Midterm I: Basic Micro (Ben) Flashcards
1
Q
List the facultative structures of bacterial cells.
(6)
A
- Flagella
- Fimbriae / Pili
- Capsule
- Spores
- Granules
FFPCSG
2
Q
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)
A
- 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
3
Q
What is the medical significance of flagella?
(3 main points)
A
- 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
4
Q
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?)
A
- 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
5
Q
What is the function + medical importance of fimbriae/pili?
And what is the distinction btwn fimbriae and pili?
A
- 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
6
Q
What are bacterial capsules and what are most capsules made of?
How do capsule + similar structures differ in structural integrity?
A
- 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
7
Q
What are 4 reasons bacterial capsules can be medically important?
A
- 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
8
Q
What are bacterial spores made up of?
(6 important components)
When/where are they made?
How are they “germinated”?
A
- 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
9
Q
What is the medical importance of spores?
What are the common medically important spore-forming genuses?
A
- Spores can resist:
- heat (common sterilization practices)
- chemicals (common disinfectants)
- antibiotics (due to lack of metabolic activity)
- Bacillus and Clostridia species are spore-forming
10
Q
What is the purpose of intracellular granules in bacteria?
Give an example of a certain kind of granule?
A
- 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
11
Q
What are 3 categories of protein synthesis-inhibiting antibiotics, based on their target?
Give examples of groups that fall into each category.
A
-
30s ribosomal subunit inhibitors
- aminoglycosides
- tetracyclines
-
50s ribosomal subunit inhibitors
- chloramphenicol
- macrolides
- lincosamides
- oxazolidinones
- streptogramins
- Isoleucyl tRNA synthetase inhibitor - mupirocin
12
Q
Aminoglycosides
Mechanisms? Results? Examples?
A
-
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)
13
Q
Aminoglycosides
Uses/efficacy spectrum?
Toxicity/side-effects?
A
-
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)
14
Q
Tetracyclines
Mechanism/result? Examples?
A
-
Mechanism: reversible 30s binding (which blocks AA-tRNA binding)
- bacteriostatic only due to reversibility
- Examples: tetra-/doxy-/minocyclin (similar spectra, different kinetics)
15
Q
Tetracyclines
Spectrum?
Side effects?
A
-
Spectrum:
- good against Chlamydia, Rickettsia, Borellia, Yersinia, Mycoplasma
- some MRSA activity
- Side effects: tooth discoloration, calcium chelation
16
Q
Chloramphenicol
Mechanism? Side Effects?
A
- Mechanism: reversible 50s peptidyl transferase binding + elongation inhibition (mostly bacteriostatic)
-
Side Effects:
- bone marrow toxicity (aplastic anemia, etc.)
17
Q
Chloramphenicol
Spectrum?
A
Is mostly bacteriostatic, but…
can be bactericide against H. influenzae, S. pneumoniae, Rickettsia + N. meningitidis
18
Q
Macrolides
Mechanism + result? Examples?
A
-
Mechanism: reversible 23s rRNA binding (on 50s subunit) + translocation/elongation inhibition
- bacteriostatic only
-
Examples:
- Erythro-/Azithro-/Clarithromycin
19
Q
Macrolides
Spectrum?
A
- effective against Mycoplasma, Mycobacterium, Chlamydia + Legionella
- good for S. Pyogenes in penicillin allergic patients
20
Q
Lincosamides
Mechanism + result? Example?
A
- Mechanism: reversible 50s subunit binding + elongation inhibition (bacteriostatic)
- Example: clindamycin
21
Q
Lincosamides
Spectrum?
A
- both Gram+ and Gram- anaerobes
- inactive against Gram- aerobes
22
Q
Oxazolidinones
Mechanism + result? Example?
A
- Mechanism: binds 50s subunit, distorts tRNA binding site + inhibits 70s initiation complex formation
-
Example:
- linezolid
23
Q
Oxazolidinones
Spectrum?
A
- narrow-spectrum for strepto-/staphylo-/enterococci
- usually reserved for multi-drug resistant cocci (including MRSA)
24
Q
Streptogramins
Mechanism + result? Spectrum?
A
-
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
25
**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)
26
What are the two categories of antibiotic susceptibility tests and the specific tests within each?
1. _Dilution Tests_
* __**macrodilution test**
* **microdilution test**
* **agar dilution test**
2. _Diffusion Tests_
* **disc diffusion test**
* **punching test**
* **E-test**
27
Describe the process of a _macrodilution test_ for antibiotic susceptibility.
1. 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)
2. **_Inoculate_** tubes with same amt bacterial suspension
3. **_Incubate_** at 37 C overnight
4. Determine **_MIC_**, the lowest drug conc. @ which no bacterial growth was observed (no cloudiness in tube)
5. Determine **_MBC_** by "subculturing" tubes without growth
* add loopful of each solution to agar + observe min. conc. showing no growth after further incubation
28
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
29
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
30
Describe the _disc diffusion test_.
1. 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
2. Use _sterile forceps_ to place antibiotic-infused discs at equal distances apart around the plate
3. **Incubate** at 37 C overnight
4. Measure the diameter of the "**_inhibition zone_**"
* can check reference info to determine if this diameter correlates to _sensitivity_, _resistance_, etc.
31
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.
32
Describe the **E-test** for antibiotic susceptibility.
* combines diffusion + dilution methods
1. A pre-made strip containing a sequential dilution is applied to an agar plate inoculted with a "bacterial lawn".
2. Incubation at 37 C overnight
3. Where the elliptical zone of inhibition meets the test strip, the MIC can be read.
33
How can the efficacy of actual concentrations of antibiotics found in clinical samples be evaluated?
* a comparative punch test can be performed
1. Prepare wells in inoculated agar plate via punching
2. To some wells, add standard volume of known dilutions of antibiotic in use in patient
3. To one well, add standard volume of clinical sample (ie serum, urine, etc.)
4. Compare inhibition zone diameters of known dilutions to that of clinical sample
34
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
35
# 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.
36
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
37
# 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
38
What are the parameters for _hot air sterilization_?
* normal pressure
* 3 hrs @ 140 C
* 2 hrs @ 160 C
* 1 hr @ 180 C
39
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**
40
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
41
# 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
42
What are the antibiotics categories which disrupt cell wall synthesis?
3 types based on action, each with 2-3 categories
1. _Beta-lactams_ - bind PBPs and peptidoglycan synthesis enzymes
* **Penicillins**, **Cephalosporins, Carbapenams, Monobactams**
2. _Cross-linkage inhibitors_ - inhibit peptidoglycan cross-linking
* **Vancomycin, Cycloserine**
3. _Others_ - various mechanisms of action
* **Isoniazid/Ethionamid, Ethambutol, Bacitracin** (IEEB)
43
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
44
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 **_mur_**amic --\> 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
45
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
46
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**
47
What is the core structure of **cephalosporins**?
**7-aminocephalosporanic acid**
- has _beta-lactam_ ring (square) and two -R groups
48
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**
49
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)
50
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
51
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**)
52
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 ****_myco_**lic acid**, so are good against ***_Myco_**bacterium tuberculosis*
53
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*
54
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.
55
What are the _obligate_ structures of bacterial cells?
1. **Cell Wall**
2. **Ribosome**
3. **Cytoplasm**
4. **Nucleoid**
5. **Periplasmic Space** (Gram-negative only)
6. **Mesosome**
WRCNPM
56
What are 4 functions of the cell wall in bacteria?
1. **Maintain cell shape**
2. **Counter osmotic pressure**
3. **Provide attachment for appendages** - flagella, fimbriae, pili
4. **Provide attachment for bacteriophages** - via teichoic acid in Gram+. not exactly a function, but...
57
How is the Gram+ bacterial cell wall different from Gram negative?
1. **Peptidoglycan** **L****ayer** - thicker and multilayered
2. **Teichoic Acid** - protrude out of PG layer, not in Gram-neg
3. _Do not have_:
* outer membrane
* periplasmic space
* LPS
58
Where is **LPS** found and what is it composed of (3 parts) ?
**LPS** is found in the _outer membrane_ of _Gram-negative_ bacteria.
1. **O-antigen** - outermost polysaccharide, antigenic + strain-determining
* some mucosa-colonizers (*Neisseria, Haemophilus*) have short O-antigen "_lipooligosaccharide_"
2. **Polysaccharide Core** - between O-antigen and lipid A
3. **Lipid A** - toxic portion, anchored in outer membrane, is a P-ated glucosamine disaccharide with FAs attached
59
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
60
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**
61
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)
62
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)
63
What is the **mesosome**?
What does it do?
an invagination of plasma membrane
participates in _cell division_ and _secretion_
64
What are **Koch's Postulates**?
1. The microbe must be found abundantly in all diseased hosts and not in healthy ones.
2. The microbe must be isolated and cultured.
3. The cultured microbe must be able to induce disease in a susceptible experimental host.
4. The microbe must be re-isolated from the experimental host and verified as identical to the original microbe
65
# 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_
66
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)
67
Give a couple examples of exotoxins.
1. **_Cholera enterotoxin_** - from *Vibrio cholerae*, causes overactive P-ation of CFTR by PKA in enterocytes, leading to GI electrolyte secretion + fluid loss (diarrhea)
2. **_Diphtheria toxin_** - from *Corynebacterium diphtheriae*, ADP ribosylation of EF2 disrupts protein synthesis, leads to death (via hepatic/cardiac necrosis)
68
**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
69
**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
70
**Minimum Inhibitory Concentration**
the lowest concentration of an antibiotic able to inhibit bacterial growth and proliferation when applied in vitro
71
**Selective Toxicity**
toxicity of an antibacterial agent against bacteria only, with relatively low or no toxicity to the host/patient
72
**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
73
Define **disinfection**.
treatment which reduces the number of potentially pathogenic microbes in an environment (without destroying all microbes)
74
What are the steps of Gram staining?
## Footnote
(maybe this is only for the practical exam, but I think I remember her saying it could be on the midterm)
1. Stain fixed specimen with **crystal violet + Na-oxalate** for _2 minutes_ + rinse.
2. Stain with **Lugol iodine** for _1 minute_ + rinse.
3. _Differentiate_ with **96% alcohol** until drips are colorless + rinse.
4. Stain with **fuchsine** or **safranin** for _1 minute_.
5. _Dry_ between filter paper
6. Examine under _oil immersion_
