Normal Flora

1. Normal Flora Faculty: Samuel Aguazim ( MD) LANGE CHAPTER 6

2. Normal flora: • bacteria and fungi that are permanent residentsof certain body sites, especially the skin, oropharynx, colon, and vagina. • The viruses and parasites which are the two other major groups of microorganisms, are usually not considered members of the normal flora,

3. Normal FloraAdd GI Tract, Urinary Tract and Heart

4. Normal Flora of the Intestinal Tract • In normal fasting people, the stomach contains few organisms because of its low pH and its enzymes. • The small intestine usually contains small numbers of streptococci, lactobacilli, and yeasts, particularly Candida albicans. • Larger numbers of these organisms are found in the terminal ileum.

5. Location of bacteria • The colonis the major location of bacteria in the body. • Roughly 20% of the feces consists of bacteria, approximately 1011 organisms/g

6. The normal flora of the GI tract plays a large role in extraintestinal disease. • E. coliis the leading cause of urinary tract infections • Bacteroidesfragilis (most common normal flora in gastrointestinal tract)is an important cause of peritonitis associated with perforation of the intestinal wall following trauma or appendicitis. • Streptococcus faecalis - urinary tract infections and endocarditis • Pseudomonas aeruginosa - can cause various infections, particularly in hospitalized patients with decreased host defenses.

7. Antibiotic Suppression of Normal Flora • clindamycincan suppress bacteroidesfragilisthe predominant normal flora in the colon, allowing a rare organism such as the toxin-producing Clostridium difficileto overgrow and cause severe colitis!!!!!!!! • Certain antibiotics, such as neomycin orally, prior to gastrointestinal surgery to “sterilize” the gut leads to a significant reduction of the normal flora for several days followed by a gradual return to normal levels.

8. NORMAL FLORA:GENITOURINARY TRACT • vaginal flora of adult women consists primarily of Lactobacillusspecies. Lactobacilli: • produces acid that keeps the pH of the adult woman's vagina low. • Before puberty and after menopause, when estrogen levels are low, lactobacilli are rare and the vaginal pH is high. • appear to prevent the growth of potential pathogens, since their suppression by antibiotics can lead to overgrowth by C albicans.

9. Normal Flora of the Vagina • The vagina is located close to the anus and can be colonized by members of the fecal flora. • Women who are prone to recurrent urinary tract infections harbor organisms such as E. coliand Enterobacter. • About 15—20% of women of childbearing age carry group B streptococci in the vagina. • B streptococci is an important cause of sepsis and meningitis in the newborn and is acquired during passage through the birth canal.

10. Urine in the bladder is sterile in the healthy person • During passage through the outermost portions of the urethra it often becomes contaminated with Staphalococcusepidermidis, and nonhemolytic Streptococci. • The area around the urethra of women and uncircumcised men contains secretions that carry Mycobacterium smegmatis,an acid-fast organism.

11. PATHOGENESIS • LANGE CHAPTER 7

12. Adherence to cell surfaces • Pili:The piliof Neisseriagonorrhoeaeand Escherichia colimediate the attachment of the organisms to the urinary tract epithelium • Capsules or glycocalyxes: that allow them to adhere to the surface of humancells, thereby enhancing their ability to cause disease. • Glycocalyx:Staphylococcusepidermidisand certain viridans streptococci allows the organisms to adhere strongly to the endothelium of heart valves. • Curli: Surface proteins of some strains of E coliand Salmonella mediate binding of the bacteria to endothelium and to extracellular proteins. **These adherence mechanisms are essential for organisms that attach to mucous membranes; mutants that lack these mechanisms are often nonpathogenic.

13. Invasion, Inflammation & Intracellular Survival: One of the twomain mechanisms by which bacteria cause disease is: • invasion of tissue followed by • inflammation. Several enzymes secreted by invasive bacteria play a role in pathogenesis: (1) Collagenase and hyaluronidase: degrade collagen and hyaluronic acid: bacteria spreads through subcutaneous tissue. (2) Coagulase: producedby Staphylococcus aureusand accelerates the formation of a fibrin clot from its precursor, fibrinogen (this clot may protect the bacteria from phagocytosis by walling off the infected area and by coating the organisms with a layer of fibrin) (3)immunoglobulin A (IgA) protease: degrades IgA, allowing the organism to adhere to mucous membranes, and is produced chiefly by N.gonorrhoeae, Haemophilusinfluenzae, and Streptococcus pneumoniae (4)Leukocidins, which can destroy both neutrophilic leukocytes and macrophages.

14. Second group of antiphagocytic factors are the cell wall proteins of the gram-positive cocci: • M protein • group A streptococci (Streptococcus pyogenes) is antiphagocytic. • Protein A • Staphylococcus aureus • Is antiphagocytic • binds to IgG and prevents the activation of complement.

15. Protein A inhibits phagocytosis PHAGOCYTE Fc receptor Protein A immunoglobulin BACTERIUM

16. “Intracellular” pathogens • Intracellular survival is an important attribute of certain bacteria that enhances their ability to cause disease. • “intracellular” pathogens: commonly cause granulomatous lesions. • Best-known bacteria belong to the genera Mycobacterium, Legionella, Brucella, and Listeria. • Best-known fungus is Histoplasma.

17. “Yops” (Yersinia outer-membrane proteins) • produced by several Yersiniaspecies • important examples of bacterial virulence factors that act primarily after invasion of cells by the organism. • These plasmid-encoded enzymes aggregate on the bacterial surface and, once inside the host cells, mediate various activities. • The most important effect of the Yops proteins is to limit phagocytosis by neutrophils and macrophages.

18. 4. Toxin Production Bacteria cause disease by two major mechanisms: (1) toxin production (2) invasion andinflammation Exotoxins: • Is secreted by several gram-positive and gram-negative bacteria. • polypeptides whose genes are frequently located on plasmids or lysogenic bacterial viruses (bacteriophages). Endotoxins: • present only in gram-negative bacteria. • Are lipopolysaccharides (LPS),whereasexotoxins are polypeptides. • are integral parts of the cell walls of both gram-negative rods and cocci, in contrast to exotoxins, which are released from the cell. In addition, several other features distinguish these substances. • Endotoxins are weakly antigenic; they induce protective antibodies so poorly that multiple episodes of toxicity can occur. • No toxoids have been produced from endotoxins • not used as antigens in any available vaccine. • fever and hypotension are salient features of septic shock. • The endotoxins of gram -negative bacteria are the best-established causes of septic shock.

19. Exotoxin polypeptides • induce the synthesis of protective antibodies called antitoxins (antibodies) • some of which are useful in prevention or treatment of diseases such as botulism and tetanus. • When treated with formaldehyde(or acid or heat), the exotoxin polypeptides are converted into toxoids, whichare used in protective vaccines because they retain their antigenicity but have lost their toxicity.

20. Gram-positive bacteria toxins (1) Diphtheria toxin, • produced by Corynebacteriumdiphtheriae • Inhibits protein synthesis by ADP-ribosylation of elongation factor (EF-2). • exotoxin activity depends on two functions mediated by different domains of the molecule. • A single proteolytic “nick” plus reduction of the sulfhydryl bonds yields two active polypeptides: • Fragment A:catalyzes the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to EF-2 thereby inactivating it. • Fragment B: binds to receptors on the outer membrane of eukaryotic cells and mediates transport of fragment A into the cells.

22. (2) Tetanus toxin • produced by Clostridium tetani • is a neurotoxin • prevents release of the inhibitory neurotransmitter glycine. • When the inhibitory neurons are nonfunctional, the excitatory neurons are unopposed • muscle spasm specially, spasm of the jaw and neck muscles (lockjaw).

24. (3) Botulinum toxin • produced by Clostridium botulinum • neurotoxin • blocks the release of acetyleholineat the synapse • flaccid paralysis. • Approximately 1g is lethal for human; it is one of the most toxic compounds known. • toxin is composed of two polypeptide subunitsheld together by disulfide bonds

26. (4)Two exotoxins are produced by Clostridium difficile Both are involved in the pathogenesis of pseudomembranous colitis. • Exotoxin A:enterotoxin that causes watery diarrhea. • Exotoxin B:cytotoxin thatdamages the colonic mucosa and causes pseudomembranes to form.

27. (5) Exotoxins produced by Bacillus anthracis: • Edema factor:which is an adenylatecyclase • Protective antigen:mediates the entry of the other two components into the cell • Lethal factor: is a proteasethat cleaves thephosphokinase leading to the inhibitions of cell growth and activates the mitogen activated protein kinase (MAPK) signal transduction pathway. This pathway controls the growth of human cells, and cleavage of the phosphokinaseinhibits cell growth.

28. Anthrax: Clinical Findings Cutaneous anthrax: • painless ulcer with a black crust. • Local edema is striking • lesion is called a “malignant pustule”. • Untreated cases progress to bacteremia and death. Inhalation anthrax • "Woolsorter's disease" (pulmonary anthrax) • life-threatening pneumonia caused by inhalation of spores. Gastrointestinal anthrax: • can occur if contaminated meat is ingested

29. (6) Toxic shock syndrome toxin (TSST) • Superantigen • produced by certain strains of Staphylococcus aureusand Streptococcus pyogenes. • TSST binds directly to class II major histocompatibility(MHC) proteins without intracellular processing. • This complex interacts with the -chain of the T cell receptor of many helper T cells. • Release of large amounts of interleukins, especially interleukin­1 and interleukin-2. • Produce many of the signs and symptoms of toxic shock. • TSST is also a T-cell “mitogen”ie. it induces T cells to multiply, which contributes to the overproduction of cytokines.

30. Gram-negative bacteria • The exotoxins produced by gram-negative bacteria also have several different mechanisms of action and produce different clinical effects. • Two very important exotoxins are the enterotoxins ofE coli and V cholerea(cholera toxin) • induce an increase in the amount of cyclic AMP within the enterocyte resulting in watery diarrhea.

31. 1. heat-labile enterotoxinproduced by E coli • causes watery, nonbloody diarrheaby stimulating adenylatecyclase activity in cells in the small intestine. • increase in the concentration of cyclic adenosine monophosphate inhibition of sodium ion absorption, and significant fluid and electrolyte loss into the lumen of the gut. (cAMP) causes excretion of the chloride ion. • inactivated at 650C for 30 minutes • composed of two subunits, a B subunit, which binds to a ganglioside receptor in the cell membrane, and an A subunit, which enters the cell and mediates the transfer of ADP ribose from NAD to a stimnulatory coupling protein (Gs protein). • This locks the Gs- protein in the on position, thereby continually stimulating adenylatecyclase to synthesize cAMP.

33. 2. E. Coli: Heat-stable toxin • polypeptide that is not inactivated by boiling for 30 minutes. • affects cGMP rather than cAMP. • stimulates guanylatecyclase • increases the concentration of cGMP • inhibits the reabsorption of sodium ions and causes non bloody diarrhea.

34. 3. E.coliVerotoxin • exotoxin produced by strains of E coli with the 0157:H7 serotype. • These strains cause bloody diarrhea • cause of outbreaks associated with eating undercooked hamburger in fast-food restaurants. • The toxin is named for its cytotoxic effect on Vero (monkey) cells in culture. • (Another name for verotoxin is shiga-like toxin).

35. Shigella: Shiga toxin • Enterotoxin produced by Shigellaand the toxin ricin which is produced by the Ricinus Plant • same mode of action as does verotoxin.

36. Enterotoxin produced by Vibriocholerae • Vibriocholerae the agent of cholera • cause of diarrhea • acts in a manner similar to that of the heat-labile toxin of E coli • stimulating adenylatecyclase to synthesize cAMP

37. Septic shock: • different from toxic shock. • In septic shock, the bacteria are in the bloodstream, whereas in toxic shock, it is the toxin that is circulating in the blood. • The clinical importance of this observation is that in septic shock, blood cultures are usually positive, whereas in toxic shock, they are usually negative. • Toxic shock can cause the death of a patient even though antibiotics have killed the bacteria in the patient’s blood, ie, the blood cultures have become negative. • This occurs because septic shock is mediated by cytokines, such as tumor necrosis factor and interleukin-l, that continue to act even though the bacteria that induced the cytokines are no longer present.

38. The biological effects of endotoxin include: 1)Fever: release bymacrophages of endogenous pyrogen (interleukin- 1), which acts on the hypothalamic temperature-regulatory center (2) Hypotension, shock, and impaired perfusion of essential organs. (3)Disseminated intravascular coagulation (DIC) due to activation of the coagulation system through Hageman factor (factor XII), resulting in thrombosis, a petechial or purpuric rash, and tissue ischemia (4) Activation of the alternative pathway of the complement cascade, resulting in inflammation and tissue damage. (5) Activation of macrophages increasing their phagocytic ability, and activation of many clones of B lymphocytes, increasing antibody production. (Endotoxin is a polyclonal activator of B cells, but not T cells.)

40. A typical acute infectious disease has four stages: (1) Incubation period, which isthe time between the acquisition of the organism (or toxin) and the beginning of symptoms (2) Prodrome period, during which nonspecific symptoms suchas fever, malaise, and loss of appetite occur. (3) Specific-illness period, during which the overtcharacteristic signs and symptoms of the disease occur (4) Recovery period, during which the illness abates and the patient returns to the healthy state.