Lecture Two BIOL 533 1
Microbial Pathogenesis
and Host-Parasite Relationships
BIOL 533
Lecture 2
Medical Microbiology
Lecture Two BIOL 533 2
Normal Flora
? General aspects
– Remember definition,organisms frequently
found on or within body of healthy
individuals
– Most are bacteria,but some are viruses,
fungi,and protozoa
? We do not carry all of them all of the time
? Each person has individualized normal flora
Lecture Two BIOL 533 3
Normal Flora
? Some are found only on body; others
also found in environment
? Problem,some people have transient
normal flora (pathogens)
– Example,about 10% of population have
meningococcus or pneumococcus as
normal flora
Lecture Two BIOL 533 4
Importance
? Opportunistic infections,normal flora in
unusual sites; for example,
– Bacteriodes from intestine into deeper
tissues as a result of trauma (or surgery)
– Staphylococci from skin and nose
– Streptococci and Gram— cocci from throat
and mouth
Lecture Two BIOL 533 5
Importance
? Depends on pathogen and on defenses
of host,
– Candida (yeast) causes pneumonia in
people undergoing cancer chemotherapy
– Pneumocystis carinii (common inhabitant
of lung) causes pneumonia and death in
AIDS patients
Lecture Two BIOL 533 6
Immune Stimulation
? Antigenic stimulation by normal flora—
do not have high antibody titers
– Serve as defense mechanism even in low
concentration
– Bacterial stimulation leads to production of
IgA that is secreted through mucus
membranes
? Probably interfere with colonization of deeper
tissues
Lecture Two BIOL 533 7
Immune Stimulation
? Sometimes antibodies elicited by normal
flora cross-react with normal tissue,
– Antibodies against ABO blood group
substances,
? A - make B antibodies
? B - make A antibodies
? O - make antibodies against both
– Why? Bacteria from intestinal flora contain Ag
that cross-react with both A & B blood substances
Lecture Two BIOL 533 8
Immune Stimulation
? Cross-reactivity does not normally cause
disease
– Possible for antibodies cross-reactive to
microbial Ag to cause problem
? Lupus erythematosus—production of Ab against
host DNA
– Some evidence that Ag may be cross-reacting bacterial
LPS
? May cross-react with pathogen (meningococcus)
Lecture Two BIOL 533 9
Physical & Chemical Aspects
? Keeps out invaders
– Mechanisms,
? Physical advantage of previous occupancy
? Some produce bacteriocins or antibiotics
– Relevance to lab work,E,coli K-12 cannot
compete with intestinal flora
Lecture Two BIOL 533 10
Physical & Chemical Aspects
? Antibiotic effects,wipes out normal flora
– Both endogenous and exogenous organisms
can cause disease
? Infecting dose of Salmonella decreases one
million-fold when mice given streptomycin
? Patients treated with some potent antibiotics,
– Suffer from diarrhea due to overgrowth of yeasts,and
staphylococci
– Administration of clindmycin-Clostridium difficile (minor
member of normal flora) causes pseudomembranous
colitis
Lecture Two BIOL 533 11
Physical & Chemical Aspects
? Role in human nutrition and metabolism
– E,coli and Bacteriodes synthesize vitamin K
– Metabolism of key compounds involves
excretion from liver into intestine and their
return to the liver
Lecture Two BIOL 533 12
Physical & Chemical Aspects
? Important for sex hormones and bile
salts
– Excreted through bile in conjugated form
as glucuronides or sulfate,but cannot be
reabsorbed in this form
– Members of intestinal bacterial flora make
glucuronidases and sulfatases that can
deconjugate these compounds
? Physiological role not known
Lecture Two BIOL 533 13
Physical & Chemical Aspects
? Source of carcinogens
– Large intestinal flora
? Many potential carcinogens are only active
after being modified
– Some modifications are carried out by enzymes of
intestinal bacteria; example,cyclamate converted to
bladder carcinogen (cyclohexamine) by bacterial
sulfatases
? Importance of carcinogen production not clear
Lecture Two BIOL 533 14
Ecology of Normal Flora
? Use of germ-free animals
– Immune systems not well developed
– Have to be fed vitamins
Lecture Two BIOL 533 15
Ecology of Normal Flora
? Parts of body colonized
– Contain large numbers,
? Skin
? Respiratory tract (nose and oropharynx)
? Digestive tract (mouth and large intestine)
? Urinary tract (anterior parts of urethra)
? Genital system (vagina)
? Most are strict anaerobes
Lecture Two BIOL 533 16
Ecology of Normal Flora
? Parts of body colonized
– Contain small numbers,many in transit,
? Rest of respiratory and digestive tracts
? Bladder
? Uterus
– Finding pathogens at these sites is
suggestive of disease,but not proof
Lecture Two BIOL 533 17
Ecology of Normal Flora
? Sterile sites—pathogens in these
definitely indicate disease
– Blood
– Cerebrospinal fluid
– Synovial fluid
– Deep tissues
Lecture Two BIOL 533 18
Strategies for Studying
Microbial Pathogenesis
BIOL 533
Lecture 2
Medical Microbiology
Lecture Two BIOL 533 19
Identification of Pathogens
? Traditional,
associate disease
with organism
Lecture Two BIOL 533 20
Koch’s Postulates
1 Bacterium found in all patients having
disease and it or its products found in
all body parts affected
2 The bacterium should be isolated and
grown in pure culture
Lecture Two BIOL 533 21
Koch’s Postulates
3 Pure culture inoculated into susceptible
animal should produce disease
4 Same bacterium re-isolated in pure
culture from experimental animal
Lecture Two BIOL 533 22
Koch’s Postulates
? Some assumptions questioned in light
of more modern approaches and new
information about host-parasite
interaction
Lecture Two BIOL 533 23
Challenge to Postulate #1
? Implies virulence resides only with
pathogen and not at all with host
? Clearly,susceptibility of host is as
important
– Immuno-compromised individuals vs,
healthy adults prove the point
– Minor pathogen causes disease in immuno-
compromised individuals only
Lecture Two BIOL 533 24
Challenge to Postulate #2
? Places considerable emphasis on
culturing organisms in pure culture
? Some organisms have not been
cultured in laboratory media
Lecture Two BIOL 533 25
Challenge to Postulate #2
? For example,Treponema pallidium,
Mycobacterium leprae clearly cause
disease,
– Antibiotics cause both symptoms and
organisms from tissues to disappear
– Immune response in infected patients to
surface Ag of bacteria from infected tissue
Lecture Two BIOL 533 26
Challenge to Postulate #3
? Implies all members of a bacterial
species are equally virulent and only a
single species causes disease
– Different strains of species vary in virulence
– Different strains can cause different diseases
– Same symptoms caused by numerous
organisms
– Disease caused by multiple organisms
Lecture Two BIOL 533 27
Challenge to Postulate #3
? Well known fact that cultivation of some
pathogens can lead to loss of virulence
factors
Lecture Two BIOL 533 28
Challenge to Postulate #4
? Requires pathogen be reinoculated into
an animal and produces symptoms of
disease
– Some diseases don’t affect animals,or
cause different symptoms from human
form
? Therefore,to be practical,Koch’s
Postulates require animal models
Lecture Two BIOL 533 29
Identification of Pathogens
? Molecular
version
Lecture Two BIOL 533 30
Molecular Version
? Emphasis shifted from identification of
pathogens to identification of virulence
factors
? Not complete agreement on requirements
to prove a particular gene or product
plays a role in disease,but criteria widely
accepted
Lecture Two BIOL 533 31
Molecular Version
1 Gene or product found in strains that
cause disease and not in avirulent
bacteria
—If gene found in organisms not known to
cause disease,gene should be mutated to
less active or inactive form,or not
expressed
Lecture Two BIOL 533 32
Molecular Version
2 Disrupting gene in virulent strain
reduces or eliminates its virulence
—Introduction of cloned gene into avirulent
strain should make it virulent
— Systems with multiple genes,
? These other genes would also have to be
modified or introduced
Lecture Two BIOL 533 33
Molecular Version
3 Gene is expressed in bacteria inside
host sometime during disease process
4 Ab to gene product should be protective
or in cases where cell-mediated
immunity involved,gene product should
elicit protective immunity
Lecture Two BIOL 533 34
Identification without Culturing
? Combine PCR,
Polymerase Chain Reaction with 16S
r-RNA phylogeny
? 16S r-RNA found in all bacteria
? Conserved (domain) and variable
(particular organism) sequences
Lecture Two BIOL 533 35
Identification without Culturing
? Sizable database and similarities in
sequence correspond well to evolutionary
relationships
? Sequence will either identify it as
member of known or unknown species
Lecture Two BIOL 533 36
Identification without Culturing
? PCR primers that recognize two
conserved regions of 16S rRNA flanking
a variable region are used to amplify
and clone a DNA segment from a
clinical speciman
– If amplified segment is obtained,indicates
bacteria present in speciman
– It can be sequenced to identify bacterium
Lecture Two BIOL 533 37
Identification without Culturing
? Fluorescently labeled probe of sequence
can then visualize bacterium in clinical
speciman
? Rules out PCR amplification of
contaminating DNA from other sources
Lecture Two BIOL 533 38
Lecture Two
? Questions?
? Comments?
? Assignments..,
Microbial Pathogenesis
and Host-Parasite Relationships
BIOL 533
Lecture 2
Medical Microbiology
Lecture Two BIOL 533 2
Normal Flora
? General aspects
– Remember definition,organisms frequently
found on or within body of healthy
individuals
– Most are bacteria,but some are viruses,
fungi,and protozoa
? We do not carry all of them all of the time
? Each person has individualized normal flora
Lecture Two BIOL 533 3
Normal Flora
? Some are found only on body; others
also found in environment
? Problem,some people have transient
normal flora (pathogens)
– Example,about 10% of population have
meningococcus or pneumococcus as
normal flora
Lecture Two BIOL 533 4
Importance
? Opportunistic infections,normal flora in
unusual sites; for example,
– Bacteriodes from intestine into deeper
tissues as a result of trauma (or surgery)
– Staphylococci from skin and nose
– Streptococci and Gram— cocci from throat
and mouth
Lecture Two BIOL 533 5
Importance
? Depends on pathogen and on defenses
of host,
– Candida (yeast) causes pneumonia in
people undergoing cancer chemotherapy
– Pneumocystis carinii (common inhabitant
of lung) causes pneumonia and death in
AIDS patients
Lecture Two BIOL 533 6
Immune Stimulation
? Antigenic stimulation by normal flora—
do not have high antibody titers
– Serve as defense mechanism even in low
concentration
– Bacterial stimulation leads to production of
IgA that is secreted through mucus
membranes
? Probably interfere with colonization of deeper
tissues
Lecture Two BIOL 533 7
Immune Stimulation
? Sometimes antibodies elicited by normal
flora cross-react with normal tissue,
– Antibodies against ABO blood group
substances,
? A - make B antibodies
? B - make A antibodies
? O - make antibodies against both
– Why? Bacteria from intestinal flora contain Ag
that cross-react with both A & B blood substances
Lecture Two BIOL 533 8
Immune Stimulation
? Cross-reactivity does not normally cause
disease
– Possible for antibodies cross-reactive to
microbial Ag to cause problem
? Lupus erythematosus—production of Ab against
host DNA
– Some evidence that Ag may be cross-reacting bacterial
LPS
? May cross-react with pathogen (meningococcus)
Lecture Two BIOL 533 9
Physical & Chemical Aspects
? Keeps out invaders
– Mechanisms,
? Physical advantage of previous occupancy
? Some produce bacteriocins or antibiotics
– Relevance to lab work,E,coli K-12 cannot
compete with intestinal flora
Lecture Two BIOL 533 10
Physical & Chemical Aspects
? Antibiotic effects,wipes out normal flora
– Both endogenous and exogenous organisms
can cause disease
? Infecting dose of Salmonella decreases one
million-fold when mice given streptomycin
? Patients treated with some potent antibiotics,
– Suffer from diarrhea due to overgrowth of yeasts,and
staphylococci
– Administration of clindmycin-Clostridium difficile (minor
member of normal flora) causes pseudomembranous
colitis
Lecture Two BIOL 533 11
Physical & Chemical Aspects
? Role in human nutrition and metabolism
– E,coli and Bacteriodes synthesize vitamin K
– Metabolism of key compounds involves
excretion from liver into intestine and their
return to the liver
Lecture Two BIOL 533 12
Physical & Chemical Aspects
? Important for sex hormones and bile
salts
– Excreted through bile in conjugated form
as glucuronides or sulfate,but cannot be
reabsorbed in this form
– Members of intestinal bacterial flora make
glucuronidases and sulfatases that can
deconjugate these compounds
? Physiological role not known
Lecture Two BIOL 533 13
Physical & Chemical Aspects
? Source of carcinogens
– Large intestinal flora
? Many potential carcinogens are only active
after being modified
– Some modifications are carried out by enzymes of
intestinal bacteria; example,cyclamate converted to
bladder carcinogen (cyclohexamine) by bacterial
sulfatases
? Importance of carcinogen production not clear
Lecture Two BIOL 533 14
Ecology of Normal Flora
? Use of germ-free animals
– Immune systems not well developed
– Have to be fed vitamins
Lecture Two BIOL 533 15
Ecology of Normal Flora
? Parts of body colonized
– Contain large numbers,
? Skin
? Respiratory tract (nose and oropharynx)
? Digestive tract (mouth and large intestine)
? Urinary tract (anterior parts of urethra)
? Genital system (vagina)
? Most are strict anaerobes
Lecture Two BIOL 533 16
Ecology of Normal Flora
? Parts of body colonized
– Contain small numbers,many in transit,
? Rest of respiratory and digestive tracts
? Bladder
? Uterus
– Finding pathogens at these sites is
suggestive of disease,but not proof
Lecture Two BIOL 533 17
Ecology of Normal Flora
? Sterile sites—pathogens in these
definitely indicate disease
– Blood
– Cerebrospinal fluid
– Synovial fluid
– Deep tissues
Lecture Two BIOL 533 18
Strategies for Studying
Microbial Pathogenesis
BIOL 533
Lecture 2
Medical Microbiology
Lecture Two BIOL 533 19
Identification of Pathogens
? Traditional,
associate disease
with organism
Lecture Two BIOL 533 20
Koch’s Postulates
1 Bacterium found in all patients having
disease and it or its products found in
all body parts affected
2 The bacterium should be isolated and
grown in pure culture
Lecture Two BIOL 533 21
Koch’s Postulates
3 Pure culture inoculated into susceptible
animal should produce disease
4 Same bacterium re-isolated in pure
culture from experimental animal
Lecture Two BIOL 533 22
Koch’s Postulates
? Some assumptions questioned in light
of more modern approaches and new
information about host-parasite
interaction
Lecture Two BIOL 533 23
Challenge to Postulate #1
? Implies virulence resides only with
pathogen and not at all with host
? Clearly,susceptibility of host is as
important
– Immuno-compromised individuals vs,
healthy adults prove the point
– Minor pathogen causes disease in immuno-
compromised individuals only
Lecture Two BIOL 533 24
Challenge to Postulate #2
? Places considerable emphasis on
culturing organisms in pure culture
? Some organisms have not been
cultured in laboratory media
Lecture Two BIOL 533 25
Challenge to Postulate #2
? For example,Treponema pallidium,
Mycobacterium leprae clearly cause
disease,
– Antibiotics cause both symptoms and
organisms from tissues to disappear
– Immune response in infected patients to
surface Ag of bacteria from infected tissue
Lecture Two BIOL 533 26
Challenge to Postulate #3
? Implies all members of a bacterial
species are equally virulent and only a
single species causes disease
– Different strains of species vary in virulence
– Different strains can cause different diseases
– Same symptoms caused by numerous
organisms
– Disease caused by multiple organisms
Lecture Two BIOL 533 27
Challenge to Postulate #3
? Well known fact that cultivation of some
pathogens can lead to loss of virulence
factors
Lecture Two BIOL 533 28
Challenge to Postulate #4
? Requires pathogen be reinoculated into
an animal and produces symptoms of
disease
– Some diseases don’t affect animals,or
cause different symptoms from human
form
? Therefore,to be practical,Koch’s
Postulates require animal models
Lecture Two BIOL 533 29
Identification of Pathogens
? Molecular
version
Lecture Two BIOL 533 30
Molecular Version
? Emphasis shifted from identification of
pathogens to identification of virulence
factors
? Not complete agreement on requirements
to prove a particular gene or product
plays a role in disease,but criteria widely
accepted
Lecture Two BIOL 533 31
Molecular Version
1 Gene or product found in strains that
cause disease and not in avirulent
bacteria
—If gene found in organisms not known to
cause disease,gene should be mutated to
less active or inactive form,or not
expressed
Lecture Two BIOL 533 32
Molecular Version
2 Disrupting gene in virulent strain
reduces or eliminates its virulence
—Introduction of cloned gene into avirulent
strain should make it virulent
— Systems with multiple genes,
? These other genes would also have to be
modified or introduced
Lecture Two BIOL 533 33
Molecular Version
3 Gene is expressed in bacteria inside
host sometime during disease process
4 Ab to gene product should be protective
or in cases where cell-mediated
immunity involved,gene product should
elicit protective immunity
Lecture Two BIOL 533 34
Identification without Culturing
? Combine PCR,
Polymerase Chain Reaction with 16S
r-RNA phylogeny
? 16S r-RNA found in all bacteria
? Conserved (domain) and variable
(particular organism) sequences
Lecture Two BIOL 533 35
Identification without Culturing
? Sizable database and similarities in
sequence correspond well to evolutionary
relationships
? Sequence will either identify it as
member of known or unknown species
Lecture Two BIOL 533 36
Identification without Culturing
? PCR primers that recognize two
conserved regions of 16S rRNA flanking
a variable region are used to amplify
and clone a DNA segment from a
clinical speciman
– If amplified segment is obtained,indicates
bacteria present in speciman
– It can be sequenced to identify bacterium
Lecture Two BIOL 533 37
Identification without Culturing
? Fluorescently labeled probe of sequence
can then visualize bacterium in clinical
speciman
? Rules out PCR amplification of
contaminating DNA from other sources
Lecture Two BIOL 533 38
Lecture Two
? Questions?
? Comments?
? Assignments..,
