1. Meeting 6 Disease Manifestations Virulence Factors Antimicrobial resistance
1 Aug 2009

2. Manifestations of infectious disease
Classic
Signs
Fever, swelling, rashes, vomiting, diarrhea
Skin signs: lesions, erythema, papule (pimple), vesicles, pustule, ulcer or erosion or abscess
Symptoms
Pain, headache, nausea, malais
Inflammation
Result of Immune reactions
Hematologic ( leukocytosis, anemia )
Cardiac (tachycardia to heart failure)
Respiratory (hyperventilation)
Renal
Hepatic
Upper GI bleeding

3. Manifestations of infectious disease
                                                                       Table 1 Types of skin lesions
Flat lesions (in the plane of the skin)
Elevated lesions (above the plane  of the skin)
Depressed lesions (below the plane of  the skin)
Macule IInfarct Sclerosis• Telangiectasis†
Vesicle and bulla Pustule Abscess‡ Cyst‡ Papule Wheal plague Nodule‡ Vegetation Keratosis Desquamation     (scales) Exudate• (crusts) Lichenification
Atrophy§ Sclerosis§ Erosion Excoriation Scar† Ulcer Sinus‡ Gangrene§
• May also be below the plane of skin † May also be above the plane of skin ‡ May also be in or below the plane of skin § May also be in the plane of skin
Classic
Signs
Fever, swelling, rashes, vomiting, diarrhea
Skin signs: lesions, erythema, papule (pimple), vesicles, pustule, ulcer or erosion or abscess
Symptoms
Pain, headache, nausea, malaise
Inflammation

4. Flat lesion
MACULE
A macule is a change in the color of the skin. It is flat, if you were to close your eyes and run your fingers over the surface of a purely macular lesion, you could not detect it. A macule greater than 1 cm. may be referred to as a patch.

5. Elevated lesion PAPULE
is a solid raised lesion that has distinct borders and is less than 1 cm in diameter. Papules may have a variety of shapes in profile (domed, flat-topped, umbilicated) and may be associated with secondary features such as crusts or scales.

6. Elevated lesion
TUMOR
is a solid mass of the skin or subcutaneous tissue; it is larger than a nodule.

7. Elevated lesion PAPULE
is a solid raised lesion that has distinct borders and is less than 1 cm in diameter. Papules may have a variety of shapes in profile (domed, flat-topped, umbilicated) and may be associated with secondary features such as crusts or scales.

8. Depressed lesion ATROPHY
Atrophy is thinning or absence of the epidermis or subcutaneous fat.

9. Depressed lesion EROSION
Erosions are slightly depressed areas of skin in which part or all of the epidermis has been lost

10. Depressed lesion ULCERATION
occur when there is necrosis of the epidermis and dermis and sometimes of the underlying subcutaneous tissue.

11. FEVER
1. Fever increases the environmental temperature above the optimum growth temperature for many microorganisms. If the microorganisms are growing more slowly, the body's defenses have a better chance of removing them all.
2. Fever leads to the production of heat shock proteins that are recognized by some intraepithelial T-lymphocytes (delta gamma T-cells) resulting in the production of inflammation-promoting cytokines.
3. Fever elevates the temperature of the body increasing the rate of enzyme reactions, and speeding up metabolism within the body.
can increase the production and activity of phagocytes,
speed up the multiplication of lymphocytes,
increase the rate of antibody and cytokine production,
increase the rate at which leukocytes are released from the bone marrow into the bloodstream, and speed up tissue repair. Too high of a body temperature, however, may cause damage by denaturing the body's enzymes.

12. FEVER Fever may have certain signs in relation to its course.
Fever in infectious diseases usually is of short duration
Long duration (weeks or months) is always a very serious problem. If it is not possible to determine the cause of fever at the beginning, it is called the fever of unknown origin (FUO). This term is used to describe fever lasting at least 2 weeks, reaching temperatures above 38,2 , and the cause of the origin is uncertain.
Fever may last long in some infections with subacute or chronic course.

13. Stages in the Progression of Disease
No signs or symptoms
Prodormal illness
Mild signs or symptoms
Some chilling occur
Acute identifiable signs and symptoms
Intermittent fever
Recovery
Action of immune system
Action of antibiotics
Convalescence

14. Stages in the Progression of Disease

17. Manifestations of infectious disease
Result of Immune reactions
Hematologic ( leukocytosis, anemia )
Cardiac (tachycardia to heart failure)
Respiratory (hyperventilation)
Renal
Hepatic
Upper GI bleeding

18. Case study 1 S. T. was seen by you in the Emergency Room in December
23 y/o medical student in Southern California
c/o sudden onset of fever, chills, malaise, headache, myalgia, sore throat, runny nose, sneezing
Room-mate has same symptoms
Exam: erythematous, inflammed tonsils,
no pharyngeal exudates
Throat culture: Group A streptococci

19. Case study 1
erythematous, inflammed tonsils

20. Case study 2 P. A. is a patient in the Intensive Care Unit 65 y/o man
Intubated, on respirator: good oxygenation
Nurse says that he hasn’t had fever or purulent sputum
Exam: clear breath sounds, no rhonchi nor rales
CXR: clear without infiltrates or effusions
Sputum Gram stain: mixed flora with Gram positive cocci; thin, long Gram negative rods
Sputum culture: normal respiratory flora, Pseudomonas aeruginosa

21. Case study 3 T. M. was referred to you by the Public Health Department
38 y/o woman
Private cook in Manhattan
In the past 10 years, 7 of the 8 families she has worked for have had outbreaks of illness:
Fever, malaise, headache, myalgia, maculopapular rash, bradycardia, constipation, bloody diarrhea
T. M. denies h/o similar illness and denies current symptoms
“But, Doctor, I’m not sick!”

22. Case study 3
maculopapular rash

23. Disease state: complex interaction between pathogen and host
1. If a bacterium is present, is it causing disease?
a. Normal flora
b. Colonization
c. Carrier state
d. Infection:
i. Asymptomatic
ii. Symptomatic
2. Is the bacterium capable of causing disease?
a. Nonpathogen
b. Opportunistic pathogen
c. Primary pathogen

24. Case study 1: Viral pharyngitis + Group A strep normal respiratory flora
S. T. was seen by you in the Emergency Room in December
23 y/o medical student in Southern California
c/o sudden onset of fever, chills, malaise, headache, myalgia, sore throat, runny nose, sneezing
Room-mate has same symptoms
Exam: erythematous, inflammed tonsils,
no pharyngeal exudates
Throat culture: Group A streptococci

25. Case study 2: Colonization with Pseudomonas aeruginosa
P. A. is a patient in the Intensive Care Unit
65 y/o man
Intubated, on respirator: good oxygenation
Nurse says that he hasn’t had fever or purulent sputum
Exam: clear breath sounds, no rhonchi nor rales
CXR: clear without infiltrates or effusions
Sputum Gram stain: mixed flora with Gram positive cocci; thin, long Gram negative rods
Sputum culture: normal respiratory flora, Pseudomonas aeruginosa

26. Case study 3: Typhoid Mary Salmonella typhi carrier
T. M. was referred to you by the Public Health Department
38 y/o woman
Private cook in Manhattan
In the past 10 years, 7 of the 8 families she has worked for have had outbreaks of illness:
Fever, malaise, headache, myalgia, maculopapular rash, bradycardia, constipation, bloody diarrhea
T. M. denies h/o similar illness and denies current symptoms
“But, Doctor, I’m not sick!”

27. Outbreak 2001

30. Virulence Factors that Promote Colonization and Invasion
Virulence factors that damage the host
Exotoxins
Endotoxins
Ability to resist innate immunity
Ability to evade adaptive immunity
Ability to induce autoimmune response

31. Virulence Factors that Promote Colonization and Invasion
Basic requirements: receptor + adhesin
Receptor
Specific carbohydrate or peptide residues on the cell surface
Adhesin or adherence factor
A macromolecular component of the bacterial cell surface interacting to the receptor

32. Virulence Factors that Promote Colonization and Invasion
Surface proteins called adhesins in the bacterial cell wall bind to receptor molecules on the surface of a susceptible host cell enabling the bacterium to make intimate contact with the host cell, adhere, colonize, and resist flushing.

33. Virulence Factors that Promote Colonization and Invasion
Specific adherence of bacteria to cell and tissue surfaces
Tissue tropism
Strep mutans in dental plaque but not in the tongue
Species specificity
Neisseria gonorrheae limited to humans
Genetic specificity within a species
Mechanism of specific adherence
Reversible attachment – “docking”
Non-reversible attachment – “anchoring”

34. Virulence Factors that Promote Colonization and Invasion: Example
(Tabulate according to the ff:
Bacterium:Adherence Factor:Receptor (optional):Attachment Site: Disease)
Streptococcus pyogenes:
Protein F
Aminoterminus of fibronectin
Pharyngeal Epithelium
Sore Throat

35. S. pyogenes
F-protein
lipoteichoic acid
fibronectin

36. Virulence Factors that Promote Colonization and Invasion:
Fimbriae
Adhesins
Protein F
Capsules
Invasins
Cleaves secretory IgA (Glycosyl transferase)
Spreading factors:
Hyaluronidase – attacks the interstitial cement of connective tissue by depolymerizing hyaluronic acid
Collagenase
Neuraminidase
Streptokinase and staphylokinase
Siderophores
Low molecular weight compounds that chelate iron with very high affinity
Competing for iron and other nutrients

37. Virulence Factors that Promote Colonization and Invasion

38. Virulence Factors that Promote Colonization and Invasion

39. Virulence Factors that Promote Colonization and Invasion
Glycocalyx
Polysaccharide or peptide slime
Capsule
Slime layer
Functions:
Resists phagocytosis
Enhanced attachment

40. Virulence Factors that Promote Colonization and Invasion
A proposed model for invasion of epithelial cells of the colon. 1) The Shigella first cross the mucosa by passing through specialized cells called M cells. The M cell passes the Shigella on to a macrophage from which it subsequently escapes - possibly by inducing apoptosis, a programmed cell suicide. 2) The Shigella then uses its invasins to enter the mucosal epithelial cells from underneath. The invasins cause actin polymer rearrangements in the cell's cytoskeleton resulting in the bacterium being engulfed and placed in an endocytic vesicle in a manner similar to phagocytic cells. Once inside, the Shigella escape from the vacuole into the cytoplasm and multiply. 3) The Shigella are able to move through the host cell and spread to adjacent host cells by a unique process called actin-based motility. In this process, actin filaments polymerize at one end of the bacterium, producing comet-like tails that propel the Shigella through the cytoplasm of the host cell. 4) When they reach the boundary of that cell, the actin filaments push the Shigella across that membrane and into the adjacent cell.
Shigella Passing Through the Mucous Membrane and Invading Mucosal Epithelial Cells

41. Virulence Factors that Promote Damage to the Host: EXOTOXINS
Properties
Secreted during exponential growth
Protein toxins
High biological activity
Exhibits specificity of action
Components
A = active
B = binding

42. A-B toxins
Cell surface
Active
Binding A B

43. Binding and Entry of an A-B Toxin
A-B toxins consist of two parts, an A (active) component and a B (binding) component. The B component of the exotoxin binds to a receptor on the surface of a susceptible host cell. The exotoxin now enters the host cell, in this case by endocytosis, and causes harm by inactivating a host cell target protein through ADP-ribosylation.

44. Virulence Factors that Promote Damage to the Host: EXOTOXINS
Based on structure
A-B prototype
Botulinum toxin, diphtheria toxin, shiga toxin, tetanus toxin
Membrane disrupting toxin
Lacks A & B
Pore forming
Phospholipase
Superantigens

45. Superantigens 1. Some exotoxins are superantigens
2. Produced by bacteria and viruses
3. Action: polyclonal stimulation of subset of lymphocytes to divide and produce cytokines
4. Best known example: Staph Toxic Shock Syndrome Toxin-1 (TSST-1)
5. Also Strep exotoxins
6. Pyrogenic toxins cause fever (unlike other exotoxins)

46. 2-subunit A-B exotoxin Neurotoxin: Clostridium botulinum
Murray, P.R. et al. Medical Microbiology, 3rd edition, 1998, p. 156

47. Botulism Exotoxin Blocking Acetylcholine Release
The botulism exotoxin binds to the presynaptic neuron and blocks its release of acetylcholine. This causes a flaccid paralysis, a weakening of the involved muscles.

48. Membrane damaging exotoxins
Proteases
Phospholipases
Detergent-like action

49. Virulence Factors that Promote Damage to the Host: EXOTOXINS
Based on modes of action
Breaks down cells
Alpha toxins, hemolysins, leukocidins
Enhance microbial spread
Spreading factors: hyaluronidase, mucinase etc
Interfere in cellular metabolism
Tetanus toxin, botulinum toxin

50. Virulence Factors that Promote Damage to the Host: EXOTOXINS
3 ways this can contribute to the progression of disease:
Ingestion of preformed toxin
Colonization of wound or mucosal surface followed by exotoxin production
Colonization of wound followed by local exotoxin production

51. Neutralization of Exotoxins

52. Evasion of host immune response: IgA protease
1. Cleaves IgA, which is important for mucosal immunity
2. An enzyme produced by Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae

53. Requirement for iron 1. Most iron in the body is intracellular
In hemoglobin and myoglobin
2. There is very little free iron
Extracellular iron is bound to transferrin (in plasma) and lactoferrin (in milk and other secretions)
3. Some bacteria produce siderophores to capture iron e.g. Escherichia coli
Siderophores bind iron with high affinity

54. Virulence Factors that Promote Damage to the Host: ENDOTOXINS
Outer membrane of the gram negative cell wall
Lipid A:polysaccharide:O antigen
Toxicity is associated with Lipid A
Immunogenicity is associated with the polysaccharide component
Less potent, less specific
Does not form antitoxin
Released when the bacterial cell is damaged

55. Periplasmic binding protein
GRAM NEGATIVE CELL ENVELOPE
Outer Membrane
(Major permeability barrier)
Lipopolysaccharide
Porin
Braun lipoprotein
Periplasmic space
Degradative enzyme
Periplasmic binding protein
Permease
Inner (cytoplasmic) membrane
Cytoplasm

56. Septic shock Septic shock hypotension (tissue pooling of fluids)
disseminated intravascular coagulation
fever
lack of effective oxygenation
overall system failure
Septicemia

57. Exotoxin vs Endotoxin

58. Bacterial Pathogenesis
1. Microbial pathogens and their hosts have often co-evolved
2. Clinical disease
a. Non-adapted host/pathogen
b. May promote pathogen’s survival and transmission
c. Can result from host response to pathogen
3. Extracellular bacteria produce toxins and enzymes

59. Bacterial Pathogenesis
4. Bacteria secrete proteins into host cells to modify host cell function
5. Intracellular bacteria must bind, enter and survive inside host cells
6. Pathogenic bacteria can evade the host immune response and have mechanisms for antibiotic resistance
7. Expression of virulence factors is often regulated in response to the environment and other signals

60. Bacterial Pathogenesis: Concepts
1. Virulence genes that encode virulence factors
A virulence gene confers on a bacterium the ability to cause disease
Pathogenic strains have acquired virulence genes that allow them to exploit the host as an environmental niche
Examples of virulence factors are toxins, enzymes, type III secreted proteins, adhesins, siderophores
Virulence factors are obvious targets for prevention or treatment strategies

61. Bacterial Pathogenesis: Concepts
2. Bacteria have found many way to modulate host cell function
Exotoxins and type III secreted proteins are virulence factors with specific functions such as to kill host cells or induce the uptake of bacteria by the host cell
Knowledge of these mechanisms can help explain the clinical disease caused by a particular bacterium, and the basis for prevention and treatment strategies

62. Bacterial Pathogenesis: Concepts
3. Toxins vs. toxoids vs. antitoxin (antitoxoid)
Toxins are made by bacteria and have harmful effects on host cells
A toxoid is the inactivated form of an exotoxin that can be used as a vaccine to elicit an immune response
Toxoids were traditionally inactivated by heat or formaldehyde, but now can be genetically engineered
Antitoxin is the antisera produced in response to the toxoid (can be from animals or humans) and can be given as treatment

63. Bacterial Pathogenesis: Concepts
4. Mechanisms of pathogenesis are conserved among bacteria
Virulence genes that provide a selective advantage are spread among different bacterial genera and species by horizontal transfer
These virulence genes are often located on mobile DNA elements such as plasmids or bacteriophages

64. … refresh...

65. Antibiotics
Antibiotics and vaccines are among the biggest medical advances since (Culver Pictures)
For lecture only
BC Yang

66. A brief history of antibiotics
1495, mercury to treat syphilis.
1630, quinine (chinchona tree) for malarial fever by South American Indians.
1889, Buillemin defined antibiosis.
1910, Paul Ehrlich developed arsenical compound (Salvarsan) for syphilis, term: the chemical knife.
1929, Alexander Fleming found penicillin.
1935, Gerhard Domagk showed the value of sulfonamides.
1940, Ernst Chain and Howard Flory demonstrated the effect of penicillin.
, then searching for new antibiotics
~ recent year: modifying old drugs, finding new discipline in antibacterial combats
Early time in war: thanks penicillin, we can go home now
Now a day……….Oh eh?!

67. Thanks to work by Alexander Fleming ( ), Howard Florey ( ) and Ernst Chain ( ), penicillin was first produced on a large scale for human use in At this time, the development of a pill that could reliably kill bacteria was a remarkable development and many lives were saved during World War II because this medication was available.
E. Chain
H. Florey
A. Fleming

68. A tale by A. Fleming
He took a sample of the mold from the contaminated plate. He found that it was from the penicillium family, later specified as Penicillium notatum. Fleming presented his findings in 1929, but they raised little interest. He published a report on penicillin and its potential uses in the British Journal of Experimental Pathology.

69. An ideal antibiotic Broad-spectrum Does not induce resistance
Selective toxicity, low side effects
Preserve normal microbial flora

70. Definitions Antimicrobial Antibacterial Antibiotic
Inhibits growth of micro-organisms
Antibacterial
Inhibits growth of bacteria
Antibiotic
Made by other micro-organisms
Usually extended to include synthetic drugs

71. Susceptibility test Tube dilution method Disk diffusion method
Minimal inhibitory concentration (MIC): the smallest amount of chemotherapeutic agent required to inhibit the growth of organism in vitro
Disk diffusion method
Zone of inhibition (ZOI): the correlation of ZOI and MIC has been established by FAD
ETest. This commercially-prepared strip creates a gradient of antibiotic concentration when placed on an agar plate

72. Bacteriostatic vs Bactericidal
Reversible inhibition of growth
When the antibiotic is removed, almost all of the bacteria can replicate
Bactericidal
Irreversible inhibition of growth
When the antibiotic is removed, almost none of the bacteria (10-7 to 10-3) can replicate

73. Guidance of antimicrobial therapy
Minimum inhibitory concentration: lowest concentration of antibiotic that inhibits visible growth
Minimum bactericidal concentration: lowest concentration of antibiotic that kills 99.9% of the inoculum
Serum bactericidal title: dilution of serum that kills 99.9% of the inoculum
Synergy test: synergistic activity of multiple antibiotics

74. Use of antibiotics; is it properly applied?
Acute infections in outpatients
Acute infections in hospitalized patients
Chronic infection (tuberculosis, AIDS)
Agriculture/veterinary medicine

75. In vitro: Factors for optimal antibiotic action
pH of environment:
Nitrofurantoin is more active in acid pH; sulfonamides and aminoglycoside are more active in alkaline pH.
Components of medium:
Anionic detergents inhibit aminoglycosides, serum proteins bind to penicillin in varying degrees.
Stability of drug:
Aminoglycosides and chloramphenical are stable for long period in vivo.
Size of inoculums:
The larger the bacterial inoculum, the greater the chance for resistnat mutant to emerge.
Metablic activity of microorganisms:
Actively and rapidly growing organisms are more susceptible to drug action
BC Yang

76. Affecting factors in vivo
Abscess: circulation is blocked off.
Foreign bodies: obstruction of the urinary, biliary or respiratory tracts etc.
Immunity.

77. Sites of action
BC Yang

78. Modes of action (1)
Inhibitors of cell wall synthesis. Penicillins, cephalosporin, bacitracin, carbapenems and vancomycin.
Inhibitors of Cell Membrane. Polyenes - Amphotericin B, nystatin, and condicidin. Imidazole - Miconazole, ketoconazole and clotrimazole. Polymixin E and B.
Inhibitors of Protein Synthesis. Aminoglycosides - Streptomycin, gentamicin, neomycin and kanamycin. Tetracyclines - Chlortetracycline, oxytetracycline, doxycycline and minocycline. Erythromycin, lincomycin, chloramphenicol and clindamycin.
vancomycin
Amphotericin
Aminoglycosides
Tetracyclines
For lecture only
BC Yang

79. Modes of action (2)
Inhibitors of metabolites (Antimetabolites). Sulfonamides - Sulfanilamide, sulfadiazine silver and sulfamethoxazole. Trimethoprim, ethambutol, isoniazid.
Inhibitors of nucleic acids (DNA/RNA polymerase). Quinolones - Nalidixic acid, norfloxacin and ciprofloxacin. Rifamycin and flucytosine. 
rifamycin
For lecture only
BC Yang

80. Resistance Natural (inherent) resistance Structural barrel
Lack of target
Transport system
Acquired resistance
Mutation
Gene exchange (conjugation in most)

81. Transferable antibiotic resistance in bacteria
Reduced uptake into cell (chloramphenicol)
Active efflux from cell (tetracycline)
Modification of antibiotic targets (b-lactam, erythromycin)
inactivation of antibiotic by anzymic modification: hydrolysis (b-lactam, erythromycin); derivatization (aminoglycosides)
Sequestration of antibiotic by protein binding (b-lactam)
Metabolic bypass (sulfonamides)
Overproduction of antibiotic target (titration: sulfonamides)

82. Spread of resistance In most:
Day-care, nursing homes, correctional facilities
Sanitation, animal feeds (fecal-oral)
Sexual/ Respiratory transmission
International travel
Immunosuppression

83. Some probable overuse/misuse of antibiotics
Prophylatic use before surgery
Empiric use (blinded use)
Increased use of broad spectrum agents
Pediatric use for viral infections
Patients who do not complete course (chronic disease, eg. TB, AIDS)
Antibiotics in animal feeds
For lecture only
BC Yang

84. Policy to deal drug resistance (1)
Ideally, bacteriological management of clinical infection should involve:
Identification of causative organism
Sensitivity test
Follow-up the drug effect
Monitor antibiotic level to avoid toxicity.
In reality, most patients requiring antimicrobial therapy are treated empirically. In serious infections immediate chemotherapy may be life-saving.

85. Policy to deal drug resistance (2)
Periodic changes of antibiotics used might change selective pressure and thus avoid the emergence of resistance and retain the therapeutic value of antibiotics over a longer period.
The unnecessary prophylactic or animal feeds use should be discouraged.
Distribution of information on current/updated infectious microbes (consult microbiologists): use more targeted antibiotics
Patient education

86. New antibiotics development
Pharmaceutical industry putting resources back into discovery
Liaisons with university researches
Discoveries in microbial physiology and genetics offering new targets, new disciplines
Combinational chemistry (mass screening)
For lecture only
BC Yang