Microbial Pathogenesis and Host-Parasite Relationships

1. Medical Microbiology Microbial Pathogenesis and Host-Parasite Relationships BIOL 533 Lecture 2

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

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

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

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

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

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

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)

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

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

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

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

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

14. Ecology of Normal Flora • Use of germ-free animals • Immune systems not well developed • Have to be fed vitamins

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

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

17. Ecology of Normal Flora • Sterile sites—pathogens in these definitely indicate disease • Blood • Cerebrospinal fluid • Synovial fluid • Deep tissues

18. Medical Microbiology Strategies for Studying Microbial Pathogenesis BIOL 533 Lecture 2

19. Identification of Pathogens • Traditional: associate disease with organism

20. Koch’s 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

21. Koch’s Postulates • Pure culture inoculated into susceptible animal should produce disease • Same bacterium re-isolated in pure culture from experimental animal

22. Koch’s Postulates • Some assumptions questioned in light of more modern approaches and new information about host-parasite interaction

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

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

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

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

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

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

29. Identification of Pathogens • Molecular version

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

31. 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

32. 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

33. 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

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

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

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

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

38. Lecture Two • Questions? • Comments? • Assignments...