Pathogenic Microbiology Course
Outline
Biology 3954
Science 110-A 8:00 - 9:00 M - F
http://www.tarleton.edu/~jkonvicka/
TEXT: Salyers and Whitt, Bacterial Pathogenesis
I. History of Microbiology and Disease
A. Names
(in some cases events) and dates
1. Demonic theory of disease
2. Pandora’s box
3. Hippocrates (ca. 460-377 B.C.)
a. Father of Medicine
4. Fracastorius (ca. 1478-1553)
a. “contagium vivum”
5. Leeuwenhoek (1632-1723)
a. “animalcules”
6. Plenciz (1705-1786)
7. Bassi (1773-1856)
8. Pasteur (1822-1895)
a. fermentation
b. superheated steam
c. dry heat
d. cotton plug
e. aerobe - anaerobe
f. pasteurization (1862)
9. Koch (1843-1910)
a. anthrax - Bacillus anthracis
10. Lister
a. dilution - 1878
11. Koch
a. raw potato - 1880
b. nutrient gelatin - 1881
c. inoculating loop - 1881
12. Fanny Hesse (1882)
a. agar-agar - 1882
13. Lister (1827-1912)
a. aseptic principle - 1868
14. Ehrlich (1854-1915)
a. chemotherapy
i. compound “606” - 1910
15. Domagk (1895-1964)
a. prontosil - 1935
i. sulfonamide
16. Fleming (1881-1955)
a. lysozyme
b. penicillin - 1929
i. Florey (1898-1968) - Chain (1906-?)
II. Host Defense Mechanisms Chapter 1,2
A. First line of
innate(nonspecific) defense
Chapter 1, p. 3-14
1. Normal skin
a. microbiota
b. sebaceous glands
2. Mucous membranes
a. mucus
b. antimicrobial substances
i. lysozyme
ii. IgA
3. Anatomical structures with microbiota
a. gastrointestinal tract
i. mouth
ii. stomach
(a). Helicobacter pylori story Chapter 22, p. 273-281
iii. small intestine
iv. large intestine
4. Urogenital tract
a. male-female anatomic differences
b. pH
B. Second line of
(nonspecific) defense Chapter 2, p. 16-24
1. Phagocytes
a. monocyte-macrophage
b. polymorphonuclear neutrophil (PMN)
2. Complement
a. series of proteins in blood
b. classical pathway
i. must have antibody to activate
ii. destruction of targeted material or cell
c. alternate pathway
i. activate by various materials
(a). microbial cellular components
(i). gram negative cell wall
(ii). mannose binding proteins (see below # 6)
3. Interferons
a. class of antiviral proteins
i. interfere with viral replication
4. Inflammation
a. increased blood flow
b. fever
i. hypothalamus in brain
ii. phagocytes
(a) lipopolysaccharide (LPS)
(i). interleukin-1(IL-1)
(ii). prostaglandins
iii. benefits
(a). inhibits microbes
(b). infiltration of other defense mechanisms
(c). IL-1 activates immune cells
5. Transferrin and lactoferrin
a. iron binding compound
b. store and deliver iron to host cells
6. Mannose-binding protein
a. activates complement by alternate pathway
C. Third line of
(specific)
defense
Chapter 2, p. 24-27
1. Immunoglobulins (Ig’s) - humoral immunity
a. IgG - most prevalent
b. IgM - largest and multivalent
c. IgA - secretions
i. protection of mucosal surfaces
d. IgE - allergies
i. protection against parasitic worms and tumor cells
e. IgD - regulator of B cells
2. Cytotoxic T cells - cell mediated immunity
a. cytokines (lymphokines)
3. Immunoglobulin production(B cells or B lymphocytes)
a. antigen presenting cell (APC)
i. phagocytic cells - macrophages and PMN’s
ii. major histocompatibility complex proteins (MHC)
b. T helper cell (CD-4)
c. B cells
d. immunoglobulins
III. Virulence Factors Chapter 3 & 4
A. Promote
colonization and
survival
Chapter 3, p.30-46
1. Adherence
a. pili (fimbriae)
b. afimbrial adhesins
c. gram positive adhesins
d. biofilms
2. IgA proteases
3. Iron acquisition
a. siderophores
4. Evasion strategies
a. capsule
5. Surviving phagocytosis
a. catalase
6. Evading Ig’s
a. resembling self
i. sialic acid
ii. fibronectin
iii. protein A - S. aureus
iv. protein G- Str. pyogenes
B. Damage
host
Chapter 4, p.47-62
1. Toxins
a. exotoxins
i. A-B toxins
(a). A portion - toxicity
(b). B portion - binding
(c). diphtheria toxin example Chapter 9, p. 113-121
(i). Corynebacterium diphtheriae
(ii). lysogenized by beta or omega phage
(iii). iron concentration critical
(iv). mode of action
[a]. Hela cells and cell free extract
[i]. stopped protein synthesis
[b]. dialyzed cell free extract
[i]. insensitive to action of toxin
[ii]. add NAD (Nicotinamide Adenine
Dinucleotide)
<a>. made sensitive to toxin
<b>. toxin cleaves NAD to
ADP-ribose
+ nicotinamide
<i>. ADP-ribose ties up
elongation factor 2
which "freezes" up
the ribosome and
stops protein
synthesis
ii. membrane-disrupting toxins
(a). channel forming type
(b). phospholipase enzyme type
iii. superantigen
(a). indiscriminate binding to the major
histocompatibility complex proteins
(b). stimulate T-cells
(i). interleukin 2 (T cell mitogen)
(c). over production of numerous cytokines
(d). shock
b. endotoxin
i. gram negative cell wall
(a). Lipid A
(b). core polysaccharide
(c). O-specific side chain
ii. septic shock
(a). cytokines
(b). coagulation cascade
(c). results -- collapse of circulatory system etc.,
which leads to multiple organ failure
2. Measurement of virulence (toxicity)
a. ID50
b. MLD
c. LD50
EXAM I Wednesday, September 19
IV. Genetic
Variability in Microorganisms
A. Simple
mutation
1. 1/106 division
B. Movement
or transfer of genetic material between organisms
1. Transformation
a. naked DNA (lysed donor cell)
2. Conjugation F+ -- F-
a. fertility factor (plasmid) (episome)
b. pili
3. Transduction
a. (temperate) phage mediated
b. lysogeny
V. Antibiotic
Mechanisms of Action and Bacterial Resistance
Chap.8, p.97-110
A.
Broad-spectrum (vs narrow spectrum) antibiotics
B.
Major mechanism targets
1. Bacterial peptidoglycan
2. Bacterial ribosome
3. Coenzyme synthesis (folate metabolism)
4. DNA supercoiling
5. RNA synthesis
C. Bacterial
peptidoglycan--types of antibiotics affecting such
1. Beta lactams -- block transpeptidation
a. penicillins
b. cephalosporins
c. carbapenems
d. monobactams
i. resistance mechanism for beta lactams:
(a). beta lactamase
(b). penicillin-binding-protein (PBP)
2. Glycopeptides -- bind the tetrapeptide and block final steps
a. vancomycin
b. teichoplanin
i. resistance mechanism for glycopeptides:
(a). change in the UDP-muramyl-pentapeptide
binding to the antibiotic
D.
Bacterial ribosome--types of antibiotics affecting such
1. Aminoglycosides -- binding 30s subunits
a. gentamycin
b. kanamycin
i. resistance mechanism for aminoglycosides:
(a). inactivate antibiotic by adding chemical groups
2. Tetracyclines - bind 30s subunit
a. tetracycline
b. aureomycin
i. resistance mechanism for tetracyclines:
(a). tetracycline efflux
(b). ribosome protection
3. Macrolides -- bind 50s subunit
a. erythromycin
i. resistance mechanism for macrolides:
(a). rRNA methylase
(b). transportation of antibiotic into cell
4. Lincosamides
a. lincomycin
i. resistance mechanism for lincoamides:
(a). same as macrolides - rRNA methylase or
transport of the antibiotic
E. DNA super coils--types
of antibiotics affecting such
1.
Quinolones--binds DNA gyrase
a. fluoroquinolone (new class of quinolones)
i. ciprofloxacin
b. nalidixic acid (mainly used as laboratory tool)
i. resistance mechanism for quinolones:
(a). genetic change in the DNA gyrase due to point
mutation
F. RNA synthesis--
types of antibiotics affecting such
1. Rifampin
- binding bacterial RNA polymerase
a. rifampicin
i. resistance mechanism for rifampin:
(a). point mutation in RNA polymerase
G. Folate
metabolism--types of antimicrobials “antibiotics”
affecting such
1.
Trimethoprim and sulfonamides -- inhibitors of enzymes in
tetrahydrofolic acid synthesis pathway
a. trimethoprim similar to dihydrofolic acid
i. competitive inhibitor of the enzyme dihydrofolate
reductase, the last enzyme in the pathway
b. sulfonamide similar to para-aminobenzoic acid
i. competitive inhibitor of the first enzyme in the pathway
ii. resistance mechanism for the folate pathway inhibitors:
(a). point mutation in the individual enzymes
VI. Staphylococcus
Chapter 10,p. 122-129
Chapter 11, p. 137-138
A. The Genus Staphylococcus
1. Staphylococcus
aureus
2. Staphylococcus
epidermidis
3. Staphylococcus
saprophyticus
B.
Staphylococcus aureus
1. Morphology
2. Physiology
a. cultural characteristics
i. facultative anaerobe
ii. carotenoid pigments
iii. beta or gamma hemolysis on blood
iv. catalase
v. mannitol positive
vi. coagulase enzymes
3. Cell
composition
a. Cell wall
i. peptidoglycan
(a) pentaglycine crosslinks
ii. teichoic acid
(a). ribitol phosphate - S. aureus
(b). glycerol phosphate - S. epidermidis
iii. protein A
(a). nonspecific binding with Ig’s
4. Classification
a. serology
b. bacteriophage typing - only coagulase positive S. aureus
i. groups I, II, III
ii. epidemiological studies
iii. genetics
(a). plasmid mediated
(b). antibiotic resistance
iv. environmental resistance
5. Determinates
of pathogenicity
a. multifactorial
b. surface antigens
i. teichoic acid
(a). complement component consumption
ii. protein A
(a). interact with IgG
c. extracellular enzymes
i. coagulase
(a). coagulase reacting factor
(i). fibrinogen ----------- fibrin
ii. lipases
iii. staphylokinase
(a). plasminogen ------------ plasmin
iv. nuclease
(a). phosphodiesterase
(i). both DNA and RNA
d. toxins
i. cytolytic toxin
(a). alpha toxin
(i). dermonecrotic
(ii). transmembrane channels
(b). beta toxin
(i). sphingomyelinase ( phospholipase)
(ii). "hot-cold" RBC lysis
(c). delta toxin
(i). detergent-like
(ii). broad-spectrum
(d). Leukocidins
(i). PMN’s and macrophages only
(ii). cation permeability (pore-former)
ii. enterotoxins (may induce the superantigen
phenomenon)
(a). six groups
(i). A - E
(b). food poisoning
(i). vomiting and diarrhea
iii. exfoliative toxins (superantigen phenomenon)
(a). scalded-skin-syndrome
iv. toxic shock syndrome toxin - 1 (superantigen
phenomenon)
(a). multiple organ dysfunction
6. Clinical
manifestation
a. furnuncles and carbuncles
b. impetigo
c. scalded skin syndrome
d. pneumonia
e. osteomyetitis
f. pyoarthrosis
g. bacteremia and endocarditis
i. metastatic infections
h. food poisoning
i. toxic shock syndrome (TTS)
7. Other
medically significant staphylococci
a. Staphylococcus epidermidis
i. low virulence
ii. breached host defense
iii. glycocalyx adhesion
b. Staphylococcus saprophyticus
i. normal microbiota
ii. urinary tract infection - mainly female
EXAM II Monday, October 8
VII. Streptococcus Chapter 10, p. 122-129
A. Historical aspects
B. Properties of genus
1. Gram positive
2. Spherical but slightly ovoid
3. Pairs or chains
4. Facultative anaerobe ?
a. fermentative metabolism
5. Catalase negative
6. 27 species
7. Classification
a. Lancefield’s grouping
b. hemolytic activity
c. physiological properties
C. Streptococcus pyogenes
1. Lancefield’s group A
2. Hemolytic activity on blood agar
a. beta hemolysis
3. Increased CO2 level
4. Bacitracin test
5. Antigenic structure and pathogenicity
a. C-polysaccharide
i. Rebecca Lancefield
ii. serological classification
iii. extraction
iv. precipitation reaction
v. polymer of rhamnose and N-acetyl glucosamine
vi. not a virulence factor
b. M-protein
i. hairlike projection (fibrillar molecule)
ii. 80+ distinct serotypes
iii. resists phagocytosis
iv. anticomplementary (questionable)
(a). interfere with C3b deposition ?
c. M-like proteins (another cell wall protein)
i. F protein
ii. adhesin--binds fibronectin on host cells
d. lipoteichoic acid (LTA)
i. adhesin question
ii. attachment to buccal epithelial cells
e. hemolysins
i. streptolysin O
(a). oxygen labile
(b). in diverse species
(c). pore-forming cytotoxin
(d). antibodies-Anti-Streptolysin O (ASO)
(i). Todd units
ii. streptolysin S
(a). oxygen stable
(b). hemolytically active polypeptide attached to an
oligonucleotide
f. erythrogenic toxin
i. 90% of all Group A
ii. three serotypes (A, B, and C)
iii. phage mediated
iv. rash and fever
v. superantigen
(a). T-cell mitogen
vi. Dick test
vii. Schultz-Charlton reaction
g. nucleases
h. streptokinases
i. fibrinolysin
ii. thrombolytic agent used in humans
iii. plasmin
6. clinical
infections
Chapter 28, p. 332-338
a. pharyngitis and scarlet fever
i. “strep” throat
ii. scarlatinal rash
iii. erythrogenic toxin producing strains
iv. type specific immunity to pharyngeal infections
b. skin infections
i. impetigo
ii. cellulitis
iii. erysipelas
c. acute rheumatic fever
i. nonsuppurative
ii. antecedent to Group A infection
iii. clinical manifestation
(a). arthritis
(b). carditis (valvular heart disease)
(c). chorea (involuntary muscle twitches)
iv. serotypes - various
(a). about 10 different varieties
d. acute poststreptococcal glomerulonephritis
i. post infectious complication
(a). pharyngitis and cutaneous infections
ii. immune complexes
(a). streptolysin O - antistreptolysin O
iii. mostly in children
iv. clinical manifestations
(a). hematuria
(b). proteinuria
D. Streptococcus agalactiae
1. Group B
2. Beta hemolytic
3. CAMP positive
a. St. aureus beta lysin (sphingomyelinase)
b. complete lysis of erythrocyte
4. Commonly found
a. female vagina
b. pharynx
5. Carrier state in pregnant female
6. Clinical manifestation
a. puerperal infection
b. neonatal septicemia
c. neonatal meningitis
i. mortality of 50% or greater
E. Streptococcus equi
1. Group C
2. Disease in horses
F. Enterococcus fecalis
1. Group D
2. Clinical manifestation
a. urinary tract infection or endocarditis
3. Antibiotic resistance
G. Viridans streptococci
1. Alpha-hemolytic
2. No group specific carbohydrate
3. Mouth or upper respiratory tract
4. Str. salivarius
a. endocarditis
5. Str. mutans
a. dental plaque
i. dextran polymer from sugar sucrose
b. dental caries
i. Lactobacillus spp.
H. Streptococcus pneumoniae
Chapter 27, p. 322-331
1. Historical aspects
2. Gram-positive
3. Coccus
4. Pairs and short chains
5. Encapsulated
6. Facultative anaerobe
7. Aerobic conditions
a. H2O2 produced
b. killed quickly
i. added blood for catalase
8. Increased CO2 tension
9. Optochin sensitive
a. ethylhydrocupreine hydrochloride reagent
10. Bile solubility
a. autolytic amidase
i. cleaves bonds between muramic acid and alanine
in cell wall
ii. causing cell to lyse
11. Quellung reaction
a. serological identification
b. antibodies to capsular polysaccharide will cause the
capsule to swell or enlarge
12. Antigenic structure and pathogenicity
a. capsular antigen
i. 90 some odd serotypes
ii. basis of vaccine
(a). 23 capsular types
iii. not an adhesin
iv. antiphagocytic
(a). degrades 3Cb
b. pneumolysin
i. autolysin
ii. releases the cytoplasmic toxic proteins
13. Clinical manifestation
a. pneumonia
i. debilitated patient
b. upper respiratory tract
i. middle ear
(a). otitis media
ii. young children
VIII. Neisseria Chapter 20, p. 244-259
A. Historical background
B. Properties of Genus
1. Gram-negative
2. Cocci
3. Pairs with adjacent sides flattened
4. Nonmotile
5. Mostly aerobic
6. Cytochrome oxidase
a. oxidase reagent -- tetra-p-phenylenediamine
dihydrochloride
7. Catalase
8. Nutritionally fastidious
a. Thayer-Martin medium
C. Neisseria meningitidis
1. Nine serotypes
a. capsular polysaccharide
2. Determinants of pathogenicity
a. capsular polysaccharide
i. antiphagocytic
b. endotoxins
i. LPS containing outer membrane
(a). vascular necrosis
(b). inflammatory response
(c). endotoxic shock
c. IgA1 protease
3. Clinical infection
a. Weichselbaum first described
b. nasopharyngeal carrier
c. crowded conditions
d. some epidemic tendencies
4. Host defense
a. specific immunity to capsular polysaccharide
b. antibody-dependent complement mediated
phagocytosis and killing
c. also complement alternate pathway activation
D. Neisseria gonorrhoeae
1. Colonial forms
a. T1 and T2
i. small dome-shaped
ii. piliated
iii. virulent
b. T3 and T4
i. large - flat
ii. no pili
iii. non infectious?
2. Typing systems
a. auxotyping - nutritional requirements
i. 30+ auxotypes
b. serological
i. Ab to protein 1 of outer membrane
(a). 46 serovars
(b). epidemiology
3. Antigenicity and pathogenicity
a. Big Problem - no laboratory animal models
b. pilus
i. pilin - long thin polymer
(a). adhesins
(i). to host columnar epithelium
ii. phase variation
(a). P+ vs P-
iii. antigenic variation
(a). eight variants
c. outer membrane proteins
i. protein I (PI)
(a). 60% total weight
(b). prevent phagolysosome (fusion) formation
(c). produced as an invarible low or high molecular
weight material
(i). gonococci with low molecular weight PI
cause disseminated infections
(ii). gonococci with high molecular weight PI
cause localized infections
(d). can form porin in combination with protein III
(PIII)
ii. protein II (PII)
(a). opaque colony (Opa)
(b). adhesin
(i). mucosal cells
(ii). neutrophils
d. lipooligosaccharide
i. similar to lipopolysaccharide
(a). lower molecular weight
(b). no repeating O-side chain
ii. responsible for most of the symptoms
iii. inflammatory response
iv. serum resistance
(a). due to sialic acid content
(i). prevent complement activation
(ii). blocks Ig binding
v. superantigen
e. IgA protease
4. Clinical manifestation
a. 10% males and 33% females are asymptomatic
b. blindness in neonates
i. Crede’ method
(a). silver nitrate prophylactic measure
c. pelvic inflammatory disease (PID)
i. fallopian tube involvement
IX. Mycobacterium Chapter 26, p. 307-319
A.
General comments
1. Mycobacterium tuberculosis and Mycobacterium bovis
a. etiologic agents of tuberculosis
B.
Properties of the organisms
1. Slender, straight rod
2. Nonmotile
3. Nonencapsulated
4. Acid-fast
5. Most resistant nonspore-forming bacterium
6. Facultative intracellular parasites
7. Slow growers
a. needs 37oC with increase CO2
b. takes up to 6 weeks to grow
8. Growth media
a. Lowensteins-Jensen medium
i. potato-egg and serum agar
b. Middlebrook 7H10 medium
9. Differential characteristics
a. niacin production by M. tuberculosis
b. parasitic vs. nonparasitic mycobacteria
i. rapid growth - nonparasitic
ii. yellow pigmentation - nonparasitic
10. Cell wall
a. contains waxes and mycolic acid
i. contribute to environmental resistance
ii. responsible for intracellular growth
iii. highly stimulatory to mammalian immune system
(a). Freund’s adjuvant
C. Transmission
1. Droplet nuclei
2. Certain ethnic groups most susceptible
a. Asian, American Indian, Blacks
D. Pathogenesis and clinical manifestation
1. Primary pulmonary type tuberculosis
a. early exudative response
i. PMN infiltration
(a). multiply within PMN
(i) mycolic acid in cell wall inhibit phagolysomal
fusion
(b). macrophages and lymphokines
ii. granulomatous response
(a). large multinucleated host cells
(b). tubercles
b. later response
i. four weeks or so later CMI or DTH develop
E. Laboratory Diagnosis
1. Sputum
a. digestion and concentration
b. microscopic examination (acid-fast stain