Midterm I: Basic Micro (Ben) Flashcards
List the facultative structures of bacterial cells.
(6)
- Flagella
- Fimbriae / Pili
- Capsule
- Spores
- Granules
FFPCSG
Describe the structure + function of flagella.
(What are they composed of? How many per cell? How are they driven? What do they help bacteria do? etc)
- long, whiplike appendages of membrane-anchored, helically-coiled flagellin protein subunits (15-20 nm diameter)
- can have one or several located polarly or all over
- driven by an ATP-dependent motor protein
- help bacteria perform chemotaxis
What is the medical significance of flagella?
(3 main points)
- can be a virulence factor leading to pathogenicity (as in helping bacteria travel up urinary tract in UTIs)
- contain antigens (ex: H-antigen) that are important in diagnosistic identification of strains
- flagellin protein is a ligand for TLR-5 and thus initiates innate immune responses
Describe the structure of fimbriae/pili.
(What are they made of? How are they different from flagella? How many per cell? On what kinds/genus of bacteria? What do they help bacteria do?)
- small, hairlike structures of (usually not coiled) pilin subunits,
- smaller diameter (3-8 nm) and shorter (1-20 microns) than flagella + are not motile!
- usually many (several hundred) per cell arranged “peritrichously” (uniformly)
- mostly on Gram-negative bacteria, some on Gram+
- ex: Enterobacteriaceae and Neisseria
What is the function + medical importance of fimbriae/pili?
And what is the distinction btwn fimbriae and pili?
- mediate attachment to host cells via adhesins + are thus a virulence factor
- fimbriae tips may also contain mannose-binding lectins
-
“pili” often refers only to sexual fimbriae used for transfer of genetic material between bacteria
- sex pili are encoded by an “F plasmid” - a plasmid carrying fertility-related genes
-
“pili” often refers only to sexual fimbriae used for transfer of genetic material between bacteria
What are bacterial capsules and what are most capsules made of?
How do capsule + similar structures differ in structural integrity?
- a polymeric layer located outside the cell wall, usually polysaccharide, but sometimes polyamine
- ex: Anthrax bacillus = D-glutamic acid polymer
- “true capsules” = discrete polysaccharide layer, slime layer/biofilm is a less discrete, more amorphous layer
- both can be considered a glycocalyx
- are not vital to bacterial growth, may not be present in cultured microbes
What are 4 reasons bacterial capsules can be medically important?
- Virulence - limits phagocytosis by host cells, is poorly antigenic, and protects from host humoral defenses
- Identification - sugar components vary btwn species + determine diff. serotypes within species (ie K antigens determine diff E. Coli spp.)
- Vaccines - capsule polysacch used as vaccine antigen
-
Adherence - help bacteria stick to host tissues/implants
- ex: S. mutans –> dental plaques = dextran polymer capsules
What are bacterial spores made up of?
(6 important components)
When/where are they made?
How are they “germinated”?
- Components: DNA, cytoplasm, p-lipid membrane, peptidoglycan, dipicolinic acid (up to 20% spore weight, stabilizes DNA), keratin-like coat, water
- Made inside Gram-positive cells (never Gram-neg!) when nutrient sources are low + have no metabolic activity until…
- Germination into single living bacterial cell upon exposure to water + nutrients
What is the medical importance of spores?
What are the common medically important spore-forming genuses?
- Spores can resist:
- heat (common sterilization practices)
- chemicals (common disinfectants)
- antibiotics (due to lack of metabolic activity)
- Bacillus and Clostridia species are spore-forming
What is the purpose of intracellular granules in bacteria?
Give an example of a certain kind of granule?
- granules store nutrients
-
volutin granules are a stored form of inorganic PPi
- are “metachromatic” - will turn red when stained with the methylene blue in Neisser stain
What are 3 categories of protein synthesis-inhibiting antibiotics, based on their target?
Give examples of groups that fall into each category.
-
30s ribosomal subunit inhibitors
- aminoglycosides
- tetracyclines
-
50s ribosomal subunit inhibitors
- chloramphenicol
- macrolides
- lincosamides
- oxazolidinones
- streptogramins
- Isoleucyl tRNA synthetase inhibitor - mupirocin
Aminoglycosides
Mechanisms? Results? Examples?
-
Mechanisms:
- irreversible 30s binding leads to initial complex blockage, mRNA misreading + premature mRNA release
- results in bactericidal effect
-
Examples (in order of activity):
- Amikacin
- Gentamicin + Tobramycin
- Streptomycin (1st developed, less use now)
Aminoglycosides
Uses/efficacy spectrum?
Toxicity/side-effects?
-
Uses/Efficacy: serious infections with Gram-negative aerobic rods (few Gram+ species)
- cell entry is an aerobic process, so are ineffective with anaerobes
- Strepto-/Enterococci cell walls resist entry; co-admin with cell wall inhibitors required
-
Toxicity:
- nephro-/ototoxicity (CN VIII)
Tetracyclines
Mechanism/result? Examples?
-
Mechanism: reversible 30s binding (which blocks AA-tRNA binding)
- bacteriostatic only due to reversibility
- Examples: tetra-/doxy-/minocyclin (similar spectra, different kinetics)
Tetracyclines
Spectrum?
Side effects?
-
Spectrum:
- good against Chlamydia, Rickettsia, Borellia, Yersinia, Mycoplasma
- some MRSA activity
- Side effects: tooth discoloration, calcium chelation
Chloramphenicol
Mechanism? Side Effects?
- Mechanism: reversible 50s peptidyl transferase binding + elongation inhibition (mostly bacteriostatic)
-
Side Effects:
- bone marrow toxicity (aplastic anemia, etc.)
Chloramphenicol
Spectrum?
Is mostly bacteriostatic, but…
can be bactericide against H. influenzae, S. pneumoniae, Rickettsia + N. meningitidis
Macrolides
Mechanism + result? Examples?
-
Mechanism: reversible 23s rRNA binding (on 50s subunit) + translocation/elongation inhibition
- bacteriostatic only
-
Examples:
- Erythro-/Azithro-/Clarithromycin
Macrolides
Spectrum?
- effective against Mycoplasma, Mycobacterium, Chlamydia + Legionella
- good for S. Pyogenes in penicillin allergic patients
Lincosamides
Mechanism + result? Example?
- Mechanism: reversible 50s subunit binding + elongation inhibition (bacteriostatic)
- Example: clindamycin
Lincosamides
Spectrum?
- both Gram+ and Gram- anaerobes
- inactive against Gram- aerobes
Oxazolidinones
Mechanism + result? Example?
- Mechanism: binds 50s subunit, distorts tRNA binding site + inhibits 70s initiation complex formation
-
Example:
- linezolid
Oxazolidinones
Spectrum?
- narrow-spectrum for strepto-/staphylo-/enterococci
- usually reserved for multi-drug resistant cocci (including MRSA)
Streptogramins
Mechanism + result? Spectrum?
-
Mechanism: synergistic bactericidal action of group A + B streptogramins (ex: quinupristin-dalfopristin)
- dalfo- binds 50s subunit + induces conformational change allowing quinu- binding
- dalfo- prevents elongation, quinu- releases peptide
-
Spectrum:
- good against VRE + MRSA
Mupirocin
Mechanism + result? Spectrum?
-
mechanism: inhibits isoleucyl tRNA synthetase
- results in bactericidal effect
-
spectrum:
- topical use against Gram+ microbes, including MRSA
- (ineffective for anaerobes, mycobacteria + chlamydia)
What are the two categories of antibiotic susceptibility tests and the specific tests within each?
-
Dilution Tests
- macrodilution test
- microdilution test
- agar dilution test
-
Diffusion Tests
- disc diffusion test
- punching test
- E-test
Describe the process of a macrodilution test for antibiotic susceptibility.
- Prepare sequential dilution of drug in series of test tubes of prefered liquid culture medium for species in question
- (negative control = medium + bacteria, no abx)
- Inoculate tubes with same amt bacterial suspension
- Incubate at 37 C overnight
- Determine MIC, the lowest drug conc. @ which no bacterial growth was observed (no cloudiness in tube)
- Determine MBC by “subculturing” tubes without growth
- add loopful of each solution to agar + observe min. conc. showing no growth after further incubation
Describe the process of a microdilution test.
- very similar to macrodilution, but performed in ELISA polystyrene micro-titre plates and smaller volumes of growth medium, bacterial suspension + antibiotic
Describe the agar dilution test.
- add sequentially decreasing concentrations of the antibiotic in question to a series of agar plates
- allow agar to solidify
- inoculate all the plates with an equal amount of bacterial suspension
- incubate at 37 C overnight
- determine MIC
Describe the disc diffusion test.
- Use a sterile swab dipped in bacterial suspension to prepare an even bacterial lawn on an agar plate
- swab whole plate back and forth, turn 90 degrees, swab again, repeat several times
- Use sterile forceps to place antibiotic-infused discs at equal distances apart around the plate
- Incubate at 37 C overnight
- Measure the diameter of the “inhibition zone”
- can check reference info to determine if this diameter correlates to sensitivity, resistance, etc.
Describe the punch test for antimicrobial sensitivity.
- Very similar to disc diffusion, but rather than discs, wells are punched into the inoculated agar + standard conc. of diff abx are added before incubation + inhibition zone measurement.
Describe the E-test for antibiotic susceptibility.
- combines diffusion + dilution methods
- A pre-made strip containing a sequential dilution is applied to an agar plate inoculted with a “bacterial lawn”.
- Incubation at 37 C overnight
- Where the elliptical zone of inhibition meets the test strip, the MIC can be read.
How can the efficacy of actual concentrations of antibiotics found in clinical samples be evaluated?
- a comparative punch test can be performed
- Prepare wells in inoculated agar plate via punching
- To some wells, add standard volume of known dilutions of antibiotic in use in patient
- To one well, add standard volume of clinical sample (ie serum, urine, etc.)
- Compare inhibition zone diameters of known dilutions to that of clinical sample
Define endotoxin.
- lipopolysaccharide components of the outer membrane of Gram-negative bacteria
- elicit strong immune responses in animals, inducing cytokine release
- made up of an O antigen (glycan polymer), core (oligosaccharide) and Lipid A (toxic component)
- heat stable + non toxoid-forming
- are less specific/potent than exotoxins
Define precipitation.
Give an example of when it is seen in microbial diagnostic techniques.
when an antigen-antibody reaction results in the creation of immune complexes which are visible as sediment in a solution
ex: Elek’s test for Corynebacterium diphtheriae - A strip of diphtheria antitoxin is placed on an agar plate + unknown test sample/known toxic/known non-toxic strains are swabbed in lines perpendicular to strip. Precipitation indicates toxin formation + antitoxin-toxin complex formation.
Define chemotherapeutical index.
ratio of maximum tolerated dose to minimum effective dose
- CI = DTM/DCM (dosis tolerata maxima, dosis curativa minimal)
- higher = better
- means larger tolerated doses, smaller effective doses, or both
Define selective media.
Give an example of one.
culture media containing substances to inhibit growth of any microbes other than those desired
ex: EMB (Eosin Methylene Blue) - inhibits growth of any gram positive bacteria
What are the parameters for hot air sterilization?
- normal pressure
- 3 hrs @ 140 C
- 2 hrs @ 160 C
- 1 hr @ 180 C
What are the parameters for autoclave sterilization?
And for flash autoclave sterilization?
- Normal:
- +1 ATM pressure
- 30 min @ 121 C
- Flash:
- +2 ATM
- 3 min @ 134 C
Define antibiogram.
a record of the resistance of one or more microbial species/strains to certain antibiotics which is a report of the results of antibiotic susceptibility testing
Define sterilization.
How can it be achieved?
destruction of all microbes on a surface or object
usually achieved with physical, gas vapor or chemical methods
What are the antibiotics categories which disrupt cell wall synthesis?
3 types based on action, each with 2-3 categories
-
Beta-lactams - bind PBPs and peptidoglycan synthesis enzymes
- Penicillins, Cephalosporins, Carbapenams, Monobactams
-
Cross-linkage inhibitors - inhibit peptidoglycan cross-linking
- Vancomycin, Cycloserine
-
Others - various mechanisms of action
- Isoniazid/Ethionamid, Ethambutol, Bacitracin (IEEB)
What is the mechanism of action of beta-lactams?
And the resulting effect?
- bind to penicillin-binding proteins (PBPs), which are enzymes responsible for building peptidoglycan chains and cross-linking them
- PBP binding + cross-linkage inhbition leads to activation of autolysins which degrade the cell wall
- this results in a bactericidal effect
What is the general structure of peptidoglycan layers in bacterial cell walls that is inhibited by beta lactams?
- chain of 10-65 disaccharide resides of alternating N-acetylglucosamine and N-acetylmuramic acid(remember muramic –> cell wall)
- glycan chains are attached to a peptide chain of 3-5 AAs
- peptide chains are cross-linked to those of another layer of glycan chains via 5 glycine residue bridges
- glycine bridge formation is disrupted by beta lactams
What is the general structure of penicillins?
- the “penicillin nucleus” is 6-aminopenicillinic acid
- contains thiazolidine ring, beta-lactam ring and amino group
- -R group varies btwn penicillins
What are several different kinds of penicillins?
Any extra info about them (spectra, usage, etc.)?
-
Natural Penicillins
- Penicillin-G (inactivated by gastric acid, IV only), Penicillin-V (oral)
-
Penicillinase-resistant:
- oxa-/methicillin, etc.
-
Broad-spectrum:
- aminopenicillins
What is the core structure of cephalosporins?
7-aminocephalosporanic acid
- has beta-lactam ring (square) and two -R groups
What are the generations of cephalosporins and their general spectra?
(extra: an example of each)
-
1st gen - gram+ cocci
- ex: Cephalexin
-
2nd gen - gram- rods (and less active against gram+)
- ex: Cefprozil
-
3rd gen - better activity agains gram- (and still less active against gram+)
- ex: Cefixime
-
4th gen - broad spectrum, similar gram+ activity to 1st gen, and good gram- activity
- Cefipime
What are the spectra of carbapenams and monobactams?
-
carbapenams - wide spectrum, gram+/- and anaerobes
- (remember no “O” in “carbapenam”, “P” covers positive, “N” covers negative)
-
monobactams - narrow spectrum, aerobic gram-, good for pt w/ penicillin allergy
- (“O”s in monobactam = aerobic, “N” covers negatives)
What are the glycopeptide antibiotics?
Their mechanism of action?
Spectrum?
vancomycin
(and teicoplanin)
- mechanism: peptidoglycan synthesis inhibition (via steric interference of cross-linking bridge formation), also destroy cell membrane + inhib RNA synth
-
spectrum:
- oxacillin-resistant staph + other B-lactam resistant gram+(G in “glycopeptide” = Gram+)
- gram negatives are resistant, b/c glycopeptides too big
Other than the glycopeptides, what antibiotic specifically interferes with cross-linking of cell wall components?
Cycloserine
- an amino acid derivative
- blocks formation of D-Ala-D-Ala dipeptides necessary for peptidoglycan cross-linking
(inhibs Ala Racemase and D-Ala Ligase)
What two antibiotics interfere with synthesis of special microbial cell wall fatty acids?
Which fatty acids?
In what kind of bacteria?
Isoniazid + Ethionamide
- inhibit formation of mycolic acid, so are good against _Myco_bacterium tuberculosis
What antibiotic interferes with production of a polysaccharide component of certain bacteria’s cell walls?
(polysaccharide only, no peptides in this component as in peptidoglycan)
ethambutol
- interferes with arabinogalactan synthesis (polymer of arabinose + galactose) in mycobacterial cell walls
- is therefore good for M. tuberculosis
Which antibiotic that interferes with cell wall synthesis is used topically only?
What is its mechanism? Spectrum?
Bacitracin
- interferes with bactoprenol, a membrane carrier molecule that tranpsorts peptidoglycan components out of the inner membrane
- active against both gram+ and -, specifically staph/strep spp.
What are the obligate structures of bacterial cells?
- Cell Wall
- Ribosome
- Cytoplasm
- Nucleoid
- Periplasmic Space (Gram-negative only)
- Mesosome
WRCNPM
What are 4 functions of the cell wall in bacteria?
- Maintain cell shape
- Counter osmotic pressure
- Provide attachment for appendages - flagella, fimbriae, pili
- Provide attachment for bacteriophages - via teichoic acid in Gram+. not exactly a function, but…
How is the Gram+ bacterial cell wall different from Gram negative?
- Peptidoglycan Layer - thicker and multilayered
- Teichoic Acid - protrude out of PG layer, not in Gram-neg
-
Do not have:
- outer membrane
- periplasmic space
- LPS
Where is LPS found and what is it composed of (3 parts) ?
LPS is found in the outer membrane of Gram-negative bacteria.
-
O-antigen - outermost polysaccharide, antigenic + strain-determining
- some mucosa-colonizers (Neisseria, Haemophilus) have short O-antigen “lipooligosaccharide”
- Polysaccharide Core - between O-antigen and lipid A
- Lipid A - toxic portion, anchored in outer membrane, is a P-ated glucosamine disaccharide with FAs attached
What are the contents of bacterial cytoplasm?
- proteins / enzymes + amino acids
- vitamins / ions
- nucleic acids + nucleotides
- carbohydrates + monosaccharides
- FAs + derivatives
… basically just all the major classes of organic molecules
What is are the Svedberg (S) unit values for the bacterial ribosome?
And its two parts?
Whole ribosome = 70S
Large subunit = 50S, Small subunit = 30S
Describe the nucleoid of bacteria.
- one long molecule of double-stranded, helical, supercoiled DNA per cell
- two ends are covalently-bound, forming a circular molecule
- around 1000 um long, up to 3500 genes
- (some bacteria have linear, not circular DNA)
What is the periplasmic space?
Name several things that can be found in the periplasmic space.
A space between the inner and outer membrane of Gram-negative bacteria.
- thinner, Gram-neg PG layer
- metabolic enzymes (hydrolytic)
- DNAse
- toxins (exotoxins, for excretion)
What is the mesosome?
What does it do?
an invagination of plasma membrane
participates in cell division and secretion
What are Koch’s Postulates?
- The microbe must be found abundantly in all diseased hosts and not in healthy ones.
- The microbe must be isolated and cultured.
- The cultured microbe must be able to induce disease in a susceptible experimental host.
- The microbe must be re-isolated from the experimental host and verified as identical to the original microbe
Define exotoxin.
List some characteristics of endotoxins.
- general term for a variety of soluble, antigenic, injurious substances produced + excreted by bacteria
- usually heat-labile, active in small amounts, and protein in structure
- can be specifically or broadly cytotoxic
What is a common basic structure of exotoxins which exert their toxic effect intracellularly?
- intracellular exotoxins often have A and B subunits
- A is enzymatic/toxic + B is cell entry-mediating (via surface receptor binding)
Give a couple examples of exotoxins.
- Cholera enterotoxin - from Vibrio cholerae, causes overactive P-ation of CFTR by PKA in enterocytes, leading to GI electrolyte secretion + fluid loss (diarrhea)
- Diphtheria toxin - from Corynebacterium diphtheriae, ADP ribosylation of EF2 disrupts protein synthesis, leads to death (via hepatic/cardiac necrosis)
Agglutination
Example of agglutination used in a micro lab?
an Ag-AB reaction, resulting in visible sedimentation of the immune complex, in which the antigen is cellular or is attached to a cell-sized particle
- used for detection of antigens or ABs
- ex: E. Coli serotyping
Differential Media
culture media formulated to indicate differences between colonies of multiple species/strains of cultured microbes
- ex: eosin methylene blue (EMB) - for differentiation of lactose-digesting (purple) and non-lactose-digesting (colorless) Gram-negative colonies
Minimum Inhibitory Concentration
the lowest concentration of an antibiotic able to inhibit bacterial growth and proliferation when applied in vitro
Selective Toxicity
toxicity of an antibacterial agent against bacteria only, with relatively low or no toxicity to the host/patient
Antibiotic Resistance
the ability of a microbe to survive and multiply in the presence of an antibiotic, usually through the expression of genes transferred between microbes via plasmids
Define disinfection.
treatment which reduces the number of potentially pathogenic microbes in an environment (without destroying all microbes)
What are the steps of Gram staining?
(maybe this is only for the practical exam, but I think I remember her saying it could be on the midterm)
- Stain fixed specimen with crystal violet + Na-oxalate for 2 minutes + rinse.
- Stain with Lugol iodine for 1 minute + rinse.
- Differentiate with 96% alcohol until drips are colorless + rinse.
- Stain with fuchsine or safranin for 1 minute.
- Dry between filter paper
- Examine under oil immersion
