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Flashcards in LOs: 1-3 Deck (31):
1

1 Classifying & using Nomenclature for Micro-Organisms:

Genus & species apply to...

Organism

Genus

Species

Disease

Spp.

Bacteria, fungi, & parasites

Italics

Capitalized, can be abbreviated

Not capitalized

Normal print

Abbreviation for species, not italicized

2

1 Colonizers

Opportunists

Pathogens

More colonizers than human cells
Most colonize gut & skin
Prevent more pathogenic bacteria from hurting us
Ferment carbohydrates
Produce Vitamin K

Don't normally cause harm
Can cause disease (ex. catheter --> bacteremia)

Always harmful

3

1 How micro-organisms can be identified from body sites (7)

Appearance (naked eye, microscope, electron microscopy)

Staining of Cell Wall (gram, AFB, fungal)

Culture (bacteria & fungi on media, viruses on cell culture)

Antigen Recognition (Direct Fluorescent Antibody, serology/IgM/IgG)

Biochemical Properties (coagulase activity, lactose fermentation)

Gene Detection (PCR, BLAST)

Protein Detection (MALDI-TOF MS)

4

1 How to establish that a micro-organism causes a given infectious syndrome (Koch's Postulates)

1. The microorganism must be found in abundance in all organisms suffering from the disease, but not in healthy organisms.

2. The microorganism must be isolated from a diseased organism and grown in pure culture.

3. The cultured microorganism should cause disease when introduced into a healthy organism.

4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being the same as the original causative agent.

5

2 Koch's Postulates Purpose & Steps

Purpose: identify the organism causing an infectious disease

a. The organism should always be found in the diseased animal but not in healthy animals.

b. The organism can be isolated and grown in pure culture away from the animal.

c. The organism isolated in pure culture causes the same disease when re- inoculated into susceptible animals.

d. The same organism can be re-isolated from those diseased animals.

6

2 Molecular Koch's Postulates Purpose & Steps

Purpose: to identify "virulence factors" (whether a factor produced by a pathogen is important for causing disease)

a. The phenotype or property encoded by the virulence gene should be associated with pathogenic strains.

b. Specific inactivation of the virulence gene encoding the suspected virulence trait should lead to a measurable loss of virulence.

c. Addition of a cloned copy of the wild-type gene to the mutant should restore virulence.

7

2 How can you specifically inactivate a microbial gene in order to perform Molecular
Koch's Postulates

Transposon Mutagenesis

- Bacterial cells generally have 1 chromosome

- Can introduce a plasmid (extrachromosomal pieces of DNA, usually circular) carrying a transposon (jumping genes that can move from one place to another) into a bacterial cell

- That transposon will hop off the plasmid onto the bacterial chromosome, inactivating the virulence gene so the bacteria no longer makes the virulence factor

8

2 Infection vs. Intoxication

Infection (most common)
- Have to have a live, viable pathogen enter the body to cause disease

Intoxication (less common)
- Caused by acquiring a toxin (usually an ingestion)
- Don’t have to have the bacteria that made the toxin alive or in the body
- Ex. Food-borne botulism

9

2 Steps in the infectious cycle (6)

a. Pathogen entry into the body

b. Pathogen adherence and colonization

c. Pathogen invasion through the epithelium (sometimes)

d. Pathogen evasion of host defenses

e. Cell/tissue damage (toxins or immunopathology)

f. Dissemination of pathogen so it can infect a new host

10

2 Illustration of molecular Koch's postulates:

Hypothesis: Cholera toxin is a virulence factor for the bacterium, Vibrio cholerae
(the cause of cholera).

a. Is cholera toxin produced by most pathogenic V. cholerae isolates? Yes

b. Does inactivation of cholera toxin genes decrease the virulence of V. cholerae in animal models? Yes

c. Does addition of wild-type cholera toxin genes to that cholera toxin mutant restore virulence? Yes

d. Conclusion: cholera toxin is a virulence gene for V. cholerae.

11

2 Advantages & Disadvantages for Intracellular Growth of a Pathogen

a. Advantages: nutrients are supplied, the pathogen is protected from immune system (at least initially) and some antibiotics.

b. Disadvantages: mammalian cells are pretty good at killing invaders, so persisting/growing inside a host cell requires a good strategy, often requiring expenditure of considerable energy and requiring factors encoded by a lot of the pathogen's DNA.

12

2 Intracellular Obligate Pathogens:

Viruses

Bacteria

Fungi

Parasites

All

Mycobacterium leprae
Chlamydiae spp.
Rickettsia spp.

None

None

13

2 Intracellular Facultative Pathogens:

Viruses

Bacteria

Fungi

Parasites

None

Salmonella spp.
Shigella spp.
Listeria monocytogenes
Legionella spp.
Mycobacterium tuberculosis

Most

Most

14

2 Extracellular Pathogens:

Viruses

Bacteria

Fungi

Parasites

None

Most gram+ bacteria (except Listeria monocytogenes)
Vibrio cholerae
Terponema pallidum

Cryptococcus spp.

Giardia spp.

15

3 Viruses:

Type of pathogens

Size (small/large)

Nucleic Acid(s)

Independent energy production & protein synthesis (y/n)

Selectivity for particular types / species of host cells (y/n)

Sensitive to antibacterial / antifungal antibiotics (y/n)

Cells (y/n)

Ribosomes (y/n)

Mitochondria (y/n)

Cell Wall (y/n)

Motility (y/n)

Binary Fission (y/n)

Obligate intracellular (require host cell for replication)

Smallest

DNA or RNA (not both)

No

Yes

No

No

No

No

No

No

No

16

3 Bacteria:

DNA enclosed by a nuclear membrane (have a nucleus) (y/n)

Chromosome Number

Membrane-bound organelles (e.g., mitochondria) (y/n)

Ribosomes

Peptidoglycan in cell wall (y/n)

Sterols in plasma membrane (y/n)

Nucleic Acids

Independent energy production (y/n)

Independent protein synthesis (y/n)

Size relative to eukaryotes

No

Usually 1

No

70S

Yes (exceptions: Mycoplasma & Chlmaydiae)

No (exceptions: Mycoplasma, Helicobacter pylori, Borrelia burgdorferi)

RNA & DNA

Yes (exceptions: Rickettsiae & Chlamydiae)

Yes

Smaller (need oil immersion lens to visualize)

17

3 Eukaryotes:

DNA enclosed by a nuclear membrane (have a nucleus) (y/n)

Chromosome Number

Membrane-bound organelles (e.g., mitochondria) (y/n)

Ribosomes

Peptidoglycan in cell wall (y/n)

Sterols in plasma membrane (y/n)

Yes

>1

Yes

80S

No

Yes

18

3 Bacterial Cell:
Cytoplasm

i) DNA, ribosomes, metabolic enzymes: basic life processes

ii) site of some antibiotic action (e.g., protein synthesis inhibitors, antibiotics affecting DNA replication or RNA transcription)

iii) plasmids: antibiotic resistance and virulence

19

3 Bacterial Cell:
Cell Envelope

i) plasma membrane: permeability barrier and transport

ii) cell wall (target of some antibiotic action): resists osmotic lysis

iii) outer membrane (if present): permeability barrier (but contains pumps and pores)

20

3 Bacterial Cell:
External Structures (if present)

i) common pili: attachment to tissue

ii) capsule: antiphagocytic, can result in biofilm formation

iii) sex pilus: conjugation

iv) flagella: motility

21

3 Bacterial Cell Wall Synthesis & Antimicrobial Interference

- Starts in cytoplasm

- Attach 2 sugars to UDP carrier molecules: GlcNAc or MurNAc

- Also attach 5 amino acids onto MurNAc

- Monomers come together to form a dimer

- Dimer is attached to a lipid carrier which moves the dimer across the membrane (IMPORTANT)

- Lipid carrier is very expensive for the bacteria to make, so it’s recycled

- Dimer is inserted into growing chain outside of membrane

- Individual layers of peptidoglycan aren’t strong

- Cell wall gets strength from cross-linking different layers together (FINAL STEP)

- Two chains growing

- Peptidoglycan is a repeating disaccharide of GlcNAc & MurNAc

- 5 amino acids (sequence varies, almost always D-ala D-ala)

- Transpeptidation: form a peptide bond b/n the 2 layers (always occurs)

- Transpeptidases (Penicillin Binding Proteins, PBPs): enzymes for transpeptidation

- Penicillin blocks transpeptidation b/c of their structural similarities to D-ala D-ala

22

3 Gram+ vs. Gram- Cell Envelopes

Both
- Plasma membrane
- Capsule (sometimes)

Gram+
- Very thick wall (difficult to de-stain)
- Acids for attachment to host tissues (Lipoteichoic & Teichoic)

Gram-
- Very thin cell wall (easy to de-stain)
- Outer membrane (antibiotic resistance)
- Periplasmic space (b/n inner & outer membranes)
- Lipopolysaccharide (LPS): enteric
--> Inside: lipid A (endotoxic)
--> Outside: core polysacchardie (w/ KDO assay) & O antigen (for bile & complement resistance)
- Lipooligosaccharide (LOS): non-enteric
--> Lacks O antigen but retains core lipid A regions of LPS

23

3 Mycobacteria:

Stain

Cell Envelope

Medically Important (2)

Acid-fast stain positive

Waxy layer for resistance to desiccation & phagocytosis

- M. tuberculosis
- M. leprae

24

3 Spirochetes:

Visualized by...

Medically Important (2)

Dark-field microscopy

- Borrelia burgdoferi (Lyme Diseaes)
- Treponema pallidum (Syphilis)

25

3 Rickettsiae spp.:

Type of pathogen

Transmission

Energy

Structure

Medically Important (2)

Obligate intracellular bacteria

Vector-borne (fleas, ticks, lice, etc.)

Generate some energy but need to get some from host cell

Have gram- envelope but too small to gram-stain

- R. rickettsii (rocky mountain spotted fever)
- R. typhi (endemic typhi)

26

3 Chlamydiae & Chlamydophila spp.:

Type of pathogen

Life cycle

Energy

Structure

Medically Important (3)

Obligate intracellular bacteria

Reticulate Bodies: found inside host cells
Elementary Bodies: transmissible, infectious form

Energy parasites

2 membranes like gram-, no peptidoglycan, too small to gram-stain

- Chlamydiae trachomatis (blindness, nongonococcal urethritis, pneumoniae)
- Chlamydophila psittaci (psittacosis, form of pneumonia)
- Chlamydophila pneumoniae (pneumonia)

27

3 Mycoplasma spp.:

Structure

Antibiotics

Medically Important (1)

Small, lack a cell wall, have sterols in plasma membranes, can be grown on artificial media

Absence of cell wall affects antibiotic sensitivity (penicillins won't work against these organisms)

M. pneumoniae

28

3 Chlamydiae:

Can grow outside host cell (y/n)

Has independent protein synthesis (y/n)

Generates metabolic energy (y/n)

Has peptidoglycan-containing cell wall (y/n)

Susceptible to antibiotics (y/n)

Reproduced by binary fission (y/n)

Nucleic acids

No

Yes

No

No

Yes

Yes

DNA & RNA

29

3 Typical Bacteria:

Can grow outside host cell (y/n)

Has independent protein synthesis (y/n)

Generates metabolic energy (y/n)

Has peptidoglycan-containing cell wall (y/n)

Susceptible to antibiotics (y/n)

Reproduced by binary fission (y/n)

Nucleic acids

Yes

Yes

Yes

Yes

Yes

Yes

DNA & RNA

30

3 Rickettsiae:

Can grow outside host cell (y/n)

Has independent protein synthesis (y/n)

Generates metabolic energy (y/n)

Has peptidoglycan-containing cell wall (y/n)

Susceptible to antibiotics (y/n)

Reproduced by binary fission (y/n)

Nucleic acids

No

Yes

Sometimes

Yes

Yes

Yes

DNA & RNA

31

3 Mycoplasma:

Can grow outside host cell (y/n)

Has independent protein synthesis (y/n)

Generates metabolic energy (y/n)

Has peptidoglycan-containing cell wall (y/n)

Susceptible to antibiotics (y/n)

Reproduced by binary fission (y/n)

Nucleic acids

Yes

Yes

Yes

No

Yes

Yes

DNA & RNA