MICI 1100 Health Sciences Microbiology

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MICI 1100Health Sciences Microbiology
Welcome to

• Course Coordinator
• Dr David Haldane Rm 326 Mackenzie Building, QE II
  HSC
• David.Haldane_at_cdha.nshealth.ca

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Objectives of the Course

• To have an appreciation of the development of
  microbiology relating to infection.
• To understand the structure and physiology of
  microorganisms of different types.
• Be able to recognize genera and important species
  by name.
• To have an understanding of the types of
  infectious disease.
• To have an understanding of the role of
  particular organisms in infections, and how
  infection is caused.
• Be aware of the range of organisms causing
  disease, and how to distinguish groups of
  organisms.
• To understand the sources, and routes of
  transmission of organisms
• To have an understanding of how infectious
  diseases are manifested in the host.

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Objectives

• To understand the nature and role of the immune
  system
• To know the role of immunization in the
  prevention of infection.
• To have an understanding of the range and
  principle mode of action of antimicrobial agents.
• To have an understanding of the means by which
  organisms are resistant to antimicrobials.
• To have an understanding of the principles of
  environmental control of organisms.
• To have an understanding of the principles of
  infection control.
• To be able to provide appropriate specimens and
  understand laboratory results for microbiology.
• To have an awareness of the laboratory techniques
  used in the diagnosis of infectious disease.

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Milestones in Microbiology
Anthon van Leeuwenhoek (1632-1723)

• Ancient and Medieval Times microorganisms were
  unknown and their effects (e.g. plagues) were
  attributed to Divine judgement, magic, or
  sorcery.
• 1674 - Anton van Leeuwenhoek observes
  microorganisms - "animalcules" - and reports them
  to the British Royal Society.
• 1798 - Jenner uses the first vaccine and
  begins a process that will lead to the
  eradication of smallpox in the 1970s.

Edward Jenner (1749 - 1823)
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Milestones (contd)

• 17th-19th - The theory of spontaneous generation
  (that organisms were generated from rotting
  organic material) was slowly disproved, a process
  which was finally completed by Pasteur and
  Tyndall.
• 1850 Semelweiss shows the value of hygiene
• 1860s - Pasteur furthers the germ theory of
  disease by his work on silkworms, and develops
  pasteurization.
• 1870s - Lister uses "antisepsis" to control
  surgical infections.
• 1876 - Koch demonstrates that anthrax is caused
  by Bacillus anthracis.

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Milestones in Microbiology (contd)

• 1881 Lina Hesse suggests agar to solidify
  growth media for bacteria.
• 1880-1900 - The Golden Age of Microbiology -
  many pathogens first identified.
• 1940s - Development of antibiotics begins.
• 1940spresent - Widespread use of immunization
  leads to huge reductions in illness and death
  caused by many previously common infections, e.g.
  measles, diphtheria.
• 1980s - Development of molecular techniques for
  diagnosis and engineering begins.

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Koch's postulates - to establish if an organism
is the cause of a disease

• The same organism must be found in all cases of a
  given disease.
• The organism must be isolated and grown in pure
  culture.
• Organisms from the pure culture must reproduce
  the disease when inoculated into a healthy
  susceptible animal.
• The organism must then again be isolated from the
  experimentally infected animal.

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Organisms - Morphology (shapes)

• Cocci
• Streptococci (Strepto - chain)
• Staphylococci (Staphylo - grapes)
• Rods (bacilli)
• very short rods - coccobacilli
• curved rods - vibrio
• spiral rods - spirochaetes
• Filaments with branching
• actinomycetes

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Staphylococci
Rods
Bacterial Morphology
Streptococci
Vibrio
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Branching Filaments
Branching Rods
More Bacterial Morphology
Cocco-bacilli
Spirochaetes
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Structures of Bacteria

• Appendages - flagella - fimbriae - pili
• Surface and cell wall - capsule -
  cell wall - cell membrane
• Cytoplasm - Bacterial chromosome -
  Plasmids - Ribosomes - Inclusions
• Other structures - Endospores

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Appendages - project from the cell

• Flagella
• Long slender, structures made of protein
• Whip like structures
• Enable bacteria to move by rotating like a
  propeller
• Can only be seen using special stains or electron
  microscopy
• Can be single (monotrichous), or multiple, in
  tufts or around the cell (peritrichous).

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Flagella - Arrangements
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Appendages - project from the cell (contd)

• Fimbriae
• Shorter, thinner filaments made of protein
• Enable bacteria to attach to substances
• Pili
• Similar to fimbriae in structure
• Involved in transfer of DNA between bacteria

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Appendages to Bacterial Cells
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Surface and Cell Wall

• Capsule
• Material that is secreted by bacteria and covers
  the exterior of the cell
• Often polysaccharide
• May be a thick layer slime coating
• Cell Wall
• Differs from animal cells, or fungi
• A strong layer made of peptidoglycan
• Maintains cell shape and integrity
• A principle target for antibiotic action
• Stains using the Gram stain. Differs for Gram
  positive vs Gram negative organisms

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Surface and Cell Wall (contd)

• Gram Positive
• Thick peptidoglycan layer
• No outer membrane
• Gram Negative
• Outer membrane
• Thin peptidoglycan layer
• Space between membranes is periplasmic space

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Surface and Cell Wall (contd)

• Cell Membrane
• Lipid bilayer with proteins
• Controls the entrance and exit of substances from
  the cell
• Contains enzymes involved in cell wall
  production, cellular metabolism, and production
  of some extra-cellular materials
• In gram negatives, it contains endotoxin
• Cytoplasm
• Liquid containing a variety of substances
• It is where metabolism occurs

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Surface and Cell Wall (contd)

• Ribosomes
• Made of RNA and protein
• Structures where proteins are made
• Two subunits. Bacterial ribosomes are different
  from ribosomes in animal or plant cells
  (eukaryotic cells)
• Bacterial Chromosome
• Made of DNA
• A single long circular molecular of DNA
• Not separated from cytoplasm (as in animal or
  plant cells which have nuclei)

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Surface and Cell Wall (contd)

• Plasmids
• Small, circular pieces of DNA
• Separate from the chromosome
• Can be transferred between bacteria
• May carry genes for antibiotic resistance
• Inclusions
• Granules in the cytoplasm
• May act as storage of various substances

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Other Structures

• Endospores ("spore")
• Environmentally tough, dormant form
• Develop in cytoplasm of bacteria
• Do not grow or divide
• Can remain viable for long periods
• Only formed by certain genera of bacteria
• Germinates to form a new cell

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Bacterial Taxonomy

• How are bacteria organized and classified
• Domains
• Cells lacking nuclei (prokaryotes) vs cells with
  nuclei (eukaryotes)
• Kingdoms
• Animals
• Plants
• Fungi
• Protista
• Monera - the prokaryotic organisms
• (Note Different systems are used this one is
  convenient)

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Bacterial Taxonomy (contd)

• Classification Kingdom Phylum Class Order Fam
  ily ? Used most Genus ? frequently
  in Species ? clinical practise

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Bacterial Taxonomy ( contd)

• Characteristics used to classify organisms
• Traditional
• Size, shape, gram reaction, need for O2
• Ability to metabolize sugars
• Metabolic end products
• Supplemented by
• Comparison of 100-300 characteristics
• Nucleic acid sequence of ribosomal RNA

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General Groupings used in Taxonomy

• Aerobic (grows in air), obligate if must have O2.
  Capnophilic if needs CO2.
• Facultative anaerobe (grows in air, and can grow
  without oxygen).
• Anaerobe (grows without oxygen, and most species
  do not grow well in air as O2 is toxic for them).
• Microaerophilic (grows in a low concentration of
  oxygen, but not in its absence or in air).

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Staining Organisms

• Needed to allow us to see the organisms using
  light microscopy
• Organisms are killed in the process
• Simple stains
• stain is applied and colours the organism
• e.g. methylene blue

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Complex Stains

• stains may be combined which
  stain different structures different colours.
  e.g. giemsa stains malarial parasites nucleus red
  and cytoplasm blue
• stains may be applied in sequence with a step to
  remove stain in between. e.g. gram stain - a key
  stain in microbiology!!

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The Gram Stain

• Developed by Christian Gram in the 19th Century
• He found that a stain could be washed out of some
  organisms much more easily than others
• Technique allows differentiation of many bacteria
  into 2 groups gram positive and gram negative
  corresponding to cell wall type.
• Continues to be used extensively and is important!

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Method for Gram Stain

• Crystal violet stains all the bacteria
  dark purple
• Iodine binds to crystal violet and fixes it
  (acts as a mordant)
• Alcohol/Acetone washes out the stain from gram
  negative bacteria
• (Gram originally stopped here, so that organisms
  that stained purple were positive because they
  could be seen subsequently the fourth step was
  added so that both the positive and the negative
  organisms could be seen.)
• Safranin stains the gram negative bacteria pink.

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a
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Acid Fast Stain

• Some bacteria cannot be stained by the gram stain
  because of lipids in the cell walls. (e.g.
  Mycobacterium tuberculosis, the tuberculosis
  bacterium) These bacteria may be stained by an
  acid fast method.
• involves - staining with a
  strong red stain (to force
  the stain into the cell)
• washing out the stain with
  a mixture of acid and alcohol
  
• restaining (counterstaining)
  with a blue or green stain.
• Acid Fast organisms are Red. These are sometimes
  called AFB (acid fast bacilli).
• Other organisms are the colour of the counter
  stain (blue or green).

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Bacterial Growth Requirements and Metabolism
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Requirements for Bacterial Growth

• Carbon source
• Nitrogen source
• Essential nutrients
• Temperature
• Atmosphere
• Inorganic ions, iron
• pH
• Water

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Requirements for Bacterial Growth (contd)

• Carbon Sources
• Simple carbohydrates, sugars, proteins
• Some organisms can fix CO2
• Nitrogen Sources
• Protein, amino acid, peptides
• Nitrates, ammonium salts
• Some organisms can fix N2

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Requirements for Bacterial Growth (cont'd)

• Essential Nutrients
• Bacteria vary in their requirements
• Some can synthesize all their needs
• Others need complex organic molecules, blood,
  vitamins to grow. These are called fastidious.
• Temperature
• Bacteria (like humans) grow best at certain
  temperatures.
• Mesophiles grow best between 20-40C. Other
  types are best adapted to growing below 15?, or
  above 40-45?.
• Human pathogens are usually mesophiles.

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Requirements for Bacterial Growth (cont'd)

• Oxygen
• Acts as a final electron accepter in aerobic
  organisms.
• The superoxide radical (O2-) is toxic and must be
  rendered safe for cells to survive. Anaerobic
  organisms lack the means to detoxify O2-.
• Iron
• Required for enzyme action.
• Fe3 is insoluble.
• Bacteria produce siderophores, which bind to Fe3
  and make it possible to import it.

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Requirements for Bacterial Growth (contd)

• pH
• Most organisms prefer neutral conditions.
• Bacteria tend to die in acidic conditions (pH
  lt6).
• Water
• Bacteria require soluble nutrients for diffusion
  into the cell.
• Growth is inhibited in strong solutions.
• Bacteria with defective cell walls burst in very
  weak solutions.

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Growth of Bacteria

• Bacteria multiply by binary fission (a single
  cell separates to form two new cells of equal
  size).
• The rate of growth is limited by
• the availability of nutrients
• temperature
• ability to remove toxic products
• The time required to divide is called the
  generation time.
• for most organisms, it is measured in minutes

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Phases of Growth of Microbial Populations

• Lag phase
• Adaption to environment
• Active synthesis of enzymes and other
  constituents
• Log (i.e. logarithmic) phase
• Rapid reproduction
• Antibiotics most active

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Phases of Growth of Microbial Populations (contd)

• Stationary Phase
• Rate of reproduction equals rate of cell death
• Nutrients depleted
• Toxic metabolites accumulate
• Death Phase
• Death rate exceeds reproduction

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Phases of Bacterial Growth
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Metabolism
ATP

• Anabolism
• building organic molecules using small molecules
  energy
• Catabolism
• breakdown of chemical nutrients with release of
  energy
• Cells store energy as adenosine triphosphate
  (ATP) as substrates are oxidized

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Metabolism (cont'd)

• Anabolism
• Energy consuming process of building cell
  components.
• Protein synthesis by polymerization of amino
  acids.
• Glycogen and cell wall by polymerization of
  glucose.
• Lipids synthesis.
• Nucleic acid synthesis.

Starch
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Importation of Nutrients

• Active Transport - enzymes move substrate into
  the cell, requiring energy.
• Concentration inside the cell higher than outside
• No modification of substrate
• Group Translocation - enzymes modify a substance
  as it comes into the cell.
• Diffusion of altered substrate is reduced
• Energy required

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Importation of Nutrients (contd)

• Facilitated Diffusion - enzymes aid diffusion but
  no energy required.
• No modification of substrate
• Concentration does not exceed exterior conc.

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Glycolysis

• Gycolysis - glucose is broken down to pyruvic
  acid, the pyruvic acid is further broken down,
  and the products differ for different bacteria,
  but include organic acids and alcohols.

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Respiration

• Krebs cycle (also called tricarboxylic acid
  cycle, citric acid cycle)
• pyruvate is degraded to CO2 and H2O.
• Only used in aerobic organisms
• Results in much more energy production

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Catabolism

• Respiration
• electrons pass to O2 eventually (oxidative
  phosphorylation)

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• Fermentation
• anaerobic process, electrons are transferred to
  form other organic compounds, e.g. ethanol

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Other Catabolism

• lipase Lipids ?? glycerol ?
  glycolysis fatty acids ? oxidized
• protease Proteins ?? amino acids
  ? protein synthesis or ? further breakdown

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Sterilization and Disinfection
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Disinfection

• Disinfection using Chemicals.
• Antiseptics - "disinfectants" that can be used on
  skin.
• Disinfectant - usually used on inanimate objects.
• May kill bacteria (bactericide) or prevent growth
  (bacteriostatic agent).
• Pasteurization
• Preservation - drying, osmotic methods, etc.

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Disinfection (contd)

• Factors important in disinfectant activity
• Disinfectant concentration
• Time of exposure
• Number and type of microbes present
• Nature of material to be disinfected
• Mode of action
• Disruption of cell membrane (e.g. detergents).
• Denaturation of proteins (e.g. alcohol).

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Examples of Disinfectants

• Phenol based - disrupt cell membranes and
  precipitate proteins.
• As phenol is toxic, chemically altered
  (substituted) phenols phenolics- are used.
• Cresol - similar action to phenol. e.g. Lysol.
• Biguanides disrupt plasma membrane. Nontoxic.
• e.g. chlorhexidine used for skin disinfection.
• Alcohols - denature proteins.
• 70 is more effective than 100
• Requires adequate time for activity

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Examples of Disinfectants (contd)

• Halogens (fluorine, chlorine, iodine) - acts by
  oxidation of enzymes.
• Hypochlorite (javex) is commonly used
• Inactivated by organic material
• Activity of preparations drops after opening
• Quarternary ammonium compounds - possibly disrupt
  membranes
• Often combined with detergents
• Commonly used for environmental cleaning

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Examples of Disinfectants (contd)

• Detergents - disrupt cell membranes.
• Heavy metals. (e.g. copper, lead)

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High Level Disinfectants

• Substances able to kill spores, tubercle bacilli,
  and viruses given enough time.
• Examples
• Glutaraldehyde
• Formaldehyde

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Sterilization

• Elimination of viable organisms.
• Used for substances/devices to be inoculated into
  or to enter patients.

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Methods

• Heat
• moist (autoclaving)
• dry (oven, less effective)
• Gas
• ethylene oxide
• Oxidizing agents
• ozone, H2O2
• Irradiation
• Filtration (does not eliminate viruses)

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Autoclaving

• Moist heat (steam) at increased pressure for a
  defined time.
• Can be used for most items (e.g. surgical
  instruments, fabrics, etc.).
• Ability to kill spores should be checked weekly.

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Gas

• Used for objects damaged by heat or radiation.
• Requires aeration step after sterilization

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Radiation

• Used in industry for plastic objects, fluids, etc.

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Bacterial Pathogenicity Virulence Factors and
Genetics
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Microbial Ecology

• Relationships between host and microbes.
• Commensal - Microbe received benefit, but there
  is no harm to the host.
• Opportunist - Microbe received benefit, and is
  able to cause disease if host defenses are
  weakened.
• Pathogenicity - The ability of an organism to
  cause disease.

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Microbial Ecology (contd)

• Virulence - The extent to which an organism can
  cause severe disease.
• Normal Flora - The community of organisms that
  normally exist on a body surface, the
  constituents vary according to the site.

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Transmission of Infection

• Sources may be
• from the normal flora
• from other sources
• Other sources
• people
• animals (direct or via food)
• environment
• vectors and fomites

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Transmission of Infection (contd)

• Vector a small organism (e.g. insect) that
  transmits an infectious agent.
• Fomite an inanimate object that transmits
  infection when contaminated. e.g. doorknob.
• For further details, see the Infection Control
  lecture.

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Virulence Factors

• The properties that an organism has to enable it
  to cause infection.
• May enable an organism to evade host defenses.
• May improve access to the body's nutrients.
• Colonization factors, e.g. fimbriae
• Allow an organism to adhere to cells.
• Adhesions are proteins that allow organisms to
  stick to cells.

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Virulence Factors (contd)

• Antiphagocytic mechanisms, e.g. capsule
• Body's immune cells are unable to engulf
  organisms.
• Exotoxins (toxins excreted from the bacterial
  cell).
• A wide variety of enzymes and toxic proteins are
  released.

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Virulence Factors (contd)

• Substances that help organisms invade
• Hemolysins - cause lysis of red blood cells, and
  damage other body cells.
• Leukocidins - kill white blood cells.
• Hyaluronidase - breaks down connective tissue
  extracellular material allowing spread.
• Collagenase - breaks down collagen, a structural
  protein.

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Virulence Factor (contd)

• Toxins that cause disease
• Enterotoxins - attack the bowel.
• Neurotoxins - inhibit normal neurological
  function.
• Protein synthesis inhibitors - can kill cells or
  damage organs, e.g. diphtheria
• Superantigens - these toxins bind to macrophages
  and short circuit the mechanism for stimulation
  of the immune system, causing a massive response
  and consequent damage to the body, e.g. Toxic
  Shock Syndrome, "Flesh eating disease".

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Virulence Factors (contd)

• Endotoxin ("Pyrogen")
• Found in the outer membrane of gram negative
  organisms.
• Causes fever, drop in blood pressure (shock).
• Acts by binding macrophages and causing release
  of active substances (cytokines).

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Bacterial Genetics

• Bacteria do not have nuclei.
• DNA in bacteria occurs as a single circular
  molecule, and sometimes as small circular
  molecules (plasmids) that are independent of the
  chromosome but are expressed.
• DNA contains the genetic code, recorded in the
  sequences of the 4 bases in DNA. Special enzymes
  cut DNA when it has the specific base sequence
  for that enzyme.

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Bacterial Genetics (contd)

• Genetic information is transferred from DNA to
  RNA and then expressed in the form of proteins.
• As the DNA sequence of an individual strain is
  unique (although parts are identical for strains
  in the same species or genus), it is the basis
  for the revolution in molecular techniques that
  you will hear about in a future lecture.

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DNA Transfer

• Free extracellular DNA can be taken up by some
  bacteria and incorporated to the bacterial genome
  (transformation).
• Transfer of genetic material by direct contact of
  cells (conjugation) especially important in gram
  negatives.
• mediated by pili
• allows transfer of plasmids

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DNA Transfer (contd)

• Genetic material is transferred via a bacterial
  virus (bacteriophage).
• Some bacteriophages rapidly destroy infected
  bacterial cells
• Others combine their DNA with the host bacteria,
  where it can be expressed. This process is
  called transduction.