Microbio Exam 1

three domains of life

archaea, bacteria, eukarya

how do you determine domain of life

compare the ribosomal RNA sequences

archaea

prokaryotic
closer to eukaryotes than bacteria
extremophiles
do NOT cause disease in humans

bacteria

prokaryotic
typically 0.2 - 20 um
may grow as single cells, filaments, communities

protozoa

eukaryotic
motile, single-celled organisms
may be free-living or parasitic (ex: malaria)

algae

eukaryotic
contain chloroplasts and conduct photosynthesis
base of the food web
*no pathogenic role in humans

fungi

eukaryotic
nonmotile
grow as single cells or as filaments
can cause disease

viruses

noncellular (chemical entities)
genetic material can cause disease
also used as biotechnology tools
not alive

eukarya

eukaryote

relative size of bacterial pathogens

range in size from 0.2 - 2 microns in diameter

cocci

spherical bacteria

bacilli

Rod shaped bacteria

spirochete

spiral shaped bacteria (usually motile)

coccobacillus

short rod shaped bacteria

vibrio

comma shaped bacteria

streptococcal species

occur in chains

Strep pneumoniae

occur in pairs; diplococci

Staphylococci

Form grape-like clusters

binary fission steps

1. cell replicates its DNA
2. cytoplasmic membrane elongates separating DNA molecules
3. cross-wall forms; membrane invaginates
4. cross-wall forms completely
5. daughter cells separate

endospores

- produced by gram + bacillus and clostridium species
- each vegetative cell transforms into ONE endospore
- each endospore germinates to form ONE vegetative cell
- constitute a defensive strategy against hostile or unfavorable conditions

endospore formation

1. DNA is replicated
2. DNA aligns along the cell's long axis
3. cytoplasmic membrane invaginates to form forespore
4. cytoplasmic membrane grows and engulfs foreshore within a second membrane. vegetative cell's DNA disintegrates
5. a cortex of Ca++ and dipicolinic acid is deposited between the membranes
6. spore coat forms around endospore
7. maturation of endospore; completion of spore coat and increase in resistance to heat and chemicals by unknown process
8. endospore released from original cell

\***THIS IS NOT MULTIPLICATION

endospore

-extremely resistant to drying, heat, radiation and disinfectants
-can remain viable for 10 - 1000 years
-serious concern to food industry & hospitals (c. Diff)
-potential biological weapons of mass destruction

types of stains

gram stain and acid-fast stain

gram stain

crystal violet, gram's iodine, decolorized (alcohol or acetone), safranin red

what is gram stain NOT used for?

mycoplasmas and achaea

why is gram staining not used for mycoplasmas?

lack cell walls/peptidoglycan
have sterols in cytoplasmic membrane

why is gram staining not used for archaea?

wall-less or walls of pseudomurein

what type of staining is used for mycobacteria?

acid-fast stain

acid-fast bacteria

resist decolorization with acid-alcohol and stain red (carbon fuchsin)
other bacteria will decolorize and stain blue (methylene blue)

acid fast bacteria examples

mycobacterium tuberculosis
mycobacterium leprae

terminal endospores

botulism and tetanus

botulism

Clostridium botulinum; caused by neurotoxin; causes flaccid paralysis (muscles are limp)

tetanus

Clostridium tetani; caused by neurotoxin; causes spastic paralysis (muscles flexed/all muscles firing)

external structures of prokaryotes

flagella
fimbria
pili
capsule

flagella

rotate to produce movement, long tails, made up of flagellin protein

flagella proteins

H antigens (can be recognized by antibodies)

fimbriae and pili

nonmotile extensions, short hair-like projections, used to adhere/attach, can join two bacterial cells together and mediate the transfer of DNA via conjugation (conjugation pili or sex pili); also virulence determinants

slime (biofilm) vs capsule

slime
-loosely attached to cell surface

capsule
-firmly attached to cell surface

capsule

polysaccharide, antiphagocytic, adherence factor, virulence factor, antigen, can be used as vaccine component

prokaryotic cell envelope

provides structure and shape and protects cell from osmotic forces
absent in animal cells
some components are targets of important antibiotics

bacterial cell envelopes

-most have peptidoglycan layer(s)
--polysaccharide polymer of NAG and NAM
-NOT found in mycoplasma species
-Gram +/-

comparison of bacterial cell envelope structures

Gram +
-simple
-thick peptidoglycan
-teichoic acid
-no outer membrane
-no LPS

Gram -
-complex
-thin peptidoglycan
-no wall teichoic acid
-outer membrane
-LPS present

structure/function of peptidoglycan

Structure:
- mesh-like coat around the cell (except mycoplasmas)
- polysaccharide polymer of alternating NAG and NAM
- cross-linking peptides

function:
- shape/protection
- antibiotic target

outer membrane

-unique for gram - bacteria
-protects from phagocytes and some antibiotics
-has LPS
-porins form channels through membrane (nutrients and antibiotics)
-outer membrane proteins

Lipopolysaccharide (LPS)

-made up of lipid A (responsible for toxicity), core oligosaccharide, O polysaccharide antigen

Lipid A portion of LPS

released from dead cells when cell wall disintegrates; causes inflammation, fever, vasodilation, shock and blood clotting; released when antimicrobial drugs kill bacteria

periplasmic space

Between outer membrane and cell membrane
Contains peptidoglycan and periplasm
Contains water, nutrients, digestive enzymes and transport proteins

prokaryotic cytoplasmic membrane

-phospholipid bilayer (charged, hydrophobic inside); function similar to eukaryotic membranes; electron transport chain (in some bacteria); energy (ATP) production (in some bacteria); transport proteins (entry of nutrients, export of waste metabolites)

cytoplasm of prokayotes

-substance enclosed by cytoplasmic membrane
-ribosomes - sites of protein synthesis
-cytoskeleton - plays role in cell shape
-antibiotic targets: nucleoid, mRNA (made by RNA polymerase), 30S & 50S ribosomal subunits
-plasmids: circular, extrachromosomal DNA; confer antibiotic resistance

mechanisms of antimicrobial action

-Key is selective toxicity
-Antibacterial drugs constitute largest number and diversity of antimicrobial agents
-Fewer drugs to treat eukaryotic infections
-Even fewer antiviral drugs

ideal antimicrobial agent

Readily available
Inexpensive
Chemically stable
Easily administered
Nontoxic and nonallergenic
Selectively toxic against wide range of pathogens

selective inhibition/toxicity

Due to the differences in structure and metabolic pathways
Harm microorganisms, but not the host

Five major sites of antibiotics

-cell wall (peptidoglycan)
-cell membrane
-ribosomes
-metabolic pathways
-DNA/nucleic acid metabolism

broad spectrum antibiotics

-effective against many pathogens
ex: tetracycline, erythromycin, sulfonamides

narrow spectrum antibiotics

-effective against very few pathogens
ex: penicillin

bactericidal

-kill bacteria
-used when the host defense mechanisms are impaired
-required in serious infections: endocarditis, meningitis, kidney infection, etc.

bacteriostatic

-inhibit bacteria (inhibit growth)
-used when the host defense mechanism are intact
-used in many infectious diseases

inhibition of cell wall synthesis

-inhibit peptidoglycan synthesis by preventing cross linkage of NAM subunits
-effective only when bacteria cells are growing
-no effect on plant or animal cells (no peptidoglycan)
-beta lactam antibiotics (penicillin family, cephalosporins)

beta lactam antibiotics

-penicillins and cephalosporins
functional group: beta lactam rings
*inhibit peptidoglycan synthesis by preventing cross-linkage of NAM by binding to transcriptase enzymes

penicillins

effective primarily against gram + organisms

ampicillin and amoxicillin

effective against both gram +/- organisms
-used in UTI, salmonellosis, listeria monocytogenes, and group A streptococcal infections

adverse effects of penicillins

allergic reactions and anaphylactic shock

vancomycin

binds to ala-ala bridges that are used to crosslink NAM subunits in peptidoglycan of gram + bacteria
-binds to substrates of cross linking enzymes
*inhibition of cell wall synthesis

disruption of cytoplasmic membranes

some drugs become incorporated into cytoplasmic membrane and damage its integrity

amphotericin B (polyene)

antifungal; attaches to ergosterol found in fungal membranes
-humans somewhat susceptible because cholesterol is similar to ergosterol (nephrotoxicity)
-most bacteria lack sterols (not susceptible)

inhibition of bacterial nucleic acid synthesis

fluoroquinolone inhibit bacterial DNA gyrase (enzyme that makes DNA supercoiled) with little effect on eukaryotes or viruses
ex: ciprofloxacin, norfloxacin, levofloxacin

other drugs bind to and inhibit action of bacterial RNA polymerase during transcription (ex: rifampin, rifamycin, rifampicin); no mRNA, tRNA, rRNA --\> no ribosomes or protein synthesis \= no growth

fluoroquinolones

inhibit bacterial DNA gyrase (essential for function); kill bacteria because cannot package DNA into cell
ex: ciprofloxacin, norfloxacin, levofloxacin

rifampin, rifamycin, rifampicin

inhibit action of bacterial RNA polymerase during transcription
(no mRNA, tRNA, or rRNA \= no ribosomes or protein synthesis \= no growth)
-works because our RNA polymerases have very different structure to those of bacteria

inhibition of protein synthesis

chloramphenicol, lincomycin, erythromycin, tetracycline, aminoglycosides

ribosomes

essential for translation of mRNA into proteins

anti-ribosomal antibiotics

impair ribosomes by binding to either 50S or 30S ribosomal subunits
-no translation \= no protein synthesis \= no growth

chloramphenicol

-binds to large subunit
-broad spectrum
-crosses the blood-brain barrier
-staphylococcus aureus

adverse effects of chloramphenicol

bone marrow suppression, aplastic anemia, grey baby syndrome, leukemia

lincomycin

aka clindamycin
-binds to large subunits
-used in anaerobic and severe aerobic infections (e.g. streptococcal toxic shock syndrome)

adverse effects of lincomycin

pseudomembranous colitis; caused by Clostridium difficile

erythromycin (macrolide antibiotic)

(azithromycin, clarithromycin)
-effective against gram +/-
mycoplasma pneumoniae, legionella pneumophila

adverse effects of erythromycin

GI disturbances: nausea, vomiting,
abdominal pain and diarrhea

tetracycline (doxycycline, minocycline)

used to treat: acne vulgarisms, non-gonococcal urethritis (NGU), Rocky Mountain spotted fever, Lyme disease
good intracellular penetration

adverse effects of tetracycline

-stains developing teeth
-inactivated by Ca++, do NOT take with milk or yogurt
-photosensitivity
-drug induced lupus and hepatitis

aminoglycosides

used to treat severe gram - infections and tuberculosis
(amikacin, gentamicin, kanamycin, tobramycin, neomycin, streptomycin)

adverse effects of aminoglycosides

nephrotoxicity (kidney failure) and ototoxicity (hearing loss)

inhibition of metabolic pathways

-When differences exist between metabolic processes of host and pathogen
-Sulfonamides and trimethoprim inhibit enzymes involved in synthesis of bacterial folic acid (cannot make building blocks for DNA/RNA)
-Humans obtain folic acid from diet thus the metabolism is unaffected

trimethoprim

blocks dihydrofolate reductase

trimethoprim/sulfamethoxazole

(bacterium, Cotrim, septra)
-used to treat otitis media, UTIs
-broad spectrum agains fungal agents
-prophylaxis and treatment of pneumocystis jiroveci pneumonia in AIDS patients

adverse effects of trimethoprim/sulfamethoxazole

rash, Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN)

mechanisms of resistance

Genetic Mechanisms
Non-Genetic Mechanisms

genetic mechanisms

bacteria mutated so antibody cannot bind to target or acquired new genes that are antibody resistant from conjugation
-chromosome-mediated
--due to spontaneous mutation in target molecule & in the drug uptake (mutation in porin)
-plasmid mediated
--common in gram - rods
--transferred via conjugation
--multidrug resistance

non genetic mechanisms

intrinsic features of organisms/how they effect host
-inaccessibility to drugs (e.g. abscess, TB lesion)
-biofilms (bacteria are less accessible to antibiotic)
-stationary phase (insusceptible to inhibitors of cell wall synthesis)
*no growth/movement \= antibiotics are ineffective/do not work

end result of genetically conferred resistance

-production of drug inactivating enzymes
-modification of target structures
-alteration of membrane permeability

resistance to beta lactams

gram +
-B lactamase (penicillinase)
**bacteria can destroy; B lactam so no longer active
-alternation of the transpeptidase enzyme

gram -
-B lactamase (penicillinase)
-alteration of the transpeptidase enzyme
-alteration of porins (stop B lactase from getting to their target)

B-lactamase

Breaks down penicillin, allows bacteria to survive treatment with B-lactam antimicrobial drugs

B-lactamases and clavulanic acid

clavulanic acid blocks the action of b-lactamase, but is not itself an antibiotic; allows the amoxicillin to remain active

inhibition of peptidoglycan cross linking by beta lactase and vancomycin - mechanisms of resistance

1. transpeptidase enzyme binds to Ala-Ala for crosslinking
2. B-lactam antibiotic binds to transpeptidase inhibiting crosslinking
3. vancomycin binds to Ala-Ala preventing binding of enzyme
4. B-lactamase cleaves B-lactam antibiotic
5. changing terminal Ala to lactate (gene change) prevents vancomycin binding

antibiotic susceptibility testing

Dilution Method and Disc Diffusion Method

dilution method

prepare two fold of antibiotic dilutions
add 1/2 a million bacterial cells per tube
incubate over night
check for turbidity
establish the minimum inhibitory concentration (MIC)

minimum inhibitory concentration (MIC)

the lowest concentration of the drug that prevents the bacterial growth (no turbidity)

disk diffusion method

seed agar plates with bacteria in question
place antibiotic-discs over the seeded plate
incubate overnight
measure the inhibition zones
relate the results to the zones given in the interpretive chart
there is an inverse relationship between the MICs and zone diameters

therapeutic index

how good antibiotic is
ratio of max safely achievable level/MIC
(penicillin has high therapeutic index; aminoglycosides have lower therapeutic index)

microbe

microscopic organism

major trait that distinguishes different types of microbes

the possession or absence of a membrane-enclosed nucleus