Microbial Pathogenesis and HostParasite Relationships

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Microbial Pathogenesis and Host-Parasite
Relationships
Medical Microbiology

• BIOL 533
• Lecture 2

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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

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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

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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

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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

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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

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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

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Immune Stimulation

• Cross-reactivity does not normally cause disease
• Possible for antibodies cross-reactive to
  microbial Ag to cause problem
• Lupus erythematosusproduction of Ab against host
  DNA
• Some evidence that Ag may be cross-reacting
  bacterial LPS
• May cross-react with pathogen (meningococcus)

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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

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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

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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

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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

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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

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Ecology of Normal Flora

• Use of germ-free animals
• Immune systems not well developed
• Have to be fed vitamins

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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

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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

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Ecology of Normal Flora

• Sterile sitespathogens in these definitely
  indicate disease
• Blood
• Cerebrospinal fluid
• Synovial fluid
• Deep tissues

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Strategies for Studying Microbial Pathogenesis
Medical Microbiology

• BIOL 533
• Lecture 2

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Identification of Pathogens

• Traditional associate disease with organism

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Kochs Postulates

• Bacterium found in all patients having disease
  and it or its products found in all body parts
  affected
• The bacterium should be isolated and grown in
  pure culture

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Kochs Postulates

• Pure culture inoculated into susceptible animal
  should produce disease
• Same bacterium re-isolated in pure culture from
  experimental animal

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Kochs Postulates

• Some assumptions questioned in light of more
  modern approaches and new information about
  host-parasite interaction

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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

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Challenge to Postulate 2

• Places considerable emphasis on culturing
  organisms in pure culture
• Some organisms have not been cultured in
  laboratory media

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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

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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

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Challenge to Postulate 3

• Well known fact that cultivation of some
  pathogens can lead to loss of virulence factors

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Challenge to Postulate 4

• Requires pathogen be reinoculated into an animal
  and produces symptoms of disease
• Some diseases dont affect animals, or cause
  different symptoms from human form
• Therefore, to be practical, Kochs Postulates
  require animal models

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Identification of Pathogens

• Molecular version

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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

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Molecular Version

• 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

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Molecular Version

• 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

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Molecular Version

• Gene is expressed in bacteria inside host
  sometime during disease process
• Ab to gene product should be protective or in
  cases where cell-mediated immunity involved, gene
  product should elicit protective immunity

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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

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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

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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

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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

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Lecture Two

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