microbio lab final

anabolism

synthesis of organic compounds

catabolism

breaking down organic compounds

metabolism

consists of anabolic and catabolic reactions

glycolysis

• Found in nearly all microbes

• Occurs in the cytoplasmic matrix of both prokaryotes and eukaryotes

• Can function with or without O2, but does not require oxygen

• Divided into 2 phases

glycolysis investment steps

• 2 phosphorylation steps

• requires ATP

glycolysis payoff steps

• 1 oxidation-reduction step

  • requires NAD+

• 2 phosphorylation steps generate ATP

• 4 ATPs produced

• 2 NADH produced

• 2 Pyruvate produced

NET 2 ATP per glucose

what pathways could happen after glycolysis?

aerobic respiration, anaerobic respiration, fermentation

respiration ATP production

• Only 4 ATP molecules synthesized directly from oxidation of glucose to CO2.

• Most ATP made when NADH and FADH2 (formed as glucose degraded) are oxidized in electron transport system (ETS) = 32-36 ATP

Electron Transport System (ETS)

• A series of electron carriers that are, in turn, oxidized and reduced as electrons are passed down the chain

  • from NADH and FADH2

  • to a terminal electron acceptor

  • Electrons flow from carriers with more negative redox potential to carriers with more positive redox potential

Energy released can be used to produce ATP

oxidase test

The presence of different electron carriers is useful for bacterial identification → this test detects the presence of cytochrome c (E. coli does not have cytochrome c)

aerobic respiration

The final electron acceptor in the electron transport chain is molecular oxygen (O2)

anaerobic respiration

The final electron acceptor in the electron transport chain is not O2, but a nitrate, sulfate, or carbonate ion

• Yields less energy than aerobic respiration

• Where do anaerobic microbes live? Swamps, soil sediments, intestinal tracts (99% of our intestinal microbiota is strictly anaerobic)

fermentation

• Releases energy from oxidation of organic molecules

• Does not require oxygen, but can occur in its presence

• Uses an organic molecule as the final electron acceptor

oxidation fermentation glucose test

distinguish oxidation of glucose (respiration) from fermentation

procedure:

• 2 test tubes (green) containing glucose used

• one is covered w/ mineral oil, the other exposed to air

If glucose is broken down, medium will turn yellow due to the change in color of the pH indicator (bromthymol blue)

phenol red fermentation test

test for fermentation of diff sugars

• Each tube contains protein, a single carbohydrate, a pH indicator, and an inverted Durham tube

• used to distinguish Salmonella from E. coli

• acid production turns red broth → yellow

mixed acid fermentation

• Several bacteria and fungi produce a mixture of fermentation products (Acetone, Butanol)

• Specific tests can tell us if microbes produce mixed acids through fermentation

• MR-VP test

pasteur effect

• Oxygen can inhibit fermentation

• Facultative microbes, such as S. cerevisiae, have both fermentation and aerobic respiration

• With O2: Consumes less glucose (more energy for same amount of glucose)

• Without O2: Consumes more glucose (less energy for same amount of glucose)

horizontal gene transfer events

transformation, conjugation, transduction, transposition

Help bacteria gain new virulence factors:

• the ability to make toxins/capsules

• acquire antimicrobial resistance

Which type of horizontal transfer requires intimate cell-cell contact?

A. transposition

B. transduction

C. conjugation

D. transformation

conjugation

transformation

uptake and incorporation of DNA from the environment

transduction

transfer of DNA from one bacterium to another by a bacteriophage

Conjugation

temporary joining of two bacterial cells to copy DNA from donor to recipient cell

plasmid

circular DNA separate from chromosome

Sex pili are to conjugation as _______ is/are to transduction

A. the host chromosome

B. plasmids

C. bacteriophages

D. genes

bacteriophages

transposon

a segment of DNA capable of cutting and pasting itself into a new position in the genome (or another genome)

• can carry antibiotic resistance genes

• Sometimes can replicate themselves and paste copy into new location

selective toxicity

• Using drugs to kill a pathogen without harming the host

• Targets unique pathogen structures/features

  • Bacterial ribosomes, cell wall, etc

• More difficult to target eukaryotic pathogens and viruses

minimum inhibitory concentration (MIC)

What good is it to be “just” inhibitory?

• Slow down microbe so immune system can

eliminate pathogen

minimum bactericidal concentration (MBC)

Why might we not want to kill the bacteria?

• Limit endotoxin release

Therapeutic index

quantifies side effects

• Low/few side effects = high therapeutic index

broth dilution test

Determines Minimum Inhibitory Concentration

broad spectrum antibiotic

effective against wide range of pathogens

narrow spectrum antibiotic

effective against only one or two types of pathogens

β-lactam antibiotics

Binds transpeptidase enzymes (Penicillin-binding protein) preventing cross-linking of peptide chains in peptidoglycan

• ex: penicillins, cephalosporins

β-lactam Antibiotic Resistance

have evolved resistance thru HGT: Acquisition of a gene for an enzyme called beta-lactamase, which breaks beta-lactam ring

Random mutation followed by natural selection in a gene for a penicillin-binding protein

• Prevents penicillin from binding to PBP and halting peptidoglycan synthesis

Glycopeptide

peptide chain with disaccharide attached

• Binds to and physically blocks peptidoglycan subunits from cross linking enzyme

vancomycin

• highly active against Gram + bacteria

• Cannot penetrate outer membrane of Gram - bacteria

• binds to terminal D-alanine-D-alanine

  • prevents these residues from being accessible to the active site of transpeptidases

Glycopeptide Antibiotic Resistance

mutating or acquiring a cluster of genes (vanH-vanA-vanX) to synthesize D-alanine-D-lactate instead of D-ala-D-ala

targeting the plasma membrane

Low selective toxicity; used sparingly

• Examples: daptomycin, gramicidin, and polymyxins

Daptomycin/gramicidin

Inserts into bacterial cell membranes and induces rapid depolarization of electrostatic potential

• Inhibits processes that exploit this potential (e.g., ATP synthesis)

polymyxin

dissolves membranes like a detergent

Sulfonamides and Trimethoprim

Inhibit folic acid biosynthesis (required for DNA and RNA synthesis)

• Structural analogs of growth factors

• In clinical use since 1930’s so resistance to single drug is common

• Combinatorial treatment now used

Quinolones

Inhibitors of DNA Replication

• Completely synthetic compounds

• Interact with DNA gyrase and prevent supercoiling

  • Cause breaks in DNA during DNA replication and separation of daughter chromosomes

rifampin/rifampicin

Inhibitors of Transcription

• Information in DNA is copied into mRNA so this antibiotic prevents this

• Binds to and inhibits bacterial RNA Polymerase activity

• Used to treat mycobacterial infections

• Can diffuse through membranes

ribosomes

Information in mRNA is used to make protein

• Ribosomes found in animal cells (80S) are structurally distinct from those found in bacterial cells (70S)

• protein biosynthesis a good selective target for antibacterial drugs

aminoglycosides

Inhibitors of Protein Synthesis at 30S

• ex: Streptomycin, Kanamycin, Neomycin

• Bind to 30S subunit

  • Inhibits proofreading and causes misreading of mRNA = resulting in short defective proteins

• These proteins insert into cytoplasmic membrane - bactericidal

• Toxic side effects – used for serious Gram- negative infection

tetracyclines

• Bind to 30S subunit

• Block binding of tRNAs

• Stops protein synthesis

  • Bacteriostatic

macrolide

ex: Macrolides, chloramphenicol, lincosamides

Bind to 50S subunit

• Prevent peptide bond formation

  • Stops protein synthesis

how bacteria are drug resistance

• spontaneous mutations

• HGT (especially of plasmids)

• Bacteria naturally produce antibiotics to help them compete for space and resources

Prevention of Cellular Uptake or Efflux

• Can decrease permeability or transport drug back out of cell (efflux pump)

Drug Modification or Inactivation

Enzymes may alter drug structure

• Cellular Drug target may be modified

  • ABX longer “fits”

• Acetylation

• phosphorylation

• Drug may be degraded

  • ex: b-lactamase enzyme

superinfection

a secondary infection in a patient having a preexisting infection

• develops when the antibacterial intended for the preexisting infection kills the protective microbiota

• allowing another pathogen resistant to the antibacterial to proliferate and cause a secondary infection

phage therapy

using bacteriophages to target specific bacterial species for destruction

capsules

coat bacterial cell walls and can prevent phagocytes from binding (hide)

cell-surface proteins

components of the cell wall that prevent detection (disguise)

• Alter their antigens to avoid antibody binding

quorum sensing

used to communicate with other pathogens about population size (hide then attack)

• Avoid expressing virulence factors until population reaches size immune system can’t readily clear

Facultative intracellular pathogens

can invade host cells but can also survive outside the host cell

Obligate intracellular pathogens

invade and reproduce inside of a host cell only

• Can’t just grow on media; needs host tissue

immunopathy

Abnormal immune response that causes a disease

• May be triggered by an infection

Direct damage of host tissues

cellular lysis as a consequence of pathogen reproduction

Indirect damage of host tissues

Release of enzymes or toxins that damage tissues

Lipopolysaccharides (LPS)

• endotoxin

• found on outer membrane of Gram-negative cells

• Triggers release of endogenous pyrogens = fever

• Can cause development of blood clots, organ failure, shock

exotoxins

Toxic proteins secreted into environment

• Each toxin has a unique structure

• Toxin production correlates with pathogen virulence

toxemia

• toxin

• buildup of toxins in the blood or lymph nodes

• disseminated throughout the body by the blood or lymph

superantigens

cell surface antigens that trigger systemic responses

Membrane-Disrupting exotoxins

Form pores in membrane or enzymatically damage membrane components

• Ex: hemolysin

Cellular-acting Exotoxins

Act within host cells; usually composed of at least two pieces, A subunit and B subunit

• Grouped by target tissue and physiological effect

• Ex: neurotoxins, enterotoxins

anthrax toxin

PA (B subunit) creates hole in membrane and transfers in edema factor (EF) and lethal factor (LF)

• EF and LF affect cell signaling

edema factor (EF)

causes edema (water to collect outside cells)

lethal factor (LF)

cleaves regulatory proteins resulting in failure to activate immune system and cell death

Pathogenicity

the harm-causing potential of a pathogen

depends on:

• Host genetics and physiology

• Pathogen virulence factors

Different strains produce (or not):

• Different defense mechanism (i.e. capsules)

• Different toxins

• Different tissue damaging enzymes

virulence

describes the level of harm caused by a pathogen following infection

• determined by pathogen virulence factors

virulence factors

• invasion: Promote attachment and colonization of host tissues

• invasiveness: Facilitate pathogen spread from the initial site of infection

• toxins: Have a toxic effect on cells/tissues/host physiology

primary pathogens

likely to cause disease after infection in a healthy host.

• Rapidly reproduce/increase in number

• Moderate to high virulence

Opportunistic pathogens

are less likely to cause disease in a healthy host.

• Low virulence

• Req. compromised barriers, immune system, or normal microbiota

Molecular Koch’s Postulates

Identify the gene that causes organism to be pathogenic

1. Symptoms should only be associated with pathogenic strains

2. Inactivating gene reduces ability to cause disease

3. Adding gene back restores ability to cause disease

Agglutination

antibodies clump together cells or particle

• Quick indicator of presence of antibodies against pathogen or presence of antigen itself