Medical Microbiology

Normal Flora
Microbial Pathogenesis and Host-Parasite Relationships

For : www.esnips.com/web/m4mn By: Mohammed M.M..Manaa

m4mn@hotmail.com

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

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

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

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

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

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 crossreact with both A & B blood substances

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)

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

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

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

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

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

Ecology of Normal Flora

Use of germ-free animals

Immune systems not well developed Have to be fed vitamins

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

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

Ecology of Normal Flora

Sterile sitespathogens in these definitely indicate disease

Blood Cerebrospinal fluid Synovial fluid Deep tissues

Medical Microbiology
Strategies for Studying Microbial Pathogenesis

Identification of Pathogens

Traditional:
associate disease with organism

Kochs Postulates
1

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

Kochs Postulates
3

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

Kochs Postulates

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

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 immunocompromised individuals only

Challenge to Postulate #2
Places considerable emphasis on culturing organisms in pure culture Some organisms have not been cultured in laboratory media

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

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

Challenge to Postulate #3
Well known fact that cultivation of some pathogens can lead to loss of virulence factors

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

Identification of Pathogens

Molecular version

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

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

Molecular Version
2

Disrupting gene in virulent strain reduces or eliminates its virulence

Introduction

of cloned gene into avirulent should make it virulent Systems with multiple genes:

strain

These other genes would also have to be or introduced

modified

Molecular Version
3

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

Identification without Culturing

Combine PCR: Polymerase Chain Reaction with 16S RNA phylogeny 16S r-RNA found in all bacteria Conserved (domain) and variable (particular organism) sequences

r-

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

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

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