inhibition of nucleotide synthesis. Sulphonamides, trimethoprin Sulphonamides compete for the enzyme required for essential metabolite PABA involved in the synthesis of folic acid needed for synthesis of nucleic acid. (b) Inhibition of DNA synthesis. Quinolones block DNA gyrase. (c) Inhibition of mRNA synthesis- Rifampin
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