Santosh Yadav

BACTERIAL ANATOMY, PHYSIOLOGY, GROWTH AND NUTRITION

ANDBACTERIAL METABOLISM,

TOXINS AND BACTERIOCINS

Santosh Yadav M.Sc. Clinical Microbiology

Dept. of MicrobiologyInstitute of Medicine

Tribhuvan Univarsity Teaching Hospital, Nepal

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Outline

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Structure and function of different bacterial cell organelles.

Growth and nutrition of bacteria.Bacterial metabolism and different

pathways.Bacterial toxins and their clasiification.Bacteriocins, their classification ,mode of

action and typing.

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ANATOMY AND PHYSIOLOGY

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Bacterial cell structure

Cell wall Internal to cell wall

(Plasmamembrane,Mesosome,Nucleoid,Plasmid,Ribosome,Inclusion body,Endospore)

External to cell wall(capsule,Flagella,Pili)

3 – 5 um ₓ 0.2 – 1.5 um

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CELL WALLEncloses the protoplast

and lies immediately external to the cytoplasmic membrane.

Relatively rigid with some elasticity, and openly porous.

Freely permeable to solute molecules smaller than 10 kDa in mass and 1 nm in diameter.

Thickness depends on

type of bacteria.

Gram positive Gram negative

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Comparision of cellwall

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Gram positive

Gram negative

Thickness Thicker Thinner Peptidoglycan 40-65 sheets 1-2 sheetsVarieties of amino acids

Few Several

Lipids Absent or scant Present Teichoic acid present Absent Lipopolysaccharides

Absent Present

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Cell wall of Gram positive bacteriaComposed of

peptidoglycan and techoic acid.

Peptidoglycan comprises up to about 50% of the cell wall material.

Peptidoglycan layer is 15-50 nm thick.

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The peptidoglycan layerComplex polymer consisting of three parts: a)Backbone of cellwall, glycan (polymer of alternating N-acetylglucosamine(NAG) and N-acetylmuramic acid (NAM); b) tetrapeptide side chains attached to(NAM); and c) peptide interbridges.

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Contd…

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Teichoic acid Major surface antigen

of Gram positive bacteria.

Chains of either ribitol-phosphate or glycerol phosphate,to which various sugars and D-alanine are usually attached.

Two types:- lipotechoic acid (attached

to cytoplasmic membrane) and

wall techoic acid ( those attached to NAM portion of peptidoglycan of cell wall)

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Contd…

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Cell wall of Gram negative bacteria• Composed of peptidoglycan and outermembrane.• Peptidoglycan comprises 5-10% of the wall

material (thickness 2-6 nm). Outer membrane contain three components:

lipoprotein ,phospholipid and lipopolysaccharide.

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Peptidoglycan Cross-Links. (a) E. coli peptidoglycanwith direct cross-linking, typical of many gram-negative bacteria.(b) Staphylococcus aureus peptidoglycan. S. aureus is a gram-positivebacterium. NAM is N-acetylmuramic acid. NAG is N-acetylglucosamine.Gly is glycine.13

Peptidoglycan of Gram negative bacteria

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Lipoprotein• lipoprotein cross-link the outer membrane

and peptidoglycan layers.• Is peptide, linked to DAP residues of the

peptidoglycan .• Function is to stabilize the outer membrane

and anchor it to the peptidoglycan layer .

Outermembrane

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Phospholipid of outer membrane

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Is distinct from all other biological membranesIts outer leaflet contains a

lipopolysaccharides.Has special channels, consisting of

protein molecules called porins.

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Lipopolysaccharide (LPS)

outermost part of cellwall of Gram negative bacteria.

Consists of lipid A, core

polysaccharide and

a terminal series

of repeat units ( O antigen).

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Bacteria with atypical cell wall

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Mycobacteria and Nocardia.

Contains high concnentration ( around 60%) of hydrophobic waxy lipid , mycolic acid.

Mycolic acid prevent the uptake of dye .

Mycolic acids are present outside the

thin peptidoglycan layer linked by polysaccharides.

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Bacteria without cell wallMycoplasma .

Plasmamembrane of Mycoplasma contain sterol that are tough to protect from lysis.

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Bacteria with defective cell wallProtoplast Spheroplast L- forms

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Functions of cell wallProvides shape to the bacterium.Give rigidity to the organism.Protects from environment.Contains receptor sites for phages.Provides attachment to complement.Contains components toxic to host.Site of action of colicin.

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Cytoplasmic membrane( plasma membrane)

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Lies beneath the cell wall and separating it from the cell cytoplasm.

5-10 nm thick, elastic and semipermeable layer and comprises about 30% of the dry weight of bacterial cell.

Composed of mainly phospholipid (20-30%) and proteins (70-80%).

Phospholipids form bilayered structure in which proteins are embedded.

Phospholipid has two parts: Hydrophillic headHydrophobic tail

Two types of proteins are found: peripheral protein integral protein

Many enzymes are also present.

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Contd…

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Functions of plasma membrane Regulates the transport of nutrients

and waste products into and out of the cell

Synthesis of cell wall components Assists in DNA replication Secretes proteins Carries on electron transport system Captures energy in the form of ATP, etc.

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Movement of Molecules through Cytoplasmic Membrane

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Simple or passive diffusionSolute molecules cross the membrane as a result

of a difference in concentration of molecules across the membrane.

Speed and direction of diffusion depends on the relative concentration of molecules on each side of the membrane.

Ref: Microbiology by M.J. Pelczar

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Facilitated diffusion

Similar to that of simple diffusion.But requires carrier protein called permease

located in the cytoplasmic membrane.Entry of glycerol.

Ref: Microbiology by M.J. Pelczar

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Group translocation• Accumulates the solute inside the cell against concentration

gradient• Solute molecule altered chemically during transport.• PEP-dependent sugar-phosphotransferase system.• A heat stable carrier protein (HPr) is first activated by transfer

of phosphate group from PEP inside the cell . • At the same time sugar combines with enzyme II at the outer

membrane surface and is transported to inner membrane surface . Here it combines with phosphate group carried by activated HPr.

Ref: Microbiology by M.J. Pelczar

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Active Transport Almost all solutes , including sugars, amino acids, peptides,

nucleosides, and ions are taken up by cells through active transport.

• Entry of solutes occurs in three steps:-1)Binding of solute to carrier protein.2)Translocation of the solute- carrier complex, and3)Coupling of translocation to an energy yielding reaction to

lower the affinity of the carrier protein for the solute at the inner membrane surface so that the carrier protein will release solute to the cell interior.

Ref: Microbiology by M.J. Pelczar

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Two primary mechanisms of active transports, each utilizing a different form of energy.

A. Transport system that use proton motive forceUniporters : (eg. Potassium enters the cell via uniporter)Antiporters: (eg. Sodium is transported out of the cell as a proton

passes in )Symporters :(eg. A lactose molecule enters a cell with one

proton)

Contd…

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B.Transport system that use ATP:-

ABC transport system: (ABC stands for ATP Binding Cassette)

The ABC transport system utilizes a binding protein that resides immediately outside of the cytoplasmic membrane to deliver a given molecule to a

specific transport complex within the membrane.

Contd…

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Mesosome

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Convoluted or multilaminated membranous bodies.

Develop by complex invagination of the cytoplasmic membrane into the cytoplasm.

Function(1)Compartment of DNA at

cell division and at sporulation.

(2)Are principal sites of respiratory enzymes.(analogous to the mitochondria of the

eukaryotic cell)

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Nucleoid (nuclear material)

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Nucleoid contains a single dsDNA , which carry genetic information for cell.

Circular thread about 1 mm long, being condensed and looped into a supercoiled state, located centrally.

Nuclear division preceeding cell

division, two DNA may be present.

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Contd…

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Chromosomes have 2,000 to 4,000 genes.Many genes that encodes virulence factors

( adhesins, invasins, exotoxins,etc) are clustered adjacent to each other on chromosome , called pathogenicity islands.

These islands range in size from 10 to 200 kB. Can be horizontally transferred between

bacteria, resulting in enhanced virulence in the recipient.

made visible under the light microscope by Feulgen staining ( specific for DNA).

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Plasmid Many bacteria possess

plasmids in addition to chromosome.

Are circular dsDNA molecules .

size from 1.5 kilobase (kb) pairs to 120 kb pairs (less than one tenth the size of the bacterial chromosome)

Can exist and replicate independently of the chromosome or may be integrated with it.

Not required for host

growth and reproduction.

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Classification of plasmid

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Relaxed plasmid : Plasmid occur free in the cytoplasm and replicate independent of bacterial genome replication

Stringent plasmid: plasmid that integrate and replicate along with bacterial chromosome.

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36 Ref:- Brock biology of microorganisms.

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Ribosome Is spherical and granular structure with diameter of

100-200 A.Present in cytoplasm and may loosely attached to the

plasma membrane.Made up of both protein and ribonucleic acid (RNA). 70s type.Each 70s has two subunit :- larger 50s and smaller

30s.

30S

50S

50S

30S

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Contd…

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Functions

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Site of protein synthesis;

Matrix ribosomes synthesize proteins to remain within the cell and Plasma membrane ribosomes make proteins for transport to the outside.

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Inclusion bodyAre reserve deposits of

bacterial cell.Cells accumulate

nutrient when they are plenty and use when deficient.

Metachromatic

granules:- Reserve of

polyphosphate used in synthesis of ATP.

Characteristics of

Corynebacterium diphtherie.

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Polysaccharide granules:- Mainly consists of glycogen and starch.Lipid inclusions:- Poly – B-hydroxybutyric acid. Found in several species of Mycobacteria, Bacillus, Azotobacter, etcSulphur granules:- Present in sulphur bacteria .eg. Thiobacillus.

Carboxysomes :- contain enzyme ribulose-1,5-diphosphate carboxylase ,helps in CO2 fixation in photosynthetic bacteria ( Cyanobacteria and Thiobacillus, etc ).

Gas vacuoles:- Hollow cavities found in aquatic procaryotes. Consists of gases covered with proteins. Magnetosomes :- Inclusion of iron oxide. (Fe3O4) Decompose hydrogen peroxide. Found in Magnetospirillum , Magnetotactium, etc.

Contd…

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Endospore

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Highly resistant phase of bacteria, formed in unfavorable condition.

Formed internal to bacterial cell.Can survive in extreme heat, lack of

water, many chemicals, radiation , etc.. STRUCTURE:-1)Core ,2)Core wall,3) Cortex,4)Coat , and5)Exosporium .

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Contd…

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Resistance property of endospore is due to

- impermeability of their cortex and outer coat,

- high content of calcium and dipicolinic acid,

- low content of water (5-20%),

- very low metabolic and enzyme activity,

- DNA-binding proteins saturate spore DNA and protect it from heat.

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Sporulation

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Contd…

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Sporulation, occurs when growth ceases due to lack of nutrients.

Steps:-• An axial filament of nuclear material forms.• An inward folding of the cell membrane to enclose part

of the DNA and produce the forespore septum. • The membrane continues to grow and engulfs the

immature spore in a second membrane.• Cortex is laid in the space between the two

membranes, and both calcium and dipicolinic acid are accumulated.

• Protein coats then are formed around the cortex.• Maturation of the spore occurs. • Lytic enzymes destroy the sporangium releasing the

spore.

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Spore germination

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Contd…

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It has been estimated that 7500- year old endospore of Thermoactinomyces vulgaris from the freezing muds of Elk lake in Minnesota have germinated when warmed and placed in a nutrient medium.

The transformation of dormant spores into active vegetative cells is complex process.It occurs in three stages: (1) Activation, (2) Germination, and (3) Outgrowth. 1)An endospore will not germinate successfully, even in a nutrient-rich medium, unless it

has been activated. Activation is a reversible process that prepares spores for germination and usually

results from heat treatments.2)It is followed by germination, the breaking of the spore’s dormant state. It is characterized by spore swelling, rupture or absorption of the spore coat,

loss of refractility, Release of spore components, and increase in metabolic activity. Many normal metabolites or nutrients (e.g., amino acids and sugars) can

trigger germination after activation. 3)Grmination is followed by the outgrowth. The spore protoplast makes new components and develops again into an active

bacterium.

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Central (eg. Clostridium bifermentans)Subterminal ( eg. Cl. Perfringens)Oval and terminal ( eg. Cl. tertium)Spherical and terminal (eg.Cl. tetani)

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Demonstration of spore Spore stain ( Shaeffer –Fulton and Dorner

method)

Modified Ziehl Neelsen stain ( resist to decolorisation by 0.25 % H2SO4)

Evidence of presence of spore can can also be obtained by Gram staining where spore remains unstained.

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Fig- spore staining

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Capsule and slime layers

Hydrophobic gelatinous material sectreted outside and lies immediately in contact with the cell wall.

Consists largely of water and small content of solids (2%).

In most species, the solid material is a complex polysaccharide, though in some species

polypeptide or protein.

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Contd…

When the material is tightly associated with cell wall it is called capsule.

Capsules too thin to seen under the light microscope is called microcapsule.

Slime is an amorphous, colloidal material secreted extracellularly by some non-capsulated bacteria and also by many capsulated bacteria outside their capsules.

Glycocalyx is the term used for any carbohydrate molecule present on surface of cell.

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Functions of capsuleProtecting the cell wall against attack by various

kinds of antibacterial agents, e.g. bacteriophages, colicins, complement, lysozyme and other lytic enzymes. Thus the capsule is an important virulence determinant.

Is usually antigenic and the capsular antigens play a very important part in determining the antigenic specificity of bacteria.

Helps to adhere bacteria to surface.

Prevents from Phagocytosis.

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Method of demonstration

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1) Negative staining.

2) Special capsule staining using CuSO4 as mordant.

3) Quellung reaction

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Contd…

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Flagella

Organ of locomotion.Long , thin filaments,

regular.15-20 nm thick and

several times the length of the bacteria cell.

Originating in the bacterial protoplasm and extruded through the cell wall.

Made up of several thousand molecules of a protein subunit called

flagellin.

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StructureA flagellum has three basic parts:- Outer filament, contains the globular protein flagellin. Filament is attached to a slightly wider hook,

consisting of different protein, and The basal body, which anchors the flagellum to the

cell wall and plasma membrane.

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Flagellar arrangements•Bacteria without flagella are called atrichous.The arrangement of flagella may be Polar flagella :- flagella at one or both end.Monotrichous:- single flagellum at one end (eg. Vibrio cholera)Amphitrichous:- single flagellum at both ends (eg. Alkaligenes

faecalis)Lophotrichous:- tuft of flagella at one or both end (eg.

Helicobacter pylori)

Peritrichous flagella:- flagella arranged all round the body (eg. Escherichia coli , Proteus , etc)

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Flagella and bacterial motility The movement of flagella

results from rotation of basal body.

As the flagella rotates ,they form a bundle that pushes against the surrounding liquid and propels the bacterium and the flagellar rotation depends on the cells countinuous generation of energy.

Bacterial cells can alter the speed and direction of rotation of flagella and thus are capable of various patterns of motility.

When a bacterium moves in one direction for a length of time ,called run or swim.

Runs are interrupted by random changes in direction called tumble,caused by reversal of flagellar rotation.

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contd… The energy required for rotation of the flagellum comes from

the proton motive force. Proton movement across the cytoplasmic membrane through

the Mot complex drives rotation of the flagellum. In this model called the proton turbine model, protons

flowing through channels in the Mot proteins exert electrostatic forces on helically arranged charges on the rotor proteins.

Attractions between positive and negative charges would then cause the basal body to rotate as protons flow though the Mot proteins.

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Demonstration of motility

Hanging drop technique, Flagella stain, Growing in semisolid agar

media, Craigie’s tube, U –tube.

Craigies tube U-tube semisolid agar

Flagella stain

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Axial filament or endoflagellaPresent in spirochetes .Are bundle of fibrils, arise at the ends of the cell and

spiral around the cell.have structure similar to that of flagella.The rotation of the filaments produces movement of

the outer sheath that propells the spirochetes in spiral motion.

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Pili or FimbriaeOrgan of adhesion.Short , hairlike appendages thinner and smaller than

flagella, originated from cellwall.About 3 -10 um in length and 0.03-o.2nm in diameter.Composed of helically arranged protein subunits ,

pilin.Some fimbriae cause hemagglutination with RBC of

guinea pig, fowl, horses, etc and can be inhibited by 0.1-0.5% D-mannose (MS).

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Types of fimbriae

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6 types of fimbriae.Type1 : relatively thick and involve in

haemagglutination (MS), (E. coli, Salmonella spp).Type2 : resembles MS type1 but non-

haemagluttinating( S. gallinarum, S. pullorum).Type3 : thin and MR and cause indirect

haemagglutination (RBC treated with tannic acid),( Proteus spp).

Type4 : thinner than MR type3 fimbriae and have MR haemagglutinating activity for fresh RBC.

Type5 : are very long ( some Klebsiella spp)Type6 : are monopolar and found only in

Pseudomonas spp.

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Function of pilli

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Commom pili (fimbriae): Playing a role in

the adherence of symbiotic and pathogenic bacteria to host cells.

(Minor proteins termed adhesins are located at the tips of pili and are responsible for the attachment properties).

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Sex pili: long and flexible structure,being responsible for the attachment of donor and recipient cells in bacterial conjugation.

Pili also help in formation of pellicle on surface of stagnant liquid medium.

Contd…

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GROWTH AND NUTRITION OF BACTERIA

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Nutrition requirements

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Bacteria have same general chemical pattern as the cells of other organism.

Principal constituent of the cell is water, proteins , polysaccharides , lipids, nucleic acids, mucopeptides and other low molecular weigth compounds.

For growth and muliplication of bacteria,the minimum requirements are

Water, Source of carbon and energy, Source of nitrogen,and Some inorganic salts.

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Macronutrients

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• Microorganisms require some elements in large quantities, because they are used to construct carbohydrates, lipids, proteins, and nucleic acids.

• 95% or more of cell dry weight is made up of a few major elements: C ,O , H, N , S and P.

• Required in large amount.

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Trace Elements

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Microbes require very small amounts of other mineral elements, such as Fe , Cu, Mo , Zn, etc .

Most are essential for activity of certain enzymes, usually as cofactors.

Eg- Mg²⁺ for Hexokinase , Ni⁴⁺ for urease, Fe²⁺ for cytochrome oxidase , etc…

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71 Ref:-Microbiology by Anderson and Nester.

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Growth Factors

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Organic compounds required in minute quantities and are not synthesized by bacteria, also called bacterial vitamins.

Eg…Neisseria spp require at least 40 additional ingredients, including 7 vitamins and all of the 20 amino acids.

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Carbon and Energy source

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Bactrial Growth

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It is an increase in number of population rather than in size.

Bacteria divide by binary fission.

The number of cell arising from a single cell is 2n after n generations.

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Binary fission

75 Ref:-Microbiology, Nester

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Generation time

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Time required for a bacterium to give rise to 2 daughter cells under optimum conditions

Also called population doubling time.

Escherichia coli – 20 mins.

Mycobacterium tuberculosis – 20 hrs.

M.leprae – 20 days.

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Bacterial Growth Curve

77 Ref:-Microbiology, Nester

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Lag phase

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Phase of adaptation in new medium.Necessary enzymes and metabolites

are built up.Bacterial cell attain maximum size and is

critical stage for multiplication.Length depends on generation time of bacteria, size of inoculum, quantity of culture medium, environmental factors, etc…

Average time:- 2-6 hours.

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Log or exponential phase

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Cell division occurs at maximum rate and number increases exponentially with time.

Generation time is constant for a given strain under same set of condition.

Becomes smaller in size.Bacteria are most sensitive to antimicrobial

substances.

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Stationary phase

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Growth ceases due to exhaustion of nutrients,

accumulation of of toxic metabolites and lowering of pH.

Produce secondary metabolites.Frequently gram variable and stain irregularly.Sporulation occurs.Total count remains same.

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Death or decline phase

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Cells begin to die due to cessation of growth, accumulation of toxic products, autolytic and heterolytic enzymes.

Rapid fall in viable count.Some cells remain survive at the expense of

nutrients released from death of others.

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Phase of prolonged decline ( survival phase)

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Factors affecting bacterial growth

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Water

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About 80% of bacterial cell is water hence it is essential requirement.

Vehicle for entry of nutrients and elimination of waste products.

Participate in metabolic reactions and form integral part of protoplasm.

Some organism like Treponema pallidum are highly sensitive while others like Staphylococci withstand drying for months.

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Temperature and Bacterial growth

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Major environmental factor controlling growth.

Minimum:- below which growth ceases.

Optimum:- at which maximum growth occur.

Maximum:- above which growth ceases.

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Classification by tempr. requirement

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Psychrophiles:- best grow at low temprature (-5 -15⁰C)Produce enzymes that functions optimally in

cold.Membrane contain high content of

polyunaturated fatty acids which maintain rigidity at low temp.

eg. Yersinia enterocolitica , Listeria monocytogens , Pseudomonas fluorescens.

Psychrotrophs grows optimally at 20-30⁰C but can grow in refrigerator temprature.

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Mesophiles

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Temprature range for growth 25-45⁰C.Optimum growth temp 37⁰C.Are mostly pathogenic bacteria.Eg..Escherichia coli , staphylococcus aureus

, etc.

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Thermophiles and Hyperthermophiles

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Range 45-70⁰CContain heat stable enzymes and proteins.Membrane contains lipids rich in saturated

fatty acids and stabilizes the membrane at high temp.

All thermophiles contain reverse gyrase ( a unique type of DNA 1 topoisomerase) that stabilizes DNA.

Eg..Bacillus sterothermophilus.Hyperthermophiles- grow at temp above 70⁰C.Eg…Thermotoga, Methanopyrus.

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Oxygen and bacterial growth

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Different bacteria have different oxygen requirement.

When it accepts electron in ETC , it forms H2O2 , O2‾ , and OH‾‾ , all of these are toxic unless broken down.

Three enzymes namely catalase , peroxidase and superoxide dismutase(SOD) prevent bacteria from oxidative damage by breakdown of these oxygen radicals.

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Classification based on O2 requirement

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Obligate aerobes:- Grow only in presence of oxygen. Have all three enzymes and they have ETC as final

electron acceptor. Eg.. Pseudomonas , Brucella , Mycobacteria ,

Nocardia spp etc. Facultative anerobes:- prefer aerobic condition but can grow without

oxygen.Also have all three enzymes.Eg..E. coli, S. aureus, etcMicroaerophiles:- Are aerobes but require little of oxygen for growth. Eg… Campylobacter jejuni, etc.

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Anaerobes

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Obligate anaerobes:- Grow only in absence of oxygen and

oxygen is lethal for them.Lacks enzymes for using oxygen in

respiration.Enzymes of anaerobes are active only in

reduced state.Eg…Clostridium sps, Bacteroides sps, etc...Aerotolerent anaerobes:-Do not utilize oxygen , but can tolerate low

amount of oxygen because they have superoxide dismutase. ( no catalase)

Eg..Lactobacilli , anaerobic streptococci , etc

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Contd…

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Carbondioxide( CO2)

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Small amount of CO2 is required by all bacteria.

Made available endogenously or exogenously.

Some organisms grow best at higher CO2 tension and are called capnophillic.

Eg..Brucella sps , gonococcus, meningococcus.

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Hydrogen ion concentration(pH)

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Most bacteria grow at neutral or slightly alkaline pH.

pH can harm organisms by disrupting the plasma membrane , inhibiting the activity of enzymes and membrane proteins and ionization of nutrient molecules.

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Classification

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Acidophiles:- grow at acidic pH. pH range ( < 5.0). Eg..Lactobacillus sps , Thiobacillus spp,etc.Neutrophiles:- pH range ( > 5.0 and <8 ).Maintains pH by exchanging potassium ion with proton by

antiport transport system.Many pathogenic bacteria are neutrophiles.Alkalophiles:-pH range ( > 8 ).Maintain pH by exchanging internal sodium ion for external

proton.Eg.. Vibrio cholera , Bacillus alkalophilus, etc…

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Osmotic pressure

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Bacteria can withstand wide range of external osmotic pressure because of protective cellwall.

0.5% of NaCl is added in most culture medium to maintain suitable environment.

Hypertonic solution cause withdrawl of water and shrinkage of protoplasm (plasmolysis).

Hypotonic solution cause swelling and hence rupture of cell (plasmoptysis).

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contd…

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Some bacteria grow at high pressure , i.e. 400-500 atm (mostly in deep sea) and are called barophiles.

They have huge amount of monounsaturated fatty acids in membrane and OmpH in cellwall outer membrane.

Eg..Photobacterium shewanella , Colwella sps, etc.

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BACTERIAL METABOLISM

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Metabolism

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Sum total of all the chemical reactions occuring in the cell ( i.e. biosynthetic and degradative)

Metabolism in bacteria is essential for their existance , for environment , and products are commercially and medically important for human beings.

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CATABOLIC AND ANABOLIC REACTIONS

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Reactions that cause breakdown of complex molecules into simpler form with relase of energy is catabolic reactions.

Energy requiring reactions that build up complex organic molecules from simpler ones is

anabolic reactions.

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Components of metabolism

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COMPONENTS FUNCTIONS

Enzymes Biological catalyst, fascilitates each step of metabolic reaction by lowering the activation energy of reaction.

Adenosine triphosphate (ATP)

serves as energy currency of cell ,

Energy source Compund that is oxidised to release energy, also called an electron donor.

Electron carriers carry the electrons that are removed during the oxidation of energy source (NAD⁺, NADP⁺ , and FAD ( their reduced form NADH , NADPH , and FADH₂) .

Precursor metabolites

Intermediate metabolite that link anabolic and catabolic pathways, like pyruvate, acetyl-coA, glucose -6-p, etc.

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Role of ATP

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Is energy currency of cell, serving as ready and immediate donor of free energy.

Energy is releases when phosphate bond is broken, hence it is called high energy phosphate bond.

Synthesis and breakdown of ATP continuously occurs in cell during degradative

and synthetic process.

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Generation of ATP

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Bacteria uses three mechanism of phosphorylation to generate ATP from ADP.

1)Substrate level phosphorylation

C-C-C-P + ADP C-C-C + ATP

In this mechanism , a high energy phosphate from a phosphorylated substrate is directly transferred to to

ADP.

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Contd…

104

2) Oxidative phosphorylation

3)Photophosphorylation

Occurs in phototrophs.Derive ATP using radiant energy of the sun.These ATP are then utilized to synthesize

mainly glucose .

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METABOLIC PATHWAYS OF ENERGY GENERATION

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GLYCOLYSIS

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Embden-Mayerhof Parnas pathway.

Stepwise Conversion of glucose to pyruvate and each step require specific enzyme.

Occurs in cytosol. Does not require oxygen and

hence occur in both aerobic and anaerobic bacteria.

Three phases :- preparatory phase, splitting phase and energy generation phase.

2 molecules of pyruvic acids are formed from each glucose .

Net gain of 2 ATP by substrate level

phosphorylation and formation of 2 reduced substrate i.e. NADH.

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Pathways alternative to glycolysis

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Many bacteria have another pathway in addition to glycolysis for degradation of glucose.

1) Pentose phosphate pathway, and2) Entner Doudoroff pathway.

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Pentose phosphate pathway

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Hexose monophosphate shunt.

Occurs simultaneously with glycolysis and provides breakdown of both pentose sugar and glucose.

Important Feature :- intermediate pentoses are used for nucleic acid synthesis, amino acid synthesis and glucose from CO2 in photosynthetics.

Important producer of reduced coenzyme i.e. NADPH , used for biosynthetic reactions.

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Entner –Doudoroff pathway

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Bacteria having enzyme for Entner –Doudoroff pathway can metabolize without glycolysis or PPP.

Found in some Gram negative bacteria like Psedomonas spp,

Rhizobium, Agrobacterium,etc.. and generally not found in Gram positive bacteria.

Produces 1 molecule NADH, 1 molecule NADPH and 1

molecule of ATP ( from 1 glucose).

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Cellular respiration and fermentation

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Pyruvate obatained from glucose breakdown are channeled either to respiration or to fermentation.

RESPIRATION:- is ATP generating process in which molecules are oxidized and the final electron acceptor is an inorganic molecules.

TYPES OF RESPIRATION :-Aerobic respiration:- final electron acceptor is O₂ and occurs in aerobes.Anaerobic respiration: final electron acceptor is inorganic molecule other than O₂ .

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Krebs cycle or TCA cycle

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Is second phase of aerobic respiration.Cytoplasmic membrane.Pyruvate formed enters TCA cycle only after

converted into acetylCoA by decarboxylation rxn ( transition phase).

Starts with condensaion of acetylCoA and oxaloacetate.

AcetylCoA is oxidized and released as CO2 and oxaloacetate is regenerated.

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Contd…

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Cntd…….

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Contd…

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From TCA cycle, oxidation of one acetylCoA molecule produce 3 NADH , 1 FADH₂ and 2 GTP.

All the NADH and FADH2 enters into electron transport chain and finally ATP are generated.

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Electron transport chain

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Last phase of respiration which generates ATP from reduced substrates.

Consists of a sequence of carrier molecules though which electrons passes.

Occurs in plasma membrane ( eukaryotic cell- inner mitochondrial membrane).

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Contd…

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Three classes of carrier molecules 1)Flavoprotein :- coenzyme derived from viatmin B2

(riboflavin) and perform alternating oxidation and reduction reaction.

3)Ubiquinones :- small non protein carriers.2)Cytochromes :-protein with iron containing group

(heme).

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• Electron transport chain is different in different bacteria , even a single bacteria have more than one type of ETC but their target is to derive energy in the form of ATP.

Contd…

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Contd…

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Anaerobic respiration

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Final electron acceptor is NO3⁻ , NO2⁻, N₂O, SO₄⁻⁻,CO₃⁻⁻, etc. and occurs in strict anaeres and facultative anaerobes.

Sometimes Pseudomonas sps and Bacillus sps can use nitrate as final e⁻ acceptor.

ATP generation varies from bacteria to bacteria and always less than aerobic respiration.

Alternative electron carriers are used in the ETC.

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Fermentation

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Used by organisms that cannot respire because of either lack of inorganic electron acceptor or absence of ETS.

Terminal electron acceptor is always organic compound.

End product depends on type of microrganisms.

Analysis of end product is valuable in identifying particular bacteria.

ATP generating pathway is usually glycolysis( 2ATP).

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Products of fermentation

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Catabolism of other substrates

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BACTERIAL TOXINS

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Toxins

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Are virulence factor of most of bacteria and one of the major cause of tissue damage.

Two types :- endotoxin and exotoxin.

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Exotoxin Endotoxin Secreted outside cell by both Gram positive and Gram negative bacteria

Relesed after lysis of Gram negative cellwall

Protein LipopolysaccharideHeat labile (except- enterotoxin of S.aureus )

Heat stable ( upto 250⁰c)

Highly antigenic Less antigenic Highly toxic in minute dose ( microgram is fatal to animals)

Moderately toxic

Filterable not so(obtained only by cell lysis )

Can be converted to toxoid cannot Often enzymatic action no

Properties of bacterial toxin

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Endotoxin

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LPS of Gram negative cell has three parts:- O- antigen , core polysaccharide and

lipid A.Released usually when

the cell is lysed but can also be released during vegetative growth.

Has same chemical composition in almost all bacteria and has same toxic effect ( no matter which bacteria produce it).

Encoded by

chromosomal gene.

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Mode of action

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Endotoxin ( lipid A)Activates macrophages

IL-1/IL-6(fever)

TNF(fever and hypotension) i

NO(hypotension)

Activates complementC3a (hypotension , edema)

C5a (neutrophil chemotaxis)

Activates tissue factorCoagulation cascade ( DIC)

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Exotoxin

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Are most powerful and active in small quantities.

Either secreted by organism or leak into the surrounding fluid after lysis of bacterial cell.

Gene for exotoxin may be present on chromosome or plasmid or bacteriophage DNA.

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A-B toxin

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Have two components (A and B)

B components binds to specific cell receptor and facilitate the internalization of A.

Component A is active (toxic) component.

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Superantigen

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Contd…

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Some bacteria directly inject exotoxin into target cell via needle like projections called injectosome.

Also called type III secretory system.

Bacteria having type III secretory system

are more virulent.

Fig:- Injectosome

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BACTERIOCIN

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BACTERIOCIN

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Produced by many bacteria, protein in nature and have bactericidal activity.

Have killing action on strains of same or closely related species.

First reported by Gratia in 1925, Escherichia coli producing a substances which is active against other strains of the same species.

Colicin – Gratia and Frederique in 1946.Bacteriocin- Jacob and Woolman in 1953.

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contd…

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Are named on the basis of their bacterial

species of origin . Some of them arei) Colicins are bacteriocins of E.coli, ii) Aeruginocin of P. aeruginosa, iii) Diphthericins - C. diphtheriaeiv) Cloacin of Enterobacter cloaceae, v) Pesticin of Y. pestis, vi) Monocin of Listeria monocytogenes, vii) Cerecin of Bacillus cereusviii) Staphylococcin of Staphylococcus aureus,ix) Warnerin of et S. warneri

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Target cells have specific receptor for attachment of bacteriocin( same as bacterophage)

Bacteria producing bacteriocin also carry gene for immunity to them on chromosome or plasmid.

Many have narrow inhibitory spectrum of activity ( but some have activity on broad class of bacteria)

Are plasmid or chromosomal mediated.

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Gram negative bacteriocin

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Gram positive bacteriocin

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Mode of action

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Importance

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Bacteriocins produced by non-pathogenic bacteria kills other pathogenic bacteria(Normal flora vs. Pathogens).

Bacteriocins have also been suggested for certain cancer treatment.

Used for food presrvation in food industry ( eg..nisin produced by Lactococcus lactis is active against many food spoiling bacteria)

Used for bacteriocin typing of clinical strains to aid in their identification and characterization.

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Bacteriocin typing

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Done for identification of isolated strains : as they are same or different.

A strain may be typed by:1) Activity of their bacteriocin against a set of

indicator strains of same or closely related species, or

2) Pattern of their susceptibility to the bacteriocin of a set of indicator strains.

If the isolates are same strains, their bacteriocin production and susceptibility patterns will be identical.

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Pyocin typing

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Gillies and Govan(1966)- cross streaking method

- The test strains are inoculated across the surface of BHI agar plates.

- After overnight incubation at 37ᴼC , culture is exposed to chloroform to kill the test strains and then the test (producing) strains are scrapped-off from the plate by using slide.

- The sensitive strains (Indicator strains) are cross-streaked at right angle to the test strains.

- Incubated at 37ᴼC for 24 hours.- Observed for inhibition of growth at each side

of the producing strain.

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Contd…

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In pyocin typing technique with cross streaking method, 105 main types and 25 subtypes can be identified on the basis of pyocin production by test strains using 13 indicators(1 to 8 and A to E).

Disadvantages of streak methodTo remove test strains growth before

application of indicator strain.Not reliable for mucoid colony of P. aeruginosa.48 hour period is needed to obtain result.

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Bibiliography

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Microbiology : A Human Perspective by Anderson and Nester

Topley and Wilsons Microbiology and Microbial infections, vol. 2

A Handbook of Clinical Microbiology ; Prof. Dr. Bharat Mani Pokhrel

Microbiology by TortoraAnanthanarayan and Paniker Text Book Of

MicrobiologyBrock Biology of Microorganisms; Medigan and

Martinko.Microbiology ; Lansing M. Prescott.Medical bacteriology ; N.C. Dey.

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THANK YOU

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