INTRODUCTION TO BACTERIOLOGY AND PATHOGENESIS

PAUL A. GULIG, PH.D.
INTRODUCTION TO BACTERIOLOGY AND PATHOGENESIS
OCTOBER 13-21
Office - R1-250, 392-0050 Lab R1-144, 392-0682
email: [email protected]

Communication is key:

Check emails and course web page for email archive and corrections

Three sections:
introduction to bacteriology
pathogenesis of infectious diseases caused by bacteria
parasitology/mycology

Note:
Virtual Microbiology Lab
Clinical Microbiology Conferences
BUGS cases
A ten-year old boy experiences a sudden onset of extremely sore throat, pain on swallowing, fever of 103˚F, swollen lymph nodes in his neck, and general malaise.
If his throat looked like this ...












what would it be?
If his throat looked like this ...
















what would it be?
Why is his throat so inflamed?


What is the mechanism of damage?


Are there any possible serious consequences to this disease? Why, why not?
What nonspecific defenses were available to fight this infection?


Why were they not effective?


Will specific immunity eventually clear this infection? Why, why not?


Will he be immune from the disease in the future? Why, why not?
Can this infection be treated with antibiotics? Why, why not?


What determines your choice of antibiotics to try?


Do you have to worry about the organism being resistant or becoming resistant to the antibiotics? Why or why not?
Are the boy’s siblings and classmates at risk for getting this disease from him?
“The fundamental differences in the structure and physiology of bacteria as infectious agents vs. us as hosts are the bases for most of the damaging effects of infectious disease and our ability to fight infectious disease with antibiotics.”


For example, the small size and simple internal structure enable the rapid growth of bacteria to contaminate food or overcome host defenses.
Bacterial structure
A. Small (1-8 microns)
B. Shapes (important for identification and making diagnosis)
Others (vibrios, filamentous, coccobacilli)
Envelope structure is unique to prokaryotes
1. Cell wall - rigid structure surrounding the cell membrane **note**
a. functions - prevent osmotic lysis, protect cell from external stresses (host), contributes to virulence, target for antimicrobials
b. Gram stain and Acid fast stain
i. gram-positive (blue)
ii. gram-negative (pink)
iii. acid fast (red on blue)
iv. wall-less (can’t stain)


Gram-positive structure

thick peptidoglycan cell wall (40+ layers of chain link fence)
resist lysis by complement, but still can be opsonized
teichoic acids and lipoteichoic acids (polymer of ribitol or glycerol – phosphates), antigenic classification
other proteins and carbohydrates (e.g., M protein fibrillar layer and Group A carbohydrate capsule of Streptococcus pyogenes contribute to virulence).
Gram-negative structure

the outer membrane - a second lipid bilayer
periplasmic space between inner (cytoplasmic) and outer membrane
single layer of peptidoglycan in periplasmic space
special outer membrane proteins (porins) enable diffusion across outer membrane
outer surface of the outer membrane contains unique lipid component - lipopolysaccharide (LPS), which is extremely important in pathogenesis

(scratch off Bayer`s junction)
Acid fast structure - (Mycobacteria)

most similar to gram-positive bacteria

cell wall composed of fatty acids and waxes which contribute to virulence

hydrophobic components difficult to stain, but once stained, retain stain (resistant to acid decolorization)

mycolic acid, Wax D, cord factor, arabinogalactans, and sulfolipids (mycobacterial virulence factors)
Peptidoglycan = murein layer

1. unique to prokaryotes

a. antimicrobials:
β-lactams: penicillins and cephalosporins, vancomycin, bacitracin

b. enzyme lysozyme hydrolyses backbone

2. composition - murein backbone with unusual peptide chain

a. N-acetyl glucosamine - N-acetyl muramic acid
b. pentapeptide with L and D amino acids
3. synthesis
a. build blocks in cytoplasm
b. transport through cytoplasmic membrane
(bacitracin-sensitive)
c. polymerize backbone
d. cross-link peptides

4. the third amino acid - NH2 side chain (lysine [gram-positives] or diaminopimelic acid [gram-negatives]) peptide bond displaces terminal amino acid (D-alanine) of adjacent peptide chain, crosslinking chains and conferring rigidity

5. Penicillin-binding proteins (PBPs) - perform crosslinking, etc.

6. some gram-positive cells - pentaglycine bridge to form cross-links

7. muramyl dipeptide - highly inflammatory and chemotactic

8. recognized by TLR-2
Lipopolysaccharide (LPS) – Endotoxin

The most important part of gram-negative bacteria

a. lipid A
i. embedded in membrane = endotoxin activity
ii. unique C14 fatty acid - β-hydroxy myristic acid, phosphates, glucosamine

b. core oligosaccharide
i. highly conserved among different bacteria
ii. unique components - KDO and heptose

c. O antigen
i. may be present or not, depending on species

ii. repeating units of 3 to 5 sugars

iii. smooth with O antigen
rough without (ending at core)
LPS of bacteria without O antigen
sometimes called lipooligosaccharide (LOS)

iv. antigenic and highly variable among species and strains
Other optional gross structural components

1. Capsule (slime layer), K antigen - not impermeable
a. Both gram-positive and gram-negative bacteria can make capsules

b. polysaccharide
(exception: Bacillus anthracis (anthrax) poly-glutamate)

c. virulence - inhibit complement and phagocytosis

d. glycocalyx - extracellular polysaccharide; biofilms; technically not a capsule
2. Flagella - H antigen
a. propeller
b. motility and chemotaxis

3. Pili/fimbriae
a. hair-like; protein; 2 unrelated functions:
b. adherence
c. genetic exchange (not related to adherence fimbriae)

4. Fibrillar layer
a. protein coat on surface
b. virulence (e.g., M protein of Streptococcus pyogenes is anti-phagocytic, others involved in adherence to host cells)
5. Spores
a. certain gram-positives only - both aerobic and anaerobic
b. metabolically inactive
c. resistant to heat (boiling), desiccation
►need autoclave (121˚C, 15 min)
d. Contain dipicolinic acid
e. developmental stage in response to stress:


stress sporulation

vegetative (growing) cell dormant spore

germination
6. Plasmids - Non-chromosomal DNA

a. usually circular

b. can be transmissible between cells by genetic exchange (conjugation)

c. some encode virulence properties, antibiotic resistance
G. Cytoplasmic/Inner Membrane

1. similar to eukaryotic plasma membrane and mitochondrial membrane

2. little usefulness as target for antibiotics

3. carries out many functions
a. transport: facilitated diffusion, active transport, group translocation (phosphotransferase – carbos)
b. electron transport and oxidative phosphorylation
c. energy production
d. motility
e. replication
H. Nucleoid - Chromosome – DNA

1. Single, circular structure (haploid genome)
Vibrios have 2 different chromosomes

2. < eukaryotic chromosomes, ~ 3,500 genes

3. Not in nucleus - no nuclear membrane. Transcription in cytoplasm with translation

4. Supercoiling - DNA gyrase - DNA replication
Nalidixic acid and other quinolones inhibit gyrase and DNA replication
Metronidazole - binds to DNA after metabolism by anaerobes, inhibiting DNA replication
I. Ribosomes - similar but different from ours

1. 70S ribosomes composed of 50S and 30S subunits

2. co-transcription-translation

3. target of many useful antimicrobials:
aminoglycosides
tetracyclines
chloramphenicol
macrolides - erythromycin
II. Metabolism (review intermediary metabolism for other exams)

A. The "meaning of life" for bacteria is growth = replication - they don`t just sit around

B. Colony Forming Unit (CFU)

C. Replication = synthesizing a bacterial cell

D. Most metabolic pathways are similar if not identical to ours, therefore not targeted by antibiotics

E. See "Breathing Problem" BUGS case
E. Oxygen and bacterial physiology and growth

1. aerobes - grow well in the presence of oxygen; they tolerate oxygen and oxidative products of metabolism

a. strict or obligate aerobes require oxygen

b. facultative anaerobes - grow well in presence or absence of oxygen (aerobes)

2. anaerobes - grow best in the absence of oxygen
a. microaerophilic or aerotolerant - tolerate ↓oxygen
b. obligate anaerobes - cannot tolerate oxygen or oxidative products of metabolism

3. processing samples and ordering culture tests
F. Unique functions

1. acquisition of iron by siderophores - important for virulence, (no antibiotics yet)

2. folic acid metabolism (1 carbon donor: DNA synthesis, etc.)

a. humans get folic acid as a nutrient
bacteria must synthesize

b. sulfanilamide is a PABA analog that inhibits dihydropteroate synthetase

c. trimethoprim inhibits dihydrofolate reductase
G. Transcription

1. RNA polymerase - ααββ’δ
ααββ’ – core
δ – binds to promoters
2. inhibited by rifampin

3. regulation of protein synthesis is primarily at level of initiation of transcription involving regulatory DNA binding proteins to turn on/off genes in response to environmental conditions (remember the Lac operon)

4. polycistronic operons, several genes transcribed from same promoter and regulated by the same conditions
5. some genes regulated in response to stress - heat shock proteins involved in survival (also involved in autoimmune reactions)

6. Quorum sensing - regulation in bacterial communities such as biofilms
a. small inducer molecules are secreted
b. when concentration in environment reaches threshold (quorum has been attained), gene expression changes

Note: Review lactose operon and Lambda phage regulation for “other” exams.
H. Translation

1. co-transcription/translation in cytoplasm

2. ribosomes - smaller / antibiotics
Growth

A. Fast - as little as 10 min. generation time (Vibrio vulnificus) as long as 24 hr. (Mycobacterium tuberculosis)

B. Phases: lag, log (exponential), stationary, death

Calculating yield: Nt = N0 x 2g
g = number of generations

Simple rule of thumb:
3 gen. = 10X increase

Note: You might be asked to
perform some simple growth
calculations on exams.
C. Biofilms - communities on solid/liquid environments
1. change metabolism
a. glycocalyx holds the cells together
b. slow metabolism and growth.
c. resistant to antibiotics and host defenses
d. planktonic bacteria are free, individual – NOT in biofilm.

2. Contaminated devices (catheters, artificial valves, etc.)

3. Body - tooth plaque, heart valves

D. Temperature:
1. Mesophiles - grow best at our body temperature - 37˚C
2. Special growth temperatures:
Campylobacter - 42˚C, Listeria - 4˚C
D. Bacterial culture: provide all necessary things for growth

1. Fastidious organisms require many nutrients
2. Simple requirements can make everything from scratch

3. Some bacteria cannot be cultured in vitro
a. Chlamydia and Rickettsia - tissue culture like viruses
b. Treponema pallidum, Mycobacterium leprae not at all, require animal infection

4. Cannot predict virulence by growth (some slow or non-culturable bugs can still kill you!).
Sterilization and disinfection

A. Sterilization - no viable organisms at all

B. Disinfection - pathogens reduced < infectious levels

C. Methods
1. sterilization – autoclave (>121°C, 15 lb/sq in, 15 min), UV irradiation, gamma irradiation, filtration, phenolics
2. disinfection - antiseptics - detergents, ethanol, halogens (Cl), peroxide, betadyne
3. which method on what (e.g. a wound, a scalpel, i.v. fluid, bacterial waste) - need for sterility vs. ability to sterilize
4. spores are very resistant

5. WASH YOUR HANDS ! WASH YOUR HANDS !