Download Microbial physiology

‫بسم ا‪..‬الرحمن الرحیم‬
Microbial Pathogenesis
Today we will talk about
How do we study Bacteria
Microscopes
Some terms and definitions
How a microorganism enters to body
Modes of infectious disease transmission
Microbial Toxins
Intracellular/ Extracellular bacteria
Invasion
How do we study Bacteria
1- Microscope
2- Cuture
3- Immunological Methods
4- Molecular Methods
Different Kinds of Microscopes:
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Compound Light Microscope (transmitted light)
Transmission Electron Microscope (transmitted electron
beam)
Scanning Electron Microscope (reflected electron beam)
Dark-field Microscopy
Phase Contrast Microscopy
Basic Principles
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Magnification: objects are made to appear larger than they
are
Resolution: the ability to see close together objects as
distinct
Early Light Microscopes:
A Compound Light Microscope
Total Magnification:
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Simply multiply the ocular magnification by the
magnification of the objective you are using
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Resolving Power:
dmin = 0.61  / N.A.
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Resolving Power:
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Human eye: about 0.2 mm
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Compound Light Microscope: about 0.2 μm
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Transmission Electron Microscope: about 0.2 nm
Microscopes
Light M
Dark Field M
Fluorescent M
Confocal M
Transmission and Scanning Electron Microscope
Phase Contrast M
Scanning Tunnelig Microscope
Ocular Lens
Body Tube
Nose Piece
Arm
Objective
Lenses
Stage
Clips
Diaphragm
Stage
Coarse Adj.
Fine Adjustment
Light Source
Base
Skip to Magnification Section
Dark-Field Microscope
Phase Contrast Microscope
Fluorescence & Microscopy:
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Fluorescence: when a molecule is excited by light at a shorter
wavelength, and emits light at a longer wavelength
Auto fluorescence: some substances, such as chlorophyll, are
naturally fluorescent
Fluorescence Microscope: a microscope that excites and detects
fluorescent materials
Fluorescent Dyes: are widely used in biology to label cell
organelles, molecules, and genes in cells
Confocal Laser Scanning Microscopy
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The higher the magnification the less “depth of field” - only a
narrow slice is in focus
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Lasers scan the specimen at different depths
A computer reconstructs a clear 3D image
Usually used with fluorescent specimens
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Confocal Scanning Laser Microscope (CLSM)
Confocal Scanning Laser Microscope (CLSM)
Transmission Electron Microscope
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Is capable of much higher resolution than the light microscope
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An electron beam is transmitted through a very thin section

Instead of glass lenses, magnetic lenses are used, which bend and
focus the electron beam much like a glass lens bends and focuses
light
Transmission Electron Microscope
TEM Image
Scanning Electron Microscope

Is capable of higher resolution than the light microscope
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An electron beam is “bounced off” the specimen to a detector,
instead of being passed through it
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It produces a detailed image of the surface of the specimen,
but not its internal structure
Scanning Electron Microscope
SEM Image
Microbial Pathogenesis
Number of Invading Microbes
LD50: Lethal dose for 50% of hosts. Number of microbes
that will kill 50% of inoculated test animals
ID50: Infectious dose for 50% of hosts. Number of microbes
that will cause a demonstrable infection in 50% of inoculated
test animals.
Definitions
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Pathogenicity
 Ability of a microorganism to cause disease by overcoming
the defenses of a host
Virulence
 The degree or extent of pathogenicity
How a microorganism enters to body
I.
Mucous Membranes
II. Skin
III. Parenteral Route
I. Mucous Membranes
Respiratory tract: Easiest and most frequently used entry site
for microbes.
Gastrointestinal tract: Enter through water, food, contaminated
fingers and fomites. Must survive stomach HCl, enzymes, and
bile.
Genitourinary tract: Entry site for most sexually transmitted
diseases (STDs).
Conjunctiva: Membrane covering eyes and eyelids.
II. Skin
u
Unbroken skin is impenetrable by most microbes.
Some microbes gain access through hair follicles and sweat
glands.
III. Parenteral Route
Injections, bites, cuts, wounds, surgery, punctures,
and splitting due to swelling or drying.
Infection/entry
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Ingestion (fecal-oral)
Inhalation (respiratory)
Trauma (burn)
Arthropod bite (zoonoses:
mosquito, flea, tick, Tsetse fly)
Sexual transmission
Needle stick (blood
transfusion)
Maternal-neonatal
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Modes of infectious disease transmission
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Contact transmission
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Vehicle transmission
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Direct contact (person-to-person): syphilis, gonorrhear, herpes
Droplet (less than 1 meter): whooping cough, strep throat
Airborne: influenza, tuberculoses, chickenpox
Water-born: (fecal-oral infection): cholera, diarrhea
Food-born: hepatitis, food poisoning, typhoid fever
Vector transmission
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Biological vectors: malaria, plaque, yellow fever
Mechanical vectors: E. coli diarrhea, salmonellosis
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Virulance Factors
Adhesion
Invasion
Toxins
Enzymes
Adherence
Biofilms

Biofilms
 Communities
of microorganisms and their
extracellular products that attach to non-living and
living surfaces
Algae
 Dental plaque
 Medical catheters

Biofilms
How Bacterial Pathogens Penetrate Host
Defenses
Capsules
Resist host defenses by impairing phagocytosis.
Host can produce antibodies to capsule, which attach to microbe
and allow phagocytosis
Cell Wall Components
M protein: Found on cell surface and fimbriae of Streptococcus
pyogenes. Mediates attachment and helps resist phagocytosis.
Waxes:
Cell wall of Mycobacterium tuberculosis helps resist digestion
after phagocytosis
How Bacterial Pathogens Penetrate
Host Defenses
Exotoxins
Proteins: Enzymes that carry out specific reactions.
Soluble in body fluids, rapidly transported throughout body in
blood or lymph.
Produced mainly by gram-positive bacteria.
Most genes for toxins are carried on plasmids or phages.
 Cytotoxins:
Kill or damage host cells.
 Neurotoxins: Interfere with nerve impulses.
 Enterotoxins: Affect lining of gastrointestinal tract
Exotoxins
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A-B toxins
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A-B toxin released by
bacteria
B part binds to receptor of
host cell
Toxin transported across
membrane into cell
A-B components separate
A component inhibits
protein synthesis and kills
cell
Toxins: cause hyperactivation
Vibrio cholerae
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Toxins: affect on nerve-muscle transmission
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Exotoxins
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Exotoxins
2
– Membrane disrupting toxin
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Causes lysis of cells by disrupting cell membrane
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Leukocidins
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Form protein channels
 Staphylococcus aureus
Disrupt phospholipids
 Clostridium perfringens
Membrane disrupting toxins that kill phagocytic WBC’s
Hemolysins
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Membrane disrupting toxins that kill erythrocyes RBC’s
Exotoxins
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Exotoxins
 3-
Superantigens
Provoke intense immune response
 Protein in nature (antigens)
 Stimulate T – cells
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Release cytokines (too much)
 Causes fever, nausea, vomiting diarrhea
Superantigens
Polyclonal T cell activation
Aberrant cytokines,
cell death
Antigen/
MHC-1
Specific T cell
activation
Anti-microbes
immunity
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Known and suspected association of superantigens
with human disease (1)
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Acute diseases
 Food poisoning: SEs
 Staph TSS
Menstrual: TSST-1
Nonmenstrual: SEB, SEC, TSST-1
 StrepTSS:SPe’s
 Sudden infant death syndrome: SEs?, SPe,s
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Known and suspected association of superantigens
with human disease (2)
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Autoimmune diseases
 Rheumatic fever, rheumatic hart disease: M proteins, SPe’s?
 Kawasaki disease: TSST-1?, SPe’s?
 Lyme disease
 Reumatoid arthritis
 Multiple sclerosis
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How Bacterial Pathogens Penetrate
Host Defenses
Endotoxins
Part of outer membrane surrounding gram-negative bacteria.
Endotoxin is lipid portion of lipopolysaccharides (LPS), called
lipid A.
Effect exerted when gram-negative cells die and cell walls
undergo lysis, liberating endotoxin.
All produce the same signs and symptoms:
Chills, fever, weakness, general aches, blood clotting and
tissue death, shock, and even death. Can also induce
miscarriage.
Events leading to fever
Gram-negative bacteria are digested by phagocytes.
LPS is released by digestion in vacuoles, causing macrophages to
release interleukin-1 (IL-1).
IL-1 is carried via blood to hypothalamus, which controls body
temperature.
IL-1 induces hypothalamus to release prostaglandins, which reset
the body thermostat to higher temperature.
Endotoxins and the Pyrogenic (Fever) Response
Endotoxins
Shock: Any life-threatening loss of blood pressure.
Septic shock: Shock caused by endotoxins of gramnegative bacteria.
Phagocytosis of bacteria leads to secretion of tumor necrosis
factor (TNF), which alters the permeability of blood
capillaries and causes them to lose large amounts of fluids.
Low blood pressure affects kidneys, lungs, and
gastrointestinal tract.
Endotoxins: heat stable
IL-6 induced in monocytes exposed to LPS and PM10-2.5 extracts from
indoor and outdoor air. Cytokines were measured after exposure of
monocytes to particle extracts for six hours.
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Endotoxin: lipopolysaccharide
IL-1
TNF
Pseudomonas aeruginosa
Fever
Disseminated intravascular coagulation
Septic shock
death
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Enzymes
1- Hyaloronidase
2- Dnase
3- Streptokinase
.
.
.
Where to find the pathogen?
Extracellular versus Intracellular Parasitism
 Extracellular parasites
 destroyed when phagocytosed.
 damaging tissues as they remain outside cells.
 inducing the production of opsonizing antibodies, they
usually produce acute diseases of relatively short
duration.
 Intracellular parasites
 can multiply within phagocytes.
 frequently cause chronic disease.
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The environment in a cell
 Cytosol: pH=7
 Phagosome: pH=6
 Phagolysosome: pH=5
http://bio.winona.msus.edu/bates/Bio241/images/figure-04-13b.jpg
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Intracellular bacteria
Listeria
Shigella
Endosomes
Phagolysosomes
Legionella
Chlamydia
Sammonella
Mycobacteria
lysosomes
Phagosomes
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Evasion/Manipulation of Host Defense
 Modulation of innate/inflammatory response
• Resistance to phagocytic killing in subepithelial space
• Serum resistance
• Antigenic variation
Resistance to phagocytic killing in
subepithelial space
• Inhibit phagocyte mobilization :(chemotaxis, complement
activation)
Inhibit chemoattractants: Streptococcus pyogenes
degrades C5a
• Avoid ingestion
kill phagocytes: Streptolysin O lyses PMNs; Staphylococcus aureus alpha, beta
and gamma toxins and leucocidin lyses PMNs
capsular protection from opsonization: M proteins, Streptococcus pyogenes
Bacterial capsules that resemble self: Neisseria meningitidis (sialic acid);
Streptococcus pyogenes (hyaluronic acid)
• Survive within phagocyte
Survival within phagocyte
Escape endosome or phagolysosome:
- Shigella, Listeria monocytogenes
Inhibit phagosome-lysosome fusion
- Legionella pneumophila, Mycobacterium tuberculosis, Salmonella
Survive within phagolysosome
Inactivate reactive oxygen species: Salmonella, via superoxide
dismutase, catalase, recA
- Resist antimicrobial peptides: Host cationic peptides complexed with
SapA peptide
Scape from Immune response con
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Antigenic variation
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Induction of apoptosis