MICROBIOLOGY UNIT EXAM 3 Pt.1 Flashcards
1
Q
Microorganisms: Bacteria is part of what kingdom. This kingdom is broken up into two.
A
Protista (single celled, or multicellular with little tissue differentiation)
Prokaryotes (no nucleus) - bacteria
Eukaryotes (true nucleus) - fungi, protozoa
2
Q
Genus and species is used when classifying a microorganism. What is helpful of the serotype and subspecies?
A
Helpful in diagnosis and treatment. Serotypes highlights the differences in antigens among the same species. Serotyping allows for the classification of subspecies.
3
Q
Gross morphology of bacteria.
spherical forms Chains Clusters Pairs rod forms intermediate of spherical and rod forms spirals comma shaped, truncated helices
A
Cocci
- Strepto (cocci)
- Staphylo (cocci)
- Diplo (cocci)
Bacilli
Coccobacilli
Spirochetes
Vibrios
4
Q
Gram negative stains _____ (color)
Gram positive stains _____ (color)
Acid-fast stains _____
A
red
dark purple
Certain bacteria (Mycobacterium tuberculosis, mycoplasma) are difficult to stain by the Gram procedure, but they can be stained by a procedure in which the stained bacteriaresist acid alcohol destaining (so-called “acid-fast”bacteria).
5
Q
Determinant of Gram and acid fast reactions and the major difference between bacteria and human cells is the _____. How does this lead to treatment and interaction with an immune response?
A
Cell Envelope
Cell envelope can be the target for antibiotics and main interface between bacteria and immune response.
Note: Differences between the cell envelopes of gram negative and positive bacteria present different challenges.
6
Q
What’s the location of cytoplasmic membrane (AKA ___) relative to the peptidoglycan in Gram +/- bacteria? Which Gram bacteria may have its cytoplasmic membrane be contiguous with outer membrane?
What is the membrane structure of the cytoplasmic membrane and its functions?
In general membrane integrity is maintained by _____ or _____. This marks a difference in mammalian cells/ specific genus of bacteria.
What cytoplasmic maneuver elicits septum and endospore formation?
A
“Plasma membrane” is interior to the peptidoglycan in both Gram (+) and Gram (-) bacteria.
In gram negative bacteria, may be contiguous with outer membrane
Typical trilaminar (protein layer, lipid bilayer, protein layer) unit membrane structure (lipid, protein and small amounts of bound carbohydrate). Functions in cell wall synthesis, protein secretion, active transport, selective permeability, energy generation.
(membrane integrity is maintained by sterols or cell wall)
Bacterial plasma membrane has No sterols!!
Mycoplasma, which, like mammalian cells, requires sterols to compensate for lack of cell wall; provides rigidity and osmotic protection.
Membrane invagination of cytoplasmic membrane during cell division.
7
Q
Bacterial Cell Envelopes: Gram-negative bacteria cell envelopes
Cell envelope is a complex outer surface the most medically important are ____(2). What is the most general difference between the outer membrane of the cell envelope and cytoplasmic membrane?
Define cell envelope.
A
rods and diplococci.
Outer membrane is asymmetrical.
Cell envelope is the membrane(s) and other structures that surround and protect the cytoplasm (i.e. outer membrane (gram-), periplasma (gram-), cell wall, cytoplasmic membrane)
8
Q
Bacterial Cell Envelopes: Gram-negative bacteria cell envelopes
The outer membrane of the cell envelope contains lipopolysaccharide (LPS) and porins. What makes up the LPS (what parts are antigenic or not- what is the endotoxin).
Define porins and what is the selectivity based on?
What’s the advantage of the outer membrane of the cell envelope to gram negative bacteria?
A
Lipopolysaccharide (LPS) consisting of [inside out] Lipid A (not antigenic), corepolysaccharide, and O antigens (antigenic; repeat 40 units). LPS is often referred to as Endotoxin, but Lipid A is the Endotoxin.
Porins –Diffusion channels that allow the membrane to act as a sieve. Selectivity is based on size. The outer membrane contributes to resistance of gram negative bacteria to peptidoglycan antibiotics.
9
Q
Discuss how the peptidoglycan is arranged (thick/think, strongly/poorly linked) for the gram negative bacteria.
In what space is the peptidoglycan hosted in and give an example of an enzyme concentrated in this space for some bacteria.
A
Thin peptidoglycan sheath with poorly linked pieces forming a gel
The gel (thin peptidoglycan sheath) is found in the periplasmic space and various enzymes -e.g. β-lactamases are concentrated here in some bacteria.
10
Q
Host response to LPS from gram negative bacteria (or _____/_____ from gram positive bacteria). Also a response to Fungi.
Discuss the Host response interaction of macrophage and endothelial cells with LPS. What molecules specifically are involved?
What’s the danger of the levels of molecules being produced during this type of host response?
A
- teichoic acid / peptidoglycan
- LPS binds to LPB (LPS Binding Protein), which interacts with macrophage membrane receptors (CD14/TLR4).
- The binding stimulates production of cytokines and inflammatory mediators TNF alpha, IL-1, IL-6, IL-8 and IFN gamma from macrophages and endothelial cells.
- Low level production of these proteins is protective, but overproduction results in development of life threatening bacterial sepsis; systemic inflammatory response that can lead to organ failure.
Note the molecules being produced can activate the coagulation cascade, complement cascade, leukotrienes and/or prostaglandins.
11
Q
Clinical Definition of Sepsis (5+ categories)
A
- Suspected or documented bacteremia
- Fever or hypothermia;
- Tachycardia
- Tachypnea
- Hypotension or 2 of the following symptoms
- unexplained metabolic acidosis
- arterial hypoxia
- acute renal failure
- recent unexplained coagulation abnormalities
- sudden decrease in mental acuity
- elevated cardiac index with low systemic vascular resistance
12
Q
Gram-positive bacterial cell envelopes:
Most gram-positive bacteria are morphologically ____.
What are the characteristics of the cell wall (Discuss how the wall is held together, how many layers, and what it contains)?
A
- most are cocci (most prevalent are Staphylococciand Streptococci)
- Thick, extensively cross-linked cell wall. Up to 40 layers of peptidoglycan. Cross linking by pentaglycine bridge builds layers of sugar tetrapeptide into a three dimensional structure
- Contain teichoic acids; wall teachoic and lipoteichoic (extends into cytoplasm) acid.
13
Q
Gram-positive bacterial cell envelopes:
Define teichoic acids. What is the function of teichoic acids? What do teichoic acids contain (molecularly)? Describe two kinds of teichoic acids and how or what they attach to.
What percentage does teichoic acids contribute to the cell wall?
Give the etiology of teichoic acids (2).
A
Teichoic acids -polymers of ribitol or glycerol with phosphodiester links.
Provide further cross linking and integrity to the cell wall.
Teichoic acids contain amino acid residues or GlcNAc (N-acetylglucosamine) residues or both.
Ribitol teichoic acids usually attached to the MurNAc (N-acetylmuramic acid) residues of peptidoglycan.
Glycerol teichoic acids are anchored in the cytoplasmic membrane via linkage to a membrane glycolipid (extend from the membrane through the cell wall).
May contribute up to 50% of the cell wall
Function in disease:
- Serve as adhesins
- cause host cells to release inflammatory mediators (highly antigenic, although Lipoteichoic acid is 20X less potent than LPS in eliciting release of inflammatory mediators)
Note: GlcNAc is a glucosamine (glucose with an N-group) with with an acetyl attached to the N. MurNAc is GlcNAc with an ether of lactic acid.
14
Q
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically and frequently antigenic and/or otherwise biologically active.
Name 4 extracellular and surface components.
A
Capsules
Slime
Flagella
Pili
15
Q
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically andfrequently antigenic and/or otherwise biologically active.
Define slime.
A
Slime - Antigenic polysaccharide with no definite outer margin.
16
Q
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically andfrequently antigenic and/or otherwise biologically active.
Define flagella (6).
A
Flagella -
Long, helical filamentous appendages.
Responsible for motility.
Composed of flagellin.
Attached to basal body in cytoplasmic membrane.
Antigenic ("H" antigen).
Causes swarming of some bacteria.
17
Q
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically andfrequently antigenic and/or otherwise biologically active.
Define pili (3). Two or more functional types.
A
Pili (Fimbriae) -
Short filamentous appendages.
Composed of Pilin (Antigenic).
Originate in cell membrane.
Two or more functional types may appear independently or together on same cell:
a. Sex pili (bacteria-bacteria adhesion for genetic transfer via conjugation)
b. Common pili (bacteria-human cell adhesion or attachment = virulence)
18
Q
Fine structure of Bacteria: Extracellular and surface components: Usually clearly defined chemically andfrequently antigenic and/or otherwise biologically active.
Define capsules (6).
A
Capsules -
Not part of the cell proper.
Mainly polysaccharide, although a few polypeptidecapsules.
Gel-like, well-defined border.
Virulence factor due to antiphagocytic properties. Antigenic; sometimes vaccine (e.g.Streptococcus pneumoniae -more than 80 distinct antigenic types).
19
Q
Identify their Gram-stain and morphology identity:
Actinomyces, Klebsiella, Escherichia,Peptostreptococcus, Streptococcus, Bacillus, Bacteroides, Gemella , Enterococcus, Listeria, Streptomycin, Haemophilus, Moraxella, Corynebacterium, Clostridium, Staphylococcus, Bordetella, Enterobacter, Neisseria
A
Gram-positive cocci
Enterococcus, Gemella, Peptostreptococcus, Streptococcus, Staphylococcus
Gram-positive bacilli
Actinomyces, Bacillus, Clostridium, Corynebacterium,
Listeria, Streptomycin
Gram-negative cocci
Moraxella, Neisseria
Gram-negative bacilli
Bacteroides, Bordetella, Enterobacter, Escherichia, Haemophilus, Klebsiella
20
Q
Membrane rigidity and osmotic pressure: Sterols of mammalian cells provide membrane rigidity, which prevents H2O uptake and membrane stretching in response to osmotic pressure. To compensate for the absence of the sterols, most bacteria are surrounded by a mesh-like cell wall.
Cell wall: Peptidoglycan layer (mucopeptide layer, murein layer). List the characteristics of cell wall including the function, polymer constituents, cross linking, difference between gram +/-, and what’s out to break it up!
A
Cell wall layer found in all bacteria that have rigid walls and shape.
Provides rigidity and osmotic protection.
β-1,4 linked disaccharide units of:
N-acetylglucosamine (GlcNAc)
N-acetylmuramic acid (MurNAc).
A tetrapeptide attached to each N-acetylmuramic acid.
Polymers encircle cells and are cross-linked through the tetra peptide.
- Gram negative cell wall: thin peptidoglycan sheath (usually one layer) with poorly linked pieces forming a gel.
- Gram-positive cell wall: (most prevalent are Staphlococci and Streptococci)
a. Thick, extensively cross-linked cell wall. Up to 40 layers of peptidoglycan. Cross linking by PENTAGLYCINE BRIDGE builds layers of sugar tetrapeptide into a three dimensional structure. - Peptidoglycan synthesis (antibiotic target)
21
Q
Protoplasts, Spheroplasts, L-forms of bacteria.
Give two ways these morphological changes can occur to bacteria.
Define protoplast, spheroplast, and L-form. In disease state can you expect to see L-forms?
A
- Peptidoglycan synthesis blocked by antibiotics - result is lysis of cell if not in isoosmotic medium
- Under isosmotic pressure (10% sucrose, 3M NaCl, etc.) with blocked peptidoglycan sythesis –> protoplasts and spheroplasts can form
- Spheroplasts/protoplasts may replicate and form L forms (chronic infections)
Protoplasts: Have their cell wall entirely removed and are derived from gram + (gram-positive)
Spheroplasts: Have their cell wall only partially removed and are gram - (gram-negative)
22
Q
Endospore of bacteria: what are they, what genus makes them (2). Discuss where endospore develop and what they are resistant to and when do they germinate.
Highlight the important of dipicolinic acid.
A
Endospore: Refractile body-thick walled
- Clostridium & Bacillus
- Develops intracellularly, highly resistant to heat, drying, chemicals, oxygen
a. Ca2+ chelated by dipicolinate to dehydrate the peptidoglycan
b. Drying of cytoplasm
c. Germination in presence of nutrients (under favorable conditions)
Note: Dipicolinic acid forms a complex with calcium ions within the endospore core. This complex binds free water molecules, causing dehydration of the spore. As a result, the heat resistance of macromolecules within the core increases. The calcium-dipicolinic acid complex also functions to protect DNA from heat denaturation by inserting itself between the nucleobases, thereby increasing the stability of DNA.
23
Q
Intracytoplasmic components of bacteria
Define nucleoids. Discuss the state DNA is and the circumferential size.
Define plasmids. Function/involvement and what “harm” can they avoid?
Define composition of proteins (% breakdown), it’s function, shape, weight, svedberg unit, sensitive to ____, and can form poly_______.
A
Nucleoids: Regions of the cell containing the condensed bacterial chromosome.
a. Double-stranded circular DNA molecule - circumference of about 1 mm
b. Not bounded by membranes
- Plasmids: Plasmids are autonomously replicating, circular molecules of double-stranded DNA that can confer resistance to ANTIBIOTICS and are involved in the production of bactericidal proteins and toxins. Plasmids are also involved in BACTERIAL CONJUGATION and synthesize surface antigens and pili.
- Ribosomes: Large complex composed of protein and RNA. Protein synthesis.
a. Protein Composed of RNA (60%) and protein (40%).
b. Spherical, MW 2.8 x 106 daltons (70S divided into 50S & 30S particles).
c. Smaller than those of eukaryotes and sensitive to ANTIBIOTICS
d. form polyribosomes
24
Q
BACTERIAL NUTRITION, METABOLISM AND GROWTH
A. Environmental factors, nutrition and metabolism
pH: discuss range of tolerance and an example for the two extremes.
atmosphere: Discuss anaerobes and aerobes. What are their respective requirements, where can they be found, and different categorical terms for their atmospheric requirements.
temperature: Define mesophile and can some bacteria require different range of temperatures?
osmolarity: state the two categorical terms.
A
- Factors influencing growth and reproduction:
a. pH - range of tolerance (Acidophiles – Lactobacillus, Neutralophiles, Alkaliphiles - Vibrio)
b. Atmosphere
- Anaerobic - anaerobes don’t use free 02; strict (obligate) anaerobes are killed by atmospheric 02 (Most in GI, although some exist in the environment as spores)
- Aerobic -only grow if free 02 is present (esp. respiratory or mucosal pathogens)
- Facultative - grow with, or without, 02
c. Temperature requirements
- Mesophilic - flora of the human body (35oC - 37oC is often optimal, but some bacteria have unusual requirements).
d. Osmolarity (halophilic & non halophilic)
- Osmophilic – prefer high osmotic pressure
- Halophilic – prefer high [salt]
25
BACTERIAL NUTRITION, METABOLISM AND GROWTH
Nutritional Requirements:
Autotrophic vs heterotrophic. What type energy, carbon, nitrogen is used.
What is the clinical use of media?
Autotrophic: No organic matter used
i. Energy - Light, Chemical, Heat
ii. Carbon - CO2
iii. Nitrogen - N2, N02, N03, NH3
b. Heterotrophic
i. Energy – Chemical
ii. Carbon - (CO2 and at least 1 organic compound)
iii. Nitrogen - N2, N02, N03, NH3, organic
c. Minimal, defined media - useful in clinic for diagnosis
- API strips
26
-BACTERIAL NUTRITION, METABOLISM AND GROWTH
Metabolism: What compounds are used for metabolism? Provide two examples of how a diagnosis can be made based on metabolism.
Metabolism:
a. Metabolite utilization
i. Simple compounds, amino acids, sugars
ii. Complex molecules, proteins, polysaccharides, lipids
iii. Diagnostic in the clinic:
(a) N. meningitidis ferments maltose, glucose
(b) N. gonorrhea ferments only glucose
27
BACTERIAL NUTRITION, METABOLISM AND GROWTH
Growth:
Define the goal/result of binary fission, generation times, and growth rate.
Define growth curve. What are parameters, lag phase, log phase, stationary phase, death phase.
How is knowing the phase of growth important?
1. Binary fission - simple means of reproduction - EXPONENTIAL increase in cell numbers.
Generation time = time it takes for the bacterial population to double
Growth rate = number of generations (doublings)/hour
2. Growth curve - A growth curve is an empirical model of the evolution of a bacterial # over time.
a. Parameters - each bacterium has a characteristic generation time, although this varies considerably with the nutritional status, pH, etc.
b. Terminology
i. Lag phase - no increase in cell number; period of adaptation
ii. Log (EXPONENTIAL) phase - period of active and uniform growth
iii. Stationary phase - rate of multiplication and death balance one another; due to buildup of toxic compounds and nutrient depletion (RESISTANT TO CELL WALL ANTIBIOTICS)
iv. Death phase - cells die rapidly
c. Clinical significance - diagnosis and treatment may be affected by the phase of growth (gram stain, drug susceptibility, etc.).
28
Mechanism of action:
Bacitracin
Vancomycin
Penicillin
Bacitracin interferes with the dephosphorylation of C55-isoprenyl pyrophosphate, a molecule that carries the building-blocks of the peptidoglycan bacterial cell wall outside of the inner membrane. BACITRACIN - Inhibits regeneration of lipid carrier
The large hydrophilic molecule is able to form hydrogen bond interactions with the terminal D-alanyl-D-alanine moieties of the NAM/NAG-peptides. Under normal circumstances, this is a five-point interaction. This binding of vancomycin to the D-Ala-D-Ala prevents cell wall synthesis of the long polymers of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) that form the backbone strands of the bacterial cell wall, and it prevents the backbone polymers that do manage to form from cross-linking with each other.
Penicillin:
β-Lactam antibiotics inhibit the formation of peptidoglycan cross-links in the bacterial cell wall; this is achieved through binding of the four-membered β-lactam ring of penicillin to the enzyme DD-transpeptidase. As a consequence, DD-transpeptidase cannot catalyze formation of these cross-links, and an imbalance between cell wall production and degradation develops, causing the cell to rapidly die.
29
INFECTION AND HOST RESPONSE
Interaction with an infectious agent can lead to a number of manifestations of disease in the infected host:
Name and discuss (5) illnesses.
1. Acute illness - clinically apparent disease
2. Latent illness - organism not readily detectable but may recur; e.g. HSV-2
3. Recurrent illness - reinfection with same organism or recurrence of latent infection
4. Subclinical - acute or chronic symptomless infection
5. Chronic illness - persisting symptoms
30
INFECTION AND HOST RESPONSE
Host immune response
Define apparent absence, innate/adaptive immune response, and immunopathology.
1. "Apparently" absent- evidence of infection is difficult to document (superficial fungi)
2. Innate immunity - first line defense; may serve to limit extent of early infection prior to the development of specific immunity
3. Adaptive immune response - lifelong immunity mediated by T cells and antibody
4. Immunopathology - inappropriate (overly vigorous) immune response leading to tissue damage
5. A combination of the 4.
31
INFECTION AND HOST RESPONSE
Host response depends on: (5) factors.
1. Type or organism
2. Dose of organism
3. Site of infection
4. Natural history of infection
5. Host factors
- Age
– Immunocompetence
– Sex
– Genetics
– Nutrition
32
INFECTION AND HOST RESPONSE
Immunodeficiency syndromes- what this means in terms of opportunistic infections (give some examples).
Immunodeficiency syndromes - increased susceptibility to infections in immunocompromised hosts implicates the intact immune system in protection from these organisms
e.g: in AIDS - opportunistic infections such as pneumocystis carinii, CMV, Candida, some bacterial pathogens
33
INFECTION AND HOST RESPONSE
Course of immune response to infection: From entry to eradication of infectious agent.
1. Entry of microorganism through epithelial surface (skin or mucosal surfaces).
2. Innate immune response-helps contain infection and delivers antigen (e.g via DCs) to lymph nodes for elicitation of adaptive immunity.
- Distinct CD4 T cell subsets mediate clearance of diverse pathogens
3. "Priming and Proliferation" Adaptive immunity-Activation of T cells and B cells in the local lymph node; production of antibody and effector T cells.
4. Eradication of infectious agent.
34
INFECTION AND HOST RESPONSE
Mechanisms by which infectious agents evade immune response (4)
1. Hiding within cells without antigenic expression- MANY organisms
2. Lurk on periphery of body - e.g. superficial fungi
3. Pool of non-immune individuals - measles, recent outbreaks of whooping cough
4. Changing surface structure either at the population level (e.g. influenza virus) or during the normal life cycle - (African trypanosomes)
35
Balance between Th1 and Th2 cytokines is important in determining type of immune response to infectious organism. Discuss what cytokines Th1/2 release and what actions they tend to favor relative to an immune response.
Discuss the roles of Th17 cells, third signal, and Th cell in immune responses.
Th1 cytokines IFN gamma and IL-2 (esp. INF gamma) favor cell-mediated immunity and class switch to opsonizing antibody (macrophages, CTL, DTH, NK)
Th2 cytokines (IL-4, IL-5, IL-13) favor IgE responses and some Ig subclasses, parasitic infections, which are particularly important in immunity to worms.
Th17 cells, which produce IL17 and activate neutrophils for killing of extracellular bacteria and fungi. Produce IL-17, pro-inflammatory; important for resistance to extracellular G+ organisms, fungi at mucosal surfaces*; activate neutrophils. Are also involved in autoimmunity.
The third signal (cytokines) delivered by APC in part direct the type of T helper cell that is activated.
A fourth type of Th cell, the T regulatory cell, is involved in suppressing T cell responses. Produce regulatory cytokines to control magnitude and duration of immune response; tolerance
```
*– Trypanosomes
– Leishmania
– Tuberculosis
– Extracellular G+ bacteria
– Multiple sclerosis
```
Note: Dominance of one response may influence outcome
36
IL-12 and IFN (components of _____ immunity) tend to bias towards ____ and _____.
Which Th2 cytokines (2) are induced by helminthic infections, leading to production of IgE and recruitment of eosinophils.
What response is associated with Resistance/Exacerbation to murine infection with the protozoan parasite Leishmania major.
IL-12 and IFN- are two components of innate immunity that also tend to bias towards Th1 and CMI.
A specific example is the induction of Th2 cytokines IL-4, IL-5 by helminthic infections, leading to production of IgE and recruitment of eosinophils.
Another example is murine infection with the protozoan parasite Leishmania major. Resistance to infection is associated with a Th1 response (production of TNF- and IFN-) whereas exacerbation is associated with a dominant Th2 response (IL-4).
37
IMMUNITY TO BACTERIA
Effective immune response depends upon the mechanisms of pathogenicity. What two factors are we looking at?
Is damage caused by invasive or non-invasive (e.g. toxins) mechanisms?
Is the bacterium extracellular or intracellular?
Immunity to Bacteria depends on:
* Cell wall type (G+/G-)
* Invasiveness
* Toxin
* Intracellular
* Extracellular
38
Immunity to extracellular bacteria:
Components of both the innate and adaptive system are active. Discuss the actions taken on by both innate and adaptive system.
1. Innate aspects have been discussed earlier in the course (complement activation, phagocytosis, and the inflammatory response), production of IL-12 (to activate
Th1 responses). Phagocytes (macrophages and
neutrophils) Complement, IFN-gamma production by NK cells, Acute phase proteins: fibrinogen, CRP
2. The adaptive immune response to extracellular bacteria includes :
a. Production of antibody that neutralizes the bacterial cells or the toxins they produce, opsonizes, activates complement for direct lysis or induction of inflammation. Opsonization facilitates uptake of the microbes.
b. CD4 cell activation: production of cytokines, IFN-gamma (to activate macrophage killing), TNF (contributes to inflammation). activate antibody response. Although macrophages can respond directly to microbes and kill, this is dramatically increased with Th1 activation.
39
Mechanisms of immune evasion by extracellular (4) bacteria.
Extracellular bacteria
• Antigenic variation
– Neisseria gonorrhoeae: variation in Pilin expression
– E. coli
– S. typhimurium
• Inhibition of complement activation
– Capsule sialic acid residues
– Pseudomonas - elastase inactivates C3a and C5a
• Resistance to phagocytosis
– Pneumococcus (Streptococcus pneumoniae) - remove capsule, resistance decreases
• Scavenging of reactive oxygen intermediates
– Catalase-positive staphylococci
• IgA-ase
– Secretory component helps protect, but some organisms secrete enzyme to break down IgA
40
Immunity to intracellular bacteria:
Discuss the innate responses that get tied into the stimulation of adaptive responses. Discuss how the major protective effect is carried out.
1. Also associated with innate and adaptive responses
2. Innate responses include both phagocytes and NK cells. Phagocytes ingest these bacteria, but many are resistant to the degradation. NK cells can kill either directly, or through the production of IFN-gamma, which activates macrophages for killing. IL-12 production by APC (macrophage) can stimulate the adaptive response.
3. The major protective effect is through cell-mediated immunity – T cell activation of macrophages (again, through the production of cytokines like IFN-gamma or through CTL (CD8+ T cells).
Note: Macrophage is activated by IFN-gamma. An activated macrophage will secrete TNF, IL-1, IL-12, chemokine. Increase expression of MHC and costimulators (B7 molecules)
41
Bacterial cell walls - have a number of adjuvant effects.
How does the cell wall play a role in an effective/noneffective immune response?
List 5 effects of bacterial walls.
The effectiveness of the immune response to bacteria depends partly on the host's ability to damage the bacterial cell wall.
Adjuvant effects of bacterial cell walls include:
1. Trigger inflammatory mechanisms
2. Activation of complement
3. Activation of macrophages- TLR-4
4. Upregulation of costimulatory molecules
5. Polyclonal B cell activation
6. IL-1 dependent polyclonal T cell activation
42
Bacterial/Virus "Superantigens": What's the mode of action and what's the danger in them?
Bacterial "Superantigens" activate large numbers of T cells.
Staphlyococcal enterotoxins bind to V region of TCR, stimulating high frequency responses. In excess, this can cause toxic shock or septic shock syndrome. (Viruses
can also encode superantigens)
43
Other anti-bacterial defenses
Discuss ADCC (Antibody-dependent cell-mediated cytotoxicity; kills ____ bacteria), T cells and heat shock proteins (important in ______ infections), Balance of Th1 and Th2 (mycobacterium leprae infection - 2 outcomes)
Define ADCC mode of action.
1. ADCC – various effectors can kill gram negative bacteria this way
2. T cells and heat shock proteins – important in mycobacterial infections
3. Balance of Th1 and Th2 can affect outcome of infection:
In mycobacterium leprae infection: Th1 dominance leads to Tuberculoid leprosy (more contained), whereas Th2 dominance (or defective Th1), leads to lepromatous leprosy, which has higher bacterial counts in lesions
ADCC- antibodies bind to pathogenic antigen. NK CD16 recognize and bind Fc region of antibody. The NK degranulates into the Ag/Ab/Fc lytic synapse and lysis the antigen.
44
Bacterial immunopathology - When too much of a good thing isn’t so good!
Discuss:
endotoxic shock,
release of mediators
cross reactivity (specific example associate with strep infections)
granulomatous infection (specific persistent microbes).
1. Endotoxic shock (or septic shock) is caused by high systemic levels of TNF induced by bacterial lipopolysaccharide (LPS), characterized by vascular collapse, disseminated intravascular coagulation, organ failure.
2. Release of mediators at site of infection
3. Cross-reactivity of antigens: strep infections can lead to glomerular nephritis or rheumatic fever
4. Granulomatous inflammation: clusters of activated macrophages, particulate antigen form nodules of inflammatory tissue – granulomas. Characteristic of response to some persistent microbes such as TB and
also some fungi. Respiratory difficulty in TB associated with replacement of normal lung tissue with fibrotic tissue.
45
Types of effector T cells, Main functioning adaptive immune response, pathogens targeted.
CD8 CTL, CD4 Th1, CD4 Th2, CD4 Th17, CD4 regulatory (various types)
CD8 CTL - kill virus infected cell. Target viruses e.g influenza, rabies, vaccinia, and some INTRACELLULAR bacteria
CD4 Th1 - Activated infected macrophages and provide help for B cell production of antibodies. Target microbes that are persistent in macrophage vesicles (e.g. mycobacteria, Listeria, Leishmania donovani, Pneumocystis carinii, and EXTRACELLULAR bacteria.
CD4 Th2 - provide help to B cell for antibody production especially IgE class switch. Target are helminth parasites.
CD4 Th17 - Activate fibroblasts, epithelial cells. Enhance neutrophil response.
CD4 regulatory - Suppress T cell response. Inhibit immature DCs thus surpassing CD4 T cell.
46
Signal 3 delivery cascade: Treg, Th17, Th1, Th2 (signal 3, TF, Effector cell production signals)
Signal 3 delivered by APCs:
Treg - TGF-beta
Th17 - TGF-beta, IL-6
Th1 - IL-12, IFN-gamma
Th2 - IL-4
From signal 3 to TF - the activation of effector cell:
Treg - FoxP3
Th17 - RORgammaT
Th1 - T-bet
Th2 - GATA-3
From Activation of effector cell we now express :
Treg - TGF-beta, IL-10
Th17 - IL-6, IL-17
Th1 - IL-2, IFN-gamma,
Th2 - IL-4, IL-5
47
Viruses and some bacteria can induce a cascade between DCs and NK cells and how are naive CD4 T cells influenced?
Other pathogens (e.g. worms) cause a cascade with NK cells and how are naive CD4 T cells influenced?
- Viruses and some bacteria induce IL-12 secretion by DCs that can activate NK cells to produce IFN-gamma. In the presence of IL-12 and IFN-gamma naive CD4 T cell become activated and commit to differentiating into Th1 cells.
- Other pathogens (e.g. worms) may cause NK 1.1+T cellist synthesize and secrete IL-4. Naive CD4 T cells activated in the presence of IL-4 commit to differentiation into Th2 cells.
48
Discuss the IgA transition from plasma cell in Basal lamina side to lumen side of gut.
IgA transitions across epithelial gut cell at the base of the crypts. Polymeric IgA binds in the mucus layer, and then the IgA neutralized the pathogen and their toxins.
49
Can the same responses be used for different types of bacteria which for example differ in their capsular polysaccharide?
No new response is warranted.
50
Mechanisms of immune evasion by intracellular (3) bacteria.
Intracellular bacteria
• Inhibition of phagolysosome formation
– Mycobacterium tuberculosis
– Legionella pneumophilia
• Scavenging of reactive oxygen intermediates
– Mycobacterium leprae
• Inhibition of phagolysosome formation,
escape into cytoplasm
– Listeria monocytogenes (hemolysin protein)
51
INFECTION WITH MYCOBACTERIUM leprae
Mark the differences between Tuberculoid Leprosy and Lepromatous leprosy
Tuberculoid leprosy: organisms present at a low to undetectable levels, low infectivity, granulomas and local inflammation, peripheral nerve damage, normal serum immunoglobulin levels, Normal T cell responsiveness, specific response to M. leprae antigens.
Lepromatous leprosy: organisms show florid growth in macrophages, high infectivity, disseminated infection, bone, cartilage, and diffuse nerve damage, hypergammaglobulinemia, low or absent T cell responsiveness, No response to M leprae antigens.
52
List of bacterial superantigens: Enterotoxins (4)
SEB, SEC 2, SEE, TSST-1
SE- Staphylococcus enterotoxin
TSST- Toxic shock syndrome toxin
53
Bacterial immunopathology - When too much of a good thing isn’t so good!
TNF : low, moderate, high [plasma]
Low: local inflammation
Moderate --> systemic effects; fever (brain), acute phase proteins (liver), leukocytes (bone marrow recruitment)
High: >or equal 10^-7 --> septic shock; low cardiac output, blood vessel is of low resistance/ thrombus formation, hypoglycemia (liver)
54
Bacteria and Autoimmunity:
List two ways bacteria can cause autoimmunity.
1. Activation of APCs while expressing self antigen by microbe --> Induction of costimulators on APCs, presentation of self antigen APCs --> self reactive T cells --> autoimmunity
2. Molecular mimicry --> Activation of T cells --> Self-reactive T cells and/or antibody. (e.g. Rheumatic fever) --> autoimmunity.
55
What is the goal of vaccination?
The goal of vaccination is to mimic this effect so that the individual doesn’t have to have the initial disease yet has the protective effects of the secondary immune response.
56
Most vaccines induce antibody responses or CTL activity? How does vaccine design change accordingly to targeted pathogen?
The majority of vaccines currently in use induce predominantly antibody responses, with
some of the live vaccines inducing CTL activity.
For example, a vaccine that induces primarily antibody
responses may not be effective for a virus that never goes through a cell-free stage. There, a
development of CTL is likely to be more beneficial. Likewise, many of our current vaccines
favor development of Th2 responses, where as Th1 (cell-mediated) responses might be more
protective.
57
Passive immunity: Passive immunity provides a rapid, temporary protection.
Explain in what form passive immunity is provided and provide four examples.
What are the pros and cons to passive immunity?
What is a condition that would require passive immunity?
Typically, passive immunity is given in the form of immune globulin or monoclonal antibodies. Passive immunity is used when the exposure is acute and the risk is immediate – i.e. there is no time to actively immunize and wait for the
response.
Examples include snake bite, tetanus exposure in an un-immunized individual, some virus exposures, anti-respiratory syncytial virus antibody in premature babies.
It gives immediate effects but is short-lived (dependent upon the half-life of the antibody) and does not give memory.
Passive immunity can also be given – e.g. pooled human immunoglobulin – to individuals who cannot
mount their own antibody response (e.g. Bruton’s agammaglobulinemia)
58
Define Active immunity:
Activates the host immune response to produce an immune response that mimics the response to natural infection.
59
Discuss attenuated vaccines. Give examples of them. Give the pro/con to its use.
Attenuated (live) e.g. live, attenuated viruses (measles, mumps, rubella, polio, flumist) and bacteria (BCG, used in Europe, Asia).
Live vaccines often give the best protection and best induction of CTL activity because they mimic natural
infection, but may cause disease in immunocompromised hosts (e.g. Sabin polio vaccine)
60
Discuss inactivated and subunit vaccines. Give examples.
Inactivated viruses and bacteria: e.g. Salk polio vaccine, most flu vaccines, old pertussis vaccine.
Subunit vaccines – e.g. tetanus toxoid, diptheria toxoid. Usually administered with an adjuvant to enhance the immune response (e.g. alum)
61
Discuss conjugate and recombinant vaccines. Give examples.
4. Conjugate vaccines – conjugates a relatively weak immunogen for young children (polysaccharide) to an effective immunogen (tetanus toxoid). The Haemophilus influenzae vaccine (note that H. flu is a bacterium, not a
virus!) is the prototype for this.
5. Recombinant vaccines – hepatitis B
62
List 3 vaccines in clinical trials or development.
- Viral vectors
- ISCOM – liposome-like structures to deliver antigen to the cytoplasm and initiate MHC Class I presentation and CMI. (in development)
- DNA vaccines (in clinical trials)
63
WHAT’S NEEDED AND WHAT’S IN DEVELOPMENT for vaccines.
List the 4 types of vaccine modalities for improvement. Provide examples.
```
What’s Needed?
• Effective vaccines for HIV, HSV, EBV,
malaria etc.
• Better compliance - e.g. measles - Can it be
eradicated?
• Better adjuvants for human use
–
–
• Asthma connection
– , costimulation
•
```
Better adjuvants
Parasite vaccines
Cancer vaccines
Dendritic cell vaccines
- Better adjuvants for human use (Adjuvants increase magnitude and duration of response - stimulate expression of co-stimulatory molecules. Current adjuvant: alum activates inflammasomes) are being developed.
In addition, immunomodulators are being tested as vaccine
augmentors. For example, synthetic bacterial-like DNA sequences (Inclusion of cytokine, plasmid DNA (CpG)) that activate through TLR9 are being tested as adjuvants to induce Th1 responses *****(Need adjuvants that will favor Th1)*****.
Other strategies being tested include cytokine incorporation such as IL-12. Currently, the only adjuvant in use in humans in the US is alum, which enhances immunity through the inflammasome. (There are some other adjuvants approved in Europe and other parts of the world).
******* Most current vaccines induce Th2 responses; Asthma connection.********
- Parasite vaccines (for example, against malaria) are much-needed.
- Cancer vaccines are under development, and a papilloma virus vaccine has been recently approved – prevents cervical cancer. Recent estimates say that with the
current vaccine penetration in girls of about 45%, 50,000 cases of cervical cancer have been prevented!
- Dendritic cell vaccines are in development: let the professional APC choose the epitopes to present. Applications to tumor, infectious disease (HIV). First approved DC vaccine: Dendreon prostate cancer vaccine that uses the patient’s own DC to present the tumor antigens (unfortunately, the company has declared bankruptcy). Therapeutic melanoma vaccines are also in trials.
64
New challenges we face. List 4 examples.
NEW CHALLENGES – bioterrorism, bird flu and other emerging pathogens – rapid response. Most recent challenge: H1N1 “swine flu”.
65
A brief history of vaccination
Define the root of the word "immunity"
Concept dates to 430 B.C. when Thucydides, the
historian of the Peloponnesian War wrote _______.
Define variolation. Discuss the history.
1796: Jenner used cow pox to protect from
smallpox. What term come out from this?
• Immunity: comes from the Latin “immunis” meaning
“exempt”
• Concept dates to 430 B.C. when Thucydides, the
historian of the Peloponnesian War, wrote that those
who had recovered from Plague could care for those
with disease
• Variolation - used in ancient Asia; brought to Europe
in 1721 by Lady Mary Wortley and subsequently
used in the Revolutionary War
- Variation- Variolation or inoculation was the method first used to immunize an individual against smallpox (Variola) with material taken from a patient or a recently variolated individual in the hope that a mild, but protective infection would result.
• 1796: Jenner used cow pox to protect from
smallpox. The term “vaccination” (“vacca” is Latin
for “cow”) derives from this.
66
What did Koch prove in the 1870's?
1860's-1880's what did Louis Pasteur develop?
- 1870’s: Koch proved that infectious diseases are
caused by microorganisms- anthrax
- 1860’s-1880’s: Louis Pasteur developed vaccines
against cholera quite by accident - attenuation.
Coined “vaccine” in honor of Jenner. Also made first
anthrax and rabies vaccines.
67
Vaccines do not prevent infection
But…
They can ______.
prevent disease
68
Comparison of unimmunized donor vs immunized donor of B cells.
Compare frequency of specific B cells, isotope of antibody produced, affinity of antibody, and somatic hypermutation.
frequency of specific B cells: 1:10^4 - 1:10^5 vs 1:10^3
isotope of antibody produced: IgM > IgG vs IgG, IgA
affinity of antibody: low vs high
somatic hypermutation: low vs high
69
Polio: another success story. Discuss what occurred for this success story.
* 1952: 58,000 Americans contract polio
* Salk: inactivated polio vaccine - 1952
* Sabin: live vaccine
* 1994: Western world “free” of polio
• Success leads to modification of recommendation: now
use inactivated polio vaccine
70
List some infections for which vaccines are not yet available (8) and estimated annual mortality.
```
Malaria 889K
Schistosomiasis 41K
Intestinal worm infection 6K
Tuberculosis 1.5 million
Diarrheal disease 2.2 million
Respiratory infections 4 million
HIV/AIDS 2 million
Measles 400K
```
71
Give examples of passive immunity (3)
Natural maternal antibody, immune globulin, humanized monoclonal antibody, antitoxin.
Immune globulin - an antibody-containing solution derived from human blood, obtained by cold ethanol fractionation of large pools of plasma; available in IM and IV preparations.
Antitoxin - An antibody derived from the serum of animals that have been stimulated with specific antigens.
72
Give examples of active immunity
Natural infection
Vaccines
- attenuated organisms
- inactivated organisms
- purified microbial macromolecules
- Cloned microbial antigens : expressed as recombinant protein, as cloned DNA alone or in virus vectors
- Multivalent complexes
- Toxoid
Vaccines - A suspension of attenuated live or killed microorganisms, or antigenic portions of them, presented to a potential host to induce immunity and prevent disease.
Toxoid- A bacterial toxin that has been modified to be nontoxic but retains the capacity to simulate the formation of antitoxin
73
Percentage of adult level of serum immunoglobulins
Passively transferred maternal IgG peaks just before birth and approaches zero by month 9.
Transient low IgG levels month 3 - year 1.
IgM increases continuously from less than -6 months to 5 years (stabilizes).
IgA increases continuously from -1.5 months to more than 5 years (stabilizes)
74
Passive Cellular Immunity
Descrive LAK therapy and transfer of immune T cells; CAR-T
• Transfer of NK cells or activated NK
cells - LAK therapy
• Transfer of immune T cells - Tumor
infiltrating lymphcytes
– Newest therapies use modified chimeric T
cell receptors to more specifically target
tumors – “chimeric antigen receptor T
cells” – CAR-T
75
Discuss the features of effective vaccines (6).
Safe - vaccine must not itself cause illness of death
Protective - vaccine must protect against illness resulting from exposure to live pathogen
Gives sustained protection - protection against illness must last for several years
Induces neutralizing antibody - Some pathogens (such as polio virus) infect cells that cannot be replaced (e.g. neurons). Neutralizing antibody is essential to prevent infection of such cells
Induces protective T cells - Some pathogens, particularly intracellular, are more effectively health with by cell-mediated responses
Practical considerations - low cost per dose, biological stability, ease of administration, few side-effects.
76
Live, Attenuated vaccine
Why does it work? Discuss.
• Mimic natural infection without disease
• Can be delivered at appropriate site
• Classically done by passaging virus in foreign
host cells or by temperature
• Often work with one administration - develop
good immunological memory and long-term
protection. Major advantage in developing
world
• Now can be done by deletion of virulence
factors from the organism
77
How to produce an attenuated vaccine.
1. Isolate pathogenic virus from patient and grow in human cultured cells.
2. Infect monkey cells with cultured virus
3. While infecting the monkey cells the virus acquires many mutations allowing in to grow well in monkey cells.
4. As a result the virus no longer grows well in human cells (it is attenuated) and can be used as a vaccine.
78
Inactivated Vaccines: Advantages vs Disadvantages
* Typically chemicaly inactivated - formaldehyde treated
* Advantages: Stable; safer than live vaccines; refrigeration not required.
* Disadvantages: Weaker immune response; boosters required
• Salk vs. Sabin polio vaccines
– Why the switch?
• Reversion 1:2.4 million; may spread through water
system
79
Toxoid Vaccines : define toxoid and what's the theory behind its application?
* Disease is caused by a toxin released by the organism
* Give chemically modified toxin - “toxoid”
* E.g. tetanus, diptheria
80
Conjugate Vaccines: Define and discuss why this is done and the special population it may be considered for H. Flu.
- H. Flu (also pneumococcal conjugate)
• Haemophilus influenzae
– Infection problematic in young children
– Antibody to capsular polysaccharides is protective
– Young children respond poorly to polysaccharide
vaccines (T independent response weak; poor memory)
– Creation of polysaccharide-toxin conjugate
enables child to respond
• H flu polysaccharides conjugated with tetanus toxoid,
known to induce strong immune responses in children
Note: E.g we can take the polysaccharide from a bacterium and link it with a toxoid to make a conjugate vaccine.
81
Map out the cascade conjugate vaccine; polysaccharide-toxoid. From initiation to end result.
The polysaccharide component of the conjugate binds to BCR. The BCR-conjugate complex is internalized and degraded (peptide is processed).
B cell then presents processed toxoid peptides via MHC II to Th2 cell. The MHC-antigen binding activated B cell which will differentiate into a plasma cell and produce antibodies against the polysaccharide of the bacteria.
82
Viral vectors: describe how this is done in vitro.
recombinant DNA containing vaccinia promoter and pathogen gene transfects tissue culture cells along with the infection of the vaccinia virus.
The vaccinia virus links with the vaccinia promoter and pathogen gene portion of the recombinant DNA- homologous recombination.
BUdr selection follows. BrdU is commonly used in the detection of proliferating cells in living tissues.
After the selection we are left with recombinant vaccinia vector vaccine.
83
Dangers of vaccination: attenuated vs killed vaccines and provide examples.
Attenuate vaccines: (Current recommendation Salk)
- reversion to wild type; polio types 2 & 3
- severe disease in immunodeficient patients; vaccinia, BCG, measles
- persistent infection; varicella-zoster
- hypersensitivity to viral antigens; measles
- hypersensitivity to egg antigens; measles, mumps, flu
Killed vaccines:
- vaccine not killed; polio accidents in the past
- yeast contaminant; hepatitis B
- contamination with animal viruses; polio
- contamination with endotoxin; pertusis (acellular)
84
How do we know if someone is
| immune? (4) ways and (1) aid.
• Check for antibody - just knowing the
individual is immunized isn’t enough
– IgG vs. IgM - titer
* Measure T cell proliferation to antigen
* Measure CTL responses
* Skin test - e.g. PPD
• Boosters: stimulate memory cells, raise
affinity, raise Ab titer
85
Does Immune Response =
| Immunity? Give an example.
• Not Necessarily!
– E.g. HIV-1 initially induces a good antibody
response, but it is not protective
– HIV-1 vaccines that induce antibody
haven’t been protective
86
What’s New….. For Vaccines (5)
• Flumist - live influenza vaccine, cold
attenuated - better protection and more CTL
* Papilloma virus vaccine - cervical cancer
* Viral vectors - e.g. canarypox
* DNA vaccines - stable at room temperature
• DC vaccines - let the DC choose the
epitopes
– tumors (melanoma, others), infectious disease.
Approved Spring 2010: Dendreon prostate cancer
vaccine. 2014: declared bankruptcy
87
What qualities of vaccines are needed? (4) and public trust.
```
• Safer vaccines
• More effective vaccines
• Cheaper vaccines - fewer whole organisms?
• Stable vaccines - no refrigeration
– DNA vaccines
```
• Public trust - risks (e.g. asthma, autism)
– “…That vaccines given too early are going to cause
asthma, or anything else is like another big DUH! I mean
is ANYONE out there still swallowing whole the story that
vaccines can't be dangerous and cause harm? And of
course I am NOT against vaccines (another DUH), but like
anything, you do them in moderation and I don't care
what the 26 year old smiley faced pediatrician who just
popped out of medical school hearing the "no science to
support vaccines cause harm" mantra says, its not his/her
kid, you have to be CAREFUL with what you do to a baby
under six months…”
88
Can we eradicate more diseases
by vaccination? Smallpox vs. measles
- Herd immunity: the number of people needed to give
immunity to population depends on two things (smallpox vs. measles). Requirements for lifelong immunity and what is the host for these pathogens?
– Smallpox eradicated; goal to eradicate measles by
2010, but it’s still a major killer in developing world
– Relatively low infectivity (small pox) vs. higher infectivity (measles)
• Herd immunity: the number of people needed to give
immunity to population depends on the infectivity of the
virus and the rate of vaccine “takes”; for measles, this
required rate is much higher than for smallpox
– Man is only host for both (no animal reservoir); no
latency
– Immunity lifelong to both but measles requires two
doses
– Compliance: Timing and dosing; Should
vaccination be a choice?
89
Herd Immunity:
No one, some, most of population immunized. Contrast the three situations with healthy/ not healthy non-immunized and healthy immunized
No one: contagious disease spreads through the population.
Some: contagious disease spreads through some of the population
Most: Spread of contagious disease contained.
90
Other Vaccine Strategies: discuss therapeutic vaccination; e.g with tumors vaccines.
• Therapeutic vaccination- enhance host immune response against existing cells
– Tumor vaccines: enhance immunity to existing
tumors. E.g. Dendreon prostate cancer vaccine
– Therapeutic vaccination in infectious disease -
turn on or switch to more protective immunity
• HIV?
91
What are the three biological kingdoms "Tree of Life"?
What makes up prokaryotes?
Discuss two presumed common progenitors
Bacteria, Archaea, Eukarya.
Prokaryotes = Eubacteria + Archea
Common progenitor for all extant organisms
Common progenitor of archaebacteria and eukaryotes
92
Describe the differences between prokaryotes and eukaryotes in terms of intracellular structures, transcription and translation.
Contrast morphological features (5).
“Primitive Nucleus”; No nuclear membrane, no histones, no nucleosomes; Transcription & translation “coupled”
“True Nucleus”; nucleus, mitochondria, histones, nucleosomes; Transcription & translation “uncoupled”
Prokaryotes vs. Eukaryotes (Metazoans- animal that undergoes three tissue type development from embryonic stage)
size: 0.2 -2.0 micrometers dia. vs 10-100 micrometers dia
"nucleoid", no nucleus, no nucleoli vs true nucleus w/ membrane and nucleoli
no membrane-enclosed organelles vs many membrane-enclosed organelles (e.g. lysosomes and chloroplasts)
Cell wall usually present i.e chemically complex and includes peptidoglycan vs normally no cell wall and if present is chemically simple
Plasma membrane usually has no carbs and sterols vs sterols and carbs serving as receptors on plasma membrane
93
Informational knowledge of differences between prokaryotes and eukaryotes.
Prokaryotes: typically haploid, binary fission, no sexual reproduction (meiosis), horizontal gene transfer of DNA fragments, typically single circular dsDNA molecule, very little "non-essential" DNA, carry extrachromosomal DNA (plasmids and episomes), chromosomal condensation by supercoiling and "architectural proteins", transcription and translation are coupled, all "information transactions" are in cytoplasm, genes organized into operons (transcription unit with multiple genes), genes typically do not have introns ("intervening"), little to no post transcriptional modification to proceed to translation, ribosomes are smaller (70S).
Eukaryotes: diploid, mitosis, meiosis, multiple linear dsDNA molecules, genomes have large proportion of "non essential"/ repetitive DNA, little extrachromosomal DNA, chromosomes condensed by histones and confined by nuclear membrane, translation in cytoplasm, transcription and RNA processing in nucleus, genes typically singletons (one gene per transcription unit), genes have introns which need to be spliced and transcript is processed and exported into cytoplasm for translation, 80S ribosomes in cytoplasm, 70S in mitochondria.
94
Discuss the typical set up of bacterial DNA, variations to DNA organization, and extrachromosomal genetic elements; provide examples.
Typical: a single circular double-stranded DNA molecule [E. coli].
Variations: linear dsDNA chromosome [Borrelia burgdorferi (Lyme disease)]; two circular dsDNA chromosomes [Vibrio cholerae; (cholera)]
Extrachromosomal genetic elements: Plasmids, prophages, and transposons--important in pathogenesis
95
There exists Diversity in Bacterial Genomes in terms of bacterial strain, disease/niche, chromosome #, and size.
Just informational
96
Discuss the normal bp/turn of DNA in relaxed state, types of supercoiling, and why supercoiling occurs.
B-DNA ("normal DNA") conformation
10.6 (~10) bp/turn in relaxed (minimal energy) state
Linear ds DNA (Relaxed) 1000 bp / 100 turns
Relaxed circular ds DNA 1000 bp / 100 turns
- negatively supercoiled 1000 bp / 95 turns; “underwound” (required state)
- positively supercoiled; 1000 bp / 105 turns; “overwound”
* The stress of under-winding or over-winding forces supercoiling
note: severity of wound --> +/- # turns with same # bp.
97
What is the action of quinolones and provide two examples. Expression of what type of topoisomerase increase / decrease when?
Quinolones inhibits topoisomerase II (GYRASE). Expression of topoisomerase II increases with underwound DNA.
- Nalidixic Acid
- Ciprofloxacin
Expression of topoisomerase I increases with overwound DNA.
98
Overview of DNA Replication in Bacteria
Discuss replicon and difference between bacteria and eukaryote, direction of replication, and separation of two daughter cells.
1. The typical bacterial replicon: single origin, single termination sequence for entire chromosome
2. Typical eukaryotic replicon: multiple origins (~1 per 100,000 bp of DNA) dispersed throughout each chromosome
Replicon is the unit defined by an origin for replication and a termination site
E. coli chromosome has one* origin (oriC) and a termination site (terC); (eukaryotic chromosome has multiple origins (1 per 100-250 KB)
E. coli DNA replication is bidirectional, i.e., two replication forks start at origin, and move in opposite directions towards terC.
The last step, separation of two daughter chromosomes, requires topoisomerase function (Topo-IV)
99
Bacterial Replication is Carried Out by a Giant Enzymatic Machine
Discuss them (4) categories. DNA Pols and other protein factors.
Replication is Carried Out by a Giant Enzymatic Machine
DNA polymerases
DNA polymerase III holoenzyme (pol III) --a multiprotein complex--the major replication enzyme
DNA pol I: single polypeptide; fills-in gaps in "lagging strand“, and in DNA damage repair
DNA pol II, IV and V: specialized functions under stress conditions
Other protein factors:
Initiation factors, primase, helicases, topoisomerases, ssDNA-binding proteins, ligase...
100
Key Players and Drug Targets at Replication Fork
Currently exploited targets for antibiotics/antivirals: (2)
Potential targets for Antibiotics : (3)
Provide drugs.
Currently exploited targets for antibiotics/antivirals:
- Gyrase (Ciprofloxacin and quinolones)
- dNTP synthesis (trimethoprim, sulfonamide and other folate inhibitors)
Potential targets for Antibiotics :
- DNA polymerase, helicase, primase
101
Variability is required for survival of all organisms, but also leads to disease
Discuss the two major mechanisms generating variability in all organisms.
Evolution absolutely depends on variants
Cancer and inherited diseases
Bacterial pathogenesis, infection control
Two major mechanisms generate variability in all organisms:
1. Mutagenesis: "heritable change in nucleic acid sequence or content of an organism as compared to that of a reference organism called wild type"; basis for variability
2. Recombination: "physical exchange of DNA between two DNA molecules; mechanism for rapid reassortment of variability"
102
Recombination (overview)
Define.
Discus two types.
Definition: Physical exchange of DNA between two DNA molecules. Always involves cutting and joining of DNA.
Two Types of recombination:
Homologous: Requires sequence homology; homology means substantial sequence similarity, not necessarily identity. Also called “classical” or “legitimate” recombination
Non-homologous: Requires no homology. Mediated by transposable genetic elements, or related sequence elements. Also called “illegitimate”, or “site-specific” recombination.
103
Homologous Recombination
Discuss the 2 routes the can be taken.
5' ---> 3' resection, strand invasion by homologous parental DNA & DNA synthesis --> formation of D loop:
1. Second end capture D-loop extension (Double Holiday Junction) ----> resolution ---> Double Stranded Break Repair (crossover)
Homologous recombination: requirement for sequence homology.
i. Cutting and covalent linking of two DNA molecules to create “Holliday Junction”
ii. Branch Migration to expand amount of DNA exchanged
iii. Breaking apart (resolution) of the two recombinant molecules
2. Dissolution --> annealing --> Synthesis-dependent strand annealing (non-crossover)
This mechanism absolutely requires homology. Minimum length of homology for recombination in E. coli is >300 bp
104
Transposable Genetic Elements
Discuss the anatomy of TGE, where TGE can move to, what organisms have TGEs, discuss their mediation of "illegitimate recombination", TGE being replicative vs non-replicative.
Anatomy: one or a few genes flanked by inverted repeat (IR) sequences. A gene specifying a "transposase"; Inverted repeat sequences at ends that define boundaries of the TGE from host DNA
Whole unit can transpose (move) to a different location in the same or different host DNA molecules
Found in all examined organisms, from bacteria to the human
Mediate “illegitimate recombination”: promote cutting and joining of DNA without need for homology
TGEs can be replicative (the original copy stays in place, and a new copy is exported to a new location) or non-replicative (the original copy is cut out from one site and inserted into a second site)
105
Non-replicative vs. Replicative transposition
Cut and paste (non-replicative)
Replicative
With an RNA intermediate
Map out the process
Cut and paste (non-replicative): excision, insertion, repair
Replicative: cleavage, insertion, replication & repair
Sometimes for replicative an RNA intermediate is used: transcription, reverse transcription, cleavage, insertion, repair
106
Genetic Islands (GEI) / Pathogenicity Islands (PAI) - Discuss. What advantage may this offer and comment on the specificity of site integration.
GEI are large segments of DNA acquired from plasmids, phages or other unrelated bacterial genomes. When GEI confer pathogenicity to previously non-pathogenic bacteria, they are also called PAI.
Acquisition by site-specific recombination (integrase action), followed by mutations leading to loss of mobility
Integration sites are tRNA genes because sequences are homologous in all bacteria
Experimentally recognizable as “foreign” by differences in GC content and codon usage compared to adjacent chromosome segments
Acquisition of GEI often confers selective advantage to a new environment
107
Horizontal Gene Transfer:
Discuss the conclusion derived from the Griffith’s Classic Experiment (1928): Transfer of “Pneumococcal” Smooth Phenotype to Rough cells. (4) Treatments
R strain : - protective capsule ; S strain : + protective capsule
Treatment 1: R strain - live
Treatment 2: S strain - die
Treatment 3: heat killed S strain- die
Treatment 4: heat killed S strain + R strain - die (transformation)
Capsule allowed for the evasion from immune response.
108
Horizontal Gene Transfer Continuously Re-shapes Bacterial Genomes Example: Creation of Pathogenic Bacterial Lineages From Commensals
just informational
109
Horizontal Gene Transfer in Bacteria
Compare the results of intra-species transfer vs inter-species (and inter-genus) transfer.
Discuss the 3 major mechanisms of gene transfer.
Major Evolutionary Force in Bacteria:
- intra-species transfer allows for reassortment of traits
- inter-species (and inter-genus) transfer plays an extremely important role in bacterial resistance and virulence
Three major mechanisms
- Conjugation ("sexual reproduction"); mediated by a plasmid (prototype of a conjugative plasmid is the “fertility factor” F in E. coli); conjugation requires cell-cell contact; conjugation is the most efficient means for horizontal gene transfer
- Transduction: bacteriophage-mediated gene transfer
- Transformation: uptake of naked DNA from medium
110
Bacterial Plasmids
Describe the possible structures and three relevant criteria for classification
Structure: typically circular dsDNA; Variants: ssDNA circles; linear dsDNA
Classification: various criteria, but three relevant criteria are:
Mobility: conjugative Vs. non-conjugative plasmids
Pathogenic Significance: “virulence plasmids” encode toxins (e.g., tetanus, anthrax, enerotoxin), adhesins (pili) etc.
Antibiotic Resistance: R Factors (= R Plasmids): bear multiple antibiotic resistance genes contained within transposons embedded within plasmids
Note: virulence and R plasmids can be conjugative or non-conjugative
111
Significance of Conjugative Plasmids In Bacterial Pathogenesis
Discuss 3 categories: efficiency for transferring genes, carrying virulence determinants, and mobility vs mobilization.
MOST EFFICIENT route for transferring genes among bacteria (e.g., F+/F- mating).
CARRY VIRULENCE DETERMINANTS: encode toxins, adhesins and other virulence factors; genes specifying synthesis of antibiotics; genes specifying resistance to antibiotics. In fact, the most common mechanism for resistance is acquisition of “R” plasmids.
MOBILITY Vs. MOBILIZATION: can transfer themselves; can also “mobilize” other resident non-conjugative plasmids, or even the host chromosome (as in Hfr/F-mating).
112
“R” Factors (= “R” Plasmids)
History: Discuss
Features (3)
“R” Factors (= “R” Plasmids)
History: unusual antibiotic-resistant bacteria in hospitals in post-war Japan
Features: Traced to the acquisition of R plasmids (= R factors)
1. Simultaneous resistance to several antibiotics
2. High tolerance to antibiotics
3. Ability to spread across species and genera
113
Anatomy, Derivation and Dispersal of an R Plasmid
Discuss what the R plasmid is consisted of (2) segments. List the antibiotic resistance genes (6)
All R plasmids have transposons; consist of RD, Resistant Determinant, (transposon) and RTF, Resistant Transfer segments (contains Tra genes)
Have multiple clustered antibiotic resistance genes
- Nested insertions of Multiple TGEs create clusters of resistance genes
Conjugation spreads plasmids across species and genera
```
Cm, Chloramphenicol
Sm, Streptomycin
Su, sulfonamide
Ap, Ampicillin
Km, Kanamycin
Nm, Neomycin
```
Note: segment of the plasmid that contains the genes responsible for intercellular transfer. Contains Tra genes "transfer operon genes" some genes necessary for non-sexual transfer of genetic material in both gram-positive and gram-negative bacteria.
114
BACTERIOPHAGES: Discuss the anatomy from top to bottom and what's DNA/protein
Head (contains the protein), Collar, Tail, Long tail fibers, and base plate. Other than inside head everything else is protein.
115
Bacteriophages: Overview
Essential anatomy
Types of phages on the basis of genomic nucleic acid (5)
Types of phages on the basis of life-cycle (3)
Host range
Medical Significance
Bacteriophages: Overview
- Essential anatomy: DNA or RNA genome (not both) wrapped in a protective protein shell
- Types of phages on the basis of genomic nucleic acid:
1. Double-stranded DNA Phages (T4, l)
2. single-stranded DNA phages (M13),
3. RNA phages (MS2, Qb),
- Types of phages on the basis of life-cycle:
1. Lytic or virulent phage: classical virulent life cycle (e.g., phage T4)
2. Lysogenic or Temperate phage: virus has a choice of lytic or lysogenic pathways (e.g., phage l). Most phages (90%) in nature are lysogenic; most bacterial isolates harbor lysogenic phages!
3. Chronic infection (M13)
- Host range: narrow i.e., each virus attacks one or a few host species
- Medical Significance: encode numerous virulence factors including exotoxins (reviewed later in course); can transduce (transfer) host genes
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Plaque Assay: How to determine number of infectious virus particles in a sample
Discuss the process and what each plaques represents and what the agar does for mobility.
"bacterial lawn" confluent cells susceptible to transduction. Add dilute suspension of viruses; after infection, cover layer of cells with agar and incubate.
The plaques (holes in the lawn) represents a cell lysis and the agar doesn't allow for the viruses to move all around the plate. As a result can only infect contiguous cells.
117
Virulent (Lytic) Phage Life Cycle
List the steps (5)
1. Adsorption/injection
2. Expression of viral early proteins
3. Replication of viral DNA (Expression of viral late proteins)
4. Assembly
5. Lysis /release
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Temperate (Lysogenic) Cycle
Define prophage, what does it do inside the host, and what are it's options?
Prophage: dormant intracellular form of a temperate phage
- prophage gets incorporated into host DNA
Lysogen: host cell that harbors a prophage. A lysogen can no longer be infected by the same phage species (immunity). A lysogen can be induced to undergo lytic development and release of progeny phage Lysogeny: the temperate life cycle of a lysogenic
119
Host Restriction of Exogenous DNA
Discuss the purpose of Host restriction-modification (RM) systems. Discuss the mechanism of protection- how is exogenous DNA discriminated.
Host restriction-modification (RM) systems control fate of exogenous DNA (virus, plasmid or naked DNA)
RM consists of a “restriction methylase / restriction endonuclease” pair. The MTase methylates specific short DNA sequences (“restriction sites”; e.g., 5’-GATC) within the host DNA. RE’ase cuts at restriction sites that are not methylated. This ensures that only exogenous DNA is targeted for destruction.
120
Prokaryotic CRISPR/Cas Immunity: Clustered Regularly Interspersed Short Palindromic Repeats. A defense against viruses and plasmids
Discuss how immunization occurs.
Discuss how immunity occurs.
This defense is comparable to which eukaryotic defense system?
Discuss the value of not expressing PAM as the host.
- Immunization: Invading DNA (virus, plasmid or other) is processed into small fragments by Cas proteins, and some fragments are incorporated next to the Leader sequence in a CRISPR Array ( in cells that survive the invasion). CRISPR Array site now has insertion of a novel repeat-spacer unit
- Immunity: Short RNAs deriving from CRISPR Array transcription are used by Cas nuclease to target destruction of incoming (returning) invader DNA. Active Cas is a ribonulceoprotein. When an invader-specific cr-RNA is present, the Cas/crRNA binds to the invading DNA by using sequence homology; the invader DNA is subsequently cleaved and destroyed by Cas nucleases
- The invading nucleic acid has the photo-spacer as before along with PAM.
- The prokaryotic CRISPR/Cas defense targets DNA, as against the eukaryotic antiviral defense RNAi (RNA-interference) which targets RNA
Note : The absence of PAM (Protospacer Adjacent Motif) within CRISPR array prevents autoimmunity.
121
CRISPR/Cas-Based Genetic Engineering Stunningly Simple & Versatile New Tool
Discuss how this new tool can be applicable to future clinical treatment of disease.
By adapting from the CRISPR mechanism we can introduce our own complex of Cas 9 with and a guide RNA sequence to match a particular gene sequence of DNA in our nucleus. The Cas9/guide RNA complex will recognize a PAM site on DNA and cause cleavage/cut on two sides.
Naturally the DNA repair mechanisms in place will shuffle to repair and rejoin breaks but this process is random. The resulting mutation can highlight what a specific gene sequence does normally.
But to be more specific we can add our own DNA sequences to areas that have been cut, thus engineering new expression.
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Horizontal Gene transfer in bacteria
F Plasmid-Mediated Conjugation in E. coli (Known as “sexual reproduction” for historical reasons)
Mark the differences among "male/female" bacteria.
“Male”
F factor present (F+)
Conjugative (“Sex”) pilus
No adsorption site for pilus
Variant forms: F+, Hfr, F'
“Female”
No F factor (F-)
No conjugative pilus
Adsorption site for plus
No variant forms
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Three Types of “Male” Cells
Integration can occur at many loci on the host chromosome in either orientation. Different Hfr strains have F integrated at different sites and orientations. Only one copy of F is integrated into the chromosome.
Precise excision converts Hfr male back to F+. Equilibrium favors F+ (Hfr:F+ ratio is 1:1000)
Imprecise excision (rare event) coverts Hfr male to F'. F' is the F plasmid + some chromosomal DNA.
Note: F plasmid is an “episome” (can exist free, or as integrated form)
124
F Integration Occurs by Homologous Recombination at “Portable Sites of Homology” (TGEs)
Integration can occur at one of many different chromosomal locations (only one integration event per host chromosome) in either orientation on the E. coli chromosome.
125
F+Male/Female Mating
Discuss the mechanism, enzymes involved, and end result.
Sex pilus is a component of “Type IV secretion system” (T4SS). Pilus tip attaches to an adsorption site in F- cell. Pilus contracts to draw cells close, a cell-cell bridge is provided by the T4SS channel at the contact site
A copy of the F factor is transferred to female through a “rolling-circle” replication mechanism. At the point of transference there's DNA Pol (Old Donor), relaxasome (Old Donor), and transferasome (New Donor)
End result: Male remains male, female becomes male
126
Generalized Mechanism of Conjugative Plasmid Transfer Through the Type 4 Secretion System (T4SS)
Discuss the general mechanism of conjugative plasmid transfer (3).
OriT (origin of transfer) is bound by relaxosome assembly which contains the nicking-closing enzyme relaxase, and other proteins. The relaxase nicks and Pol III begins to add nt to the original donor plasmid.
5’-end of nicked strand is covalently bound by relaxase, and is transferred together with relaxosome through the T4SS channel into the recipient cytoplasm
Relaxase re-circularizes transferred ssDNA in recipient, followed by complementary strand synthesis by host replication apparatus to regenerate circular double-stranded plasmid DNA. DNA ligase ligates the host dsDNA
127
Hfr / F- Mating & Host Chromosome Transfer
Discuss the mechanism of transfer, what is usually successfully transferred and why, what is the end result for both donor and recipient, and what is the significance?
Hfr / F- Mating & Host Chromosome Transfer
Transfer Replication begins at OriT of the integrated F DNA
After transfer of a part of F DNA, adjoining chromosomal DNA is transferred (High frequency of recombination)
Bridge is fragile, and in nature, usually breaks off after partial transfer of donor DNA to recipient
End result: Male remains Hfr male; female usually remains female, but acquires a partial copy of male chromosome
Significance: transfer of donor (male) bacterial genes to recipient (female)
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Hfr/F- Mating: Exconjugant vs. Recombinant
Define exconjugant and what occurs as a result? Discuss the unstable intermediate and genetically stable recombinant.
exconjugant - A female bacterial cell that has just been in conjugation with a male and that contains a fragment of male DNA.
The exconjugant has an exogenote (fragment of donor DNA) and an endogenote (the recipient's cell's genome). The exogenote is an unstable intermediate and is converted to a double helix.
The exogenote undergoes double cross over. Some donor DNA is inserted to host genome and some host DNA is lost. This end result is a genetically stable recombinant.
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Transduction: Phage-mediated Gene Transfer
How is this phage-mediate gene transfer accomplished (is it common)?
Rare packaging error during lytic virus cycle when a host chromosomal DNA fragment is packaged instead of phage DNA
1. Phage inserts genetic material into bacterium. In a rare even the phage is packed with donor bacterium DNA. The transducing particle is released and attaches to another bacterium. The donor bacterium DNA is released into the host bacterium. The donor bacterium DNA inserts into the host DNA genome - transducer bacterium.
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Transformation of gram positives: Outline the steps of transformation
1. Host bacterium induces competence genes --> non-specific DNA uptake
2. Intracellular transfer of only one strand, the extracellular stand is degraded.
3. Intracellularly, recombination will occur of the DNA is homologous.
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Transformation: Gram +ve Vs. Gram -ve
compare bacterial competence, uptake of DNA, fate of DNA strands.
Gram Positive
(Streptococci)
Genetic Competence and Competence Factors: transient expression in a fraction of cells at specific growth phase
- Competence is inducible, transient
- Any DNA can be taken up: surface receptors (dedicated transport channels (ports)) non-specific
- Only one strand transferred to cytoplasm; the other degraded/dilution outside cell
Gram negative
(Haemophilus)
- Cell always competent
- Only homologous DNA: receptors for sequence motif; Basis of specificity: 11 bp recognition sequence
- Both strands transferred to periplasmic space, one strand transferred to cytoplasm, the other is degraded inside cell
- Fate: recombine, or get degraded
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Many Fates of Donor DNA After Horizontal Gene Transfer
Discuss the different ways donor DNA is taken up by host bacterium. Before fate of donor DNA what is the transient stage of the host bacterium called?
Discuss the fates of donor DNA inside of host bacterium (4)
Donor DNA can be taken up by host bacterium via conjugation, transduction, or transformation.
Transient stage - "partially diploid" cell if donor DNA is homologous to host DNA.
Stable genetic variants:
- Autonomous replication of donor DNA (e.g. plasmid)
- integration (recombination)
No change in recipient:
- host restriction of CRISPR Immunity (DNA destruction)
- no integration and no autonomous replication of donor.
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Discuss the revised Tree of life and the significance of horizontal gene transfer. What else can happen to cause gene variation?
At the top of the Eukaryotic Domain it remains tree-like and the it is acknowledged that chloroplasts and mitochondria have been acquired from bacteria.
However there are many horizontal linkages among the branches because of horizontal gene transfer among unicellular organisms. thus instead of predicting a single cell at the root it is a common ancestral community of primitive cells.
Genome reduction (deletions)
134
Bacterial insertion sequences (“IS elements”) vs. Transposons
Comment on size and significance. What is unique to the significance of transposons (3).
Size: IS elements are small (
135
Discuss “Captured TGEs”. What is phase variation and give an example of a bacterium that does that.
“Captured TGEs”:
Cellular functions that appear to have evolved from mutated versions of ancient TGEs
a. Salmonella “phase variation”: frequent surface antigen switching by site-specific recombination
136
Features of CRISPR (6)
(a) Clustered Regularly Interspersed Short PalindromicRepeats (CRISPR) occur in one or more chromosomal sites in most bacteria and archaea
(b) Interspersed among the palindromic repeats are short (~40 bp) unique sequences derived from viral/plasmid DNAs which the bacterium has encountered in the past
(c) Each CRISPR array is preceded by a promoter and a Leader sequence.
(d) Genes specifying Cas proteins (whose functions include nuclease activities) are in the vicinity, usually upstream of the arrays
(e) Each CRISPR array is transcribed to produce long “pre-crRNA”, which is processed by Cas proteins into short “crRNAs”
(f) The crRNAs are used as guides for targeting the DNA of a re-invading virus or plasmid
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F'/F- mating (“sexduction”)
Define F'
How is F' formed
Define sexduction and what is the significance of this
i. F’ is an F plasmid carrying a piece of chromosomal DNA
ii. F’ arises from occasional error in excision of integrated form of the F factor. The chromosomal piece carried is the part adjacent to the integration site.
iii. F’/F- mating is identical to F+/F mating, but is called “sexduction” because the snippet of chromosomal DNA is also transferred. Different F’ factors carry different snippets of chromosomal DNA.
iv. Significance: Efficient transfer of multiple transposons and associated antibiotic resistance genes and virulence factors from cell to cell; transfer of chromosomal genes from cell to cell.
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Definitions:
antibiotic, antimicrobial, sterile, aseptic, bactericidal, bacteriostatic.
A. Antibiotic: a natural (produced by a microorganism) or synthetic compound that KILLS or INHIBITS the growth of bacteria.
B. Antimicrobial: a synthetic compound that kills microorganisms (not limited to bacteria).
C. Sterile: an environment in which there are NO LIVING organisms.
D. Aseptic: an environment in which there are NO HARMFUL organisms.
E. Bactericidal : antimicrobial agents that KILL bacteria (unable to reproduce and thus die).
F. Bacteriostatic : antimicrobial agents that INHIBIT bacterial growth. Growth resumes if antibiotic removed)
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Principles of Killing (2)
Methods of achieving sterilization (3)
Disinfection agents (4)
** just list**
II. Principles of Killing
A. Characteristics of exponential inactivation
B. Application to sterilization
III. Methods for achieving sterilization:
A. Heat
B. Chemical gas
C. Radiation
IV. Disinfection agents
A. Detergents and alcohols (important uses for hand disinfection in health care facilities)
B. Low energy radiation
C. Chemicals and oxidizing agents (Triclosan and chlorine bleach)
D. Acids, Bases and filtration
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1900 vs 1997: compare the percentage of all deaths in the US due to infectious disease
1900- 40%
| 1997- 5%
141
The Decline of Infectious Diseases due to (3).
1.Increased hygiene and sanitation
(chlorinated drinking water, sewage treatment etc.)
2. Antibiotics
3. Childhood vaccination programs
142
Discuss the rate of killing during 1st order exponential killing.
During 1st order exponential killing, a fixed proportion of cells is killed per unit time. Can get the rate by examining the slope of line.
It takes the same amount of time to go from 10% to 1%
as it does to go from 100% to 10%
143
FORMS OF STERILIZATION:
AGENT; MECHANISM, APPLICATION (4)
AGENT; MECHANISM; APPLICATION
DRY HEAT 160 ºC (2 H); CARBONIZATION; GLASS, METAL
AUTOCLAVE 121 ºC (15 -20m); PROTEIN DENATURATION;
GLASS, METAL
IONIZING RADIATION X-Rays OR Gamma-Rays (Depends on Material); FREE RADICALS; DISPOSABLE SURGICAL SUPPLIES, HEAT-LABILE MATERIALS, FOOD
GAS (ethylene oxide); ALKYLATING AGENT; HEAT LABILE MATERIALS e.g plastic
144
DISINFECTION AGENTS
AGENT; MECHANISM, APPLICATION (5)
AGENT; MECHANISM, APPLICATION
UV-LIGHT; DNA DAMAGE; SURFACE DECONTAMINATION
INEFFECTIVE FOR STERILIZATION, LOW PENETRATION.
FILTRATION 0.23 microM; PHYSICAL REMOVAL; HEAT-LABILE LIQUIDS, DOES NOT REMOVE VIRUSES
Surface-active Disinfectants; MEMBRANE DISRUPTION;
e. g. CATIONIC QUARTENARY AMMONIUM COMPOUNDS & ANIONIC DETERGENTS
ALCOHOLS; MEMBRANE DISRUPTION; SKIN, CLINICAL THERMOMETERS, [70% is better than 99%]
TRICLOSAN; MEMBRANE DISRUPTION; CONSUMER PRODUCTS
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WHAT SHOULD AN ANTIBIOTIC DO?
1.Target Selectivity “US VS THEM”
Must kill microorganisms without harming patient
i.e High Therapeutic Index (ratio of dose toxic to patient and effective therapeutic dose)
Antibiotic target should be essential for microbial function
but absent/ significantly different in humans.
•LD50
•MIC
•MBC
2. Microbes do not easily acquire resistance
3. Broad spectrum vs. Narrow spectrum
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HOW TO CHOOSE AN ANTIBIOTIC: Two factors (just list)
MIC (Minimal Inhibitory Concentration)
MBC (Minimal Bacteriocidal Concentration) MBC >4X MIC
- MBC is ALWAYS > MIC
```
Turbid tubes (have bacterial growth)
Clear tubes (MIC and MBC)
```
147
MIC Interpretations: Compare in vitro interpretation vs clinical effectiveness
In vitro lab tests do not correspond precisely
to therapeutic results.
MIC is a rough guide for use of a drug.
Identity of the organism contributes to interpretation.
Examples of MIC for ampicillin:
>0.1 g /ml indicates clinical resistance for pneumococci
but 2.0 g/ml indicates clinical sensitivity for E. coli
148
COMBINED ANTIBIOTIC THERAPIES
Always beneficial?
Provide example of beneficial/antagonistic effects
SOMETIMES BENEFICIAL, BUT OTHER TIMES COUNTERPRODUCTIVE
1. BENEFICIAL EFFECTS
(ADDITIVE OR SYNERGISTIC)
e.g. STREPTOMYCIN + PENICILLIN IS SYNERGISTIC
2. ANTAGONISTIC EFFECTS
(SUM IS LESS EFFECTIVE THAN SINGLE ANTIBIOTIC)
e.g. CHLORAMPHENICOL + PENICILLIN
149
MODES OF ANTIBIOTIC ACTION (5) and provide the examples.
1. INHIBITION OF CELL WALL SYNTHESIS
e. g Penicillin, Cephalosporins, Vancomycin
2. INHIBITION OF PROTEIN SYNTHESIS
e. g Tetracyclines, aminoglycosides, macrocodes
3. INHIBITION OF NUCLEIC ACID METABOLISM
e. g Fluroquinoliones, sulfamethoxazole
4. INHIBITION OF BACTERIAL ENZYMES
e. g Fosphomycin, cephalosporins
5. DISRUPTION OF MEMBRANE FUNCTION
e. g Polymixins
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Cell Wall Synthesis Can be Blocked at Several Points (3) and provide examples of antibiotics.
1. CYTOPLASM; Synthesis of cell wall precursors
- FOSPHOMYCIN
- D-CYCLOSERINE
2. CYTOPLASMIC MEMBRANE; Synthesis of new cell wall subunit attached to lipid carrier
- BACITRACIN
3. PERIPLASM/ CELL WALL
Attachment of new wall unit to growing peptidoglycan chain
- Vancomycin
- beta-lactams
151
Bacterial Cell Wall: Peptidoglycan Layer
Define the functions of Transglycosidases and Transpeptidases.
Transglycosidases link the monomers into chains
Transpeptidases crosslink the chains to create a meshwork
152
Cytoplasm: Peptidoglycan synthesis
Describe how fosphomycin disrupts peptidoglycan synthesis (Cell wall synthesis)
Fosfomycin is a PEP analog. It inactivates Phosphoenolpyruvate transferase which attaches Phosphoenolpyruvate to NAG to get NAM.
153
How does Cycloserine disrupt peptidoglycan synthesis (Cell wall synthesis)? Two ways.
Cycloserine is a cyclic analogue of D-alanine. Cycloserine binds the enzymes with greater affinity than D-Ala!
Inhibits:
1. Alanine racemase (L-Alanine to D-Alanine)
2. D-alanyl-D-alanine synthetase (D-Alanine
D-Alanine to D-Alanyl D-Alanine)
Those two enzymes are vital in producing the a.a for the tetra peptide of cross linking the peptidoglycan to create a meshwork.
154
Transport of subunits from the cytoplasm to membrane:
Bacitracin functions how?
Bacitracin binds diphosphate on the lipid that carries peptidoglycan subunits through the membrane.
Prevents reactivation.
Lipid -PP -- (- iP) ---> Lipid -P
PP = diphosphate
155
Cell surface: Cross-linking of peptidoglycan
Which two antibiotics accomplish this and define the mechanism of action.
Vancomycin:
•BLOCKING THE ADDITION OF PEPTIDOGLYCAN
SUBUNITS TO THE GLYCAN CHAIN via inhibition of Transglycosylase
Beta-lactam antibiotics and Vancomycin (mechanisms are different):
•TARGETING THE D-ALA-D-ALA SUBSTRATE
FOR CROSS-LINKING via inhibition of Transpeptidase (penicillin binding proteins)
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Vancomycin
•Used against, effective against, ineffective against, mechanism of action, prevents?
Vancomycin
•Used against:
MRSA (methicillin-resistant S. aureus)
Antibiotic-associated colitis by C. difficile.
•Effective against Gram + bacteria.
•Ineffective against Gram - bacteria
- too big to get through membrane porins.
•Binds to the D-Ala-D-Ala terminus of the membrane-bound
peptidoglycan (mechanism different from Penicillin!).
•Prevents peptidoglycan chain extension.
157
Acquired Resistance to Vancomycin: how?
Vancomycin resistant bacteria alter their peptidoglycan synthesis so that there is no D-Ala-D-Ala intermediate.
Vancomycin no longer binds the growing
peptidoglycan chain strongly.
158
Beta-lactams Antibiotics:
List two antibiotics, which bacteria are they used for, and how do they work.
Penicillins used for:
Staphlococcus
Streptococcus
STDs
Cephalosporins used for:
Broader spectrum
beta- lactase have shared ring structure to D-ala-D-ala and thus are competitive inhibitors of D-ala-D-ala for binding to transpeptidases. Inhibit crosslinking of peptidoglycans.
159
What changes for penicillin to have different permeability
properties, and resistance to b-lactamases? Give examples (4) of different penicillins.
Discuss Cephalosporins in said terms.
Different R groups give derivatives different permeability
properties, and resistance to b-lactamases
Benzyl penicillin
Ampicillin
Methicillin
Piperacillin
Cephalosporins:
Different R groups lead to improved permeability
and. Increased resistance to b-lactamases.
160
Cephalosporins: Increasing spectrum of activity
against Gram - bacteria:
List the names of cephalosporins.
1stGeneration- CEF (AZOLIN)
2ndGeneration- CEF (ACLOR)
3rdGeneration- CEF (TRIAXONE)
( ) - for learning purpose not "AKA"
161
Discuss Bacterial beta lactamases to beta lactam antibiotics
beta lactam ring is essential for activity.
Some bacteria produce enzymes that destroy the
beta lactam ring (beta lactamases).
beta lactamases are specific for particular antibiotics.
beta lactamase inhibitors are designed to inhibit
beta lactamases.
162
How can we improve the results of using penicillins to
| allows treatment of otherwise resistant infections? Provide examples (3)
Combining beta-lactamase inhibitors and penicillins
allows treatment of otherwise resistant infections
Mechanism of action: Competitive Inhibition of beta-lactamases
1. Clavulanicacid
2. Sulbactam
3. Tazobactam
163
Combinations of inhibitor and penicillin are specific
| Optimum inhibition and avoid further induction of ß-lactamase. Provide 3 optimal combinations.
1. Clavulanicacid / Amoxicillin (Augmentin)
2. Sulbactam/ Ampicillin
3. Tazobactam/ Piperacillin
164
INHIBITION OF PROTEIN SYNTHESIS:
What are targets during the inhibition of protein synthesis?
Provide examples of 4 groups of agents and what they act on.
Targets: mRNA + ribosome (30S and 50S) protein
Protein synthesis can be inhibited at several steps.
1. 30S rRNAsubunit:
AMINOGLYCOSIDES
TETRACYCLINES
2. 50S rRNAsubunit:
CHLORAMPHENICOL,
MACROLIDE (ERYTHOMYCIN, AZITHROMYCIN
165
AMINOGLYCOSIDES
Used for what type of infections, provide examples that belong to said group (4), mechanism of action, what type of agent is it, comment on dosing, and how can bacteria be resistant (3)?
Discuss the specialities of Streptomycin.
AMINOGLYCOSIDES
(respiratory tract infections)
e. g Streptomycin, Neomycin, Kanamycin,Gentamicin
* Binds irreversibly to 30S rRNAsubunit of bacterial ribosome
* Inhibits translation initiation (?)
* Bactericidal.
* High doses have adverse effects (low therapeutic index).
* Resistance arises from lack of permeability, modification of the ribosome proteins or modification of critical groups on the aminoglycoside molecule.
Streptomycin interacts with the 30S subunit
- In high concentrations bind irreversibly, blocking initiation and preventing elongation of polypeptide chains
- In lower concentrations leads to translation initiation.
166
TETRACYCLINE
Used to treat what type of infections (give examples (2), effective against a specific state of cells, mechanism of action, how can bacteria be resistant (2)?
- Effective only against rapidly dividing cells
- Used to treat intracellular bacterial infections
(H. pylori, community acquired pneumonia)
- broad spectrum
- Blocks attachment of amino acyl tRNAto mRNA-ribosome complex
- Resistance usually due to reduced transport or plasmid encoded efflux pump
167
CHLORAMPHENICOL
Used for what treatment and why, what's its range of effect, what's specific about its combination to other agents, mechanism of action, what is the most common reason for bacterial resistance?
Bacteriostatic, not used in combination with bacteriocidalantibiotics
Useful in meningitis (H. influenzae) because it accumulates in cerebral-spinal fluid
Broad spectrum
Binds the 23S rRNAand blocks peptidyltransferaseactivity.
Prevents peptide bond formation.
Most common resistance is by induction of chloramphenicol acetyl transferase, encoded on a plasmid.
Acetylation of chloramphenicol prevents ribosome binding
168
ERYTHROMYCIN:
Used for what infections or diseased states, what is characteristic of it's spatial configuration, what has extended range of effect, mechanism of action, resistance due to what? Give other examples of drugs that fall in the macrolide class (2)
ERYTHROMYCIN: Macrolide
(respiratory tract infection, Whooping cough)
14-15 membered ring
Newer macrolides extend the spectrum
Blocks ribosomal translocation.
Generally bacteriostatic
Some resistance due to mutations in ribosomal proteins
Plasmid gene encoding a methylase of the the 23S RNA
-Clarithromycin, Azithromycin
169
Anti-Bacterial Agents: INHIBITION OF NUCLEIC ACID METABOLISM
List the three targets for this method and their effects.
1.Bacterial Topoisomerase/Gyrase
Block DNA Replication
2. RNA Polymerase
Block mRNA synthesis
3. Folate metabolism
Block nucleotide synthesis
170
Topoisomerase/ Gyrase Inhibitors
Name two examples of agents, what type of agents are they, mechanism of action, active against what bacteria, and resistance due to what?
Quinolone: Highly bacteriocidal, broad spectrum, low MICs
Quinolone: NalidixicAcid
Fluoroquinilones: Ciprofloxacin
Inhibit bacterial DNA Topoisomerase and Gyrase
Block DNA replication: Bactericidal
Active against Gram + and Gram – bacteria
Resistance due primarily to mutations in gyrase/topoisomerase
171
Rifampin
Mechanism of action, used in combination to treat ____, resistance due to what?
RNA Polymerase Inhibitor: Inhibits bacterial DNA dependent RNA Polymerase and blocks mRNA synthesis
Tuberculosis combination treatment
Resistance from mutations in RNA polymerase limit its
spectrum of use.
172
Fidaxomicin
Mechanism of action, range of effect, used to treat ____ , discuss the physical structure, and what type of agent is it?
Discuss exactly when Fidaxomicin has its effect during the inhibitory process.
RNA Polymerase Inhibitor: : Fidaxomicin
(newly approved)
Inhibits mRNA synthesis by binding to the DNA-RNAP
complex, prior to transcription initiation
Narrow spectrum, approved for C. difficile treatment
Macrocyclic, 18-membered ring
Bactericidal
Inhibition of bacterial RNAP by fidaxomicin occurs:
following binding of the holoenzyme to the DNA template, BUT prior to development of the open DNA-RNAP complex.
173
What two agents affect Folate metabolism?
Discuss where exactly they have their effects in the cascade:
Dihydropteroatediphosphate+ PABA ---->---->--->---> Nucleotides and Amino Acids
Folate metabolism: Sulfonamides & Trimethoprim act synergistically (alone both bacteriostatic but together may act as bactericidal)
Dihydropteroatediphosphate+ PABA -**--Dihydropterase
synthetase--**--> Dihyropteroic acid -----> Dihydrofolic acid ---***- Dihydrofolate reductase -***--> Tetrahydrofolic acid ---------------> Nucleotides and Amino Acids
**sulfonamides
***trimethoprim
* point of inhibition
174
Which drug is a substrate analog of bacteria specific PABA (para-amino-benzoic-acid)? What is the end result of this drug?
What two agents are also PABA antagonists and used for what type of infections?
Sulfanilamide is a PABA analogue
Reduction in purines, pyrimidines and amino acids
Bacteria-specific PABA ----X----> Folate
p-AMINOSALICYLIC ACID and SULFONES are also PABA antagonists used primarily in therapy of Mycobacteria infections
175
Trimethoprim
What specifically does it inhibit? What is the result of this inhibition and why, how is bacteria resistant?
DHFR inhibition causes a decrease in tetrahydrofolate thus
blocks synthesis of purines, pyrimidines and
amino acids.
Analogue of pteridine portion of folic acid
Resistance acquired by plasmid with eukaryotic type reductase
176
What drug elicits the synergistic effect of sulfanilamide and trimethoprim? Said drug is used to treat what?
BACTRIM to treat Traveler's Diarrhea
177
POLYMIXIN B
Specific for what type of bacteria, what part of the physical structure causes the desired effect and what exactly is the effect (2)?
Specific for Gram –bacteria.
Hydrophobic tail inserts into membranes.
Disrupts membranes and increases permeability.
Cationic-detergent-like effect on Cell surfaces. Low discrimination limits most use to topical applications.
178
Which are bacterocidal/bacteriostatic?
Penicillins, Tetracyclines, Cephalosporins
Aminoglycosides, Vancomycin Sulphonamides, Ciprofloxacin, Chloramphenicol
Bactericidal drugs:
```
Penicillins and Cephalosporins
Vancomycin
Aminoglycosides
Ciprofloxacin
Clindamycin
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Bacteriostatic drugs:
Sulfonamides
Tetracyclines
Chloramphenicol
generally macrocodes
Note : at different concentration ahminoglycosides can be Bactericidal/static
179
Define antibiotic resistance and the difference between innate resistance and acquired resistance.
ANTIBIOTIC RESISTANCE: Bacteria are not inhibited or killed at concentrations achievable after normal dosing.
1.Innate resistance
lack of target or impermeability.
2. Acquired resistance
plasmid based or chromosomal mutation
180
RESISTANCE MECHANISMS
Discuss 5 way that bacteria achieve resistance mechanisms.
RESISTANCE MECHANISMS
1.Antibiotic destruction or modification.
2. Intrinsic resistance due to absence or modification (e.g. D-ala D-ala to D-ala D-lactate of the target.
3. Altered metabolism to bypass antibiotic target.
4. Inability of the antibiotic to reach target.
5. Pumps that remove antibiotic from the cell (efflux pumps).
181
Chromosomally-mediated Resistance:
Describe Mutant Selection and the results.
Define clinical resistance
Chromosomal mutation arises spontaneously and in the presence of a drug will be selected for ---> cross resistance
cross resistance- resistance to a class of structurally related drugs
Rare and limited to a single bacterial species.
clinical resistance = when the max dose to be safe in vivo is no longer effective against the bacteria.
182
Plasmid-mediated Resistance: Discuss the process and the advantage (2) over chromosomally- mediated resistance.
Plasmid-mediated Resistance
Donor + Recipient ----plasmid carries resistance determinants ----> Donor + transconjugant
Spreads more easily:
plasmids can spread faster than the cells divide
Can affect multiple drug classes and cross bacterial species
183
Discuss Plasmid-mediated Resistance on a Transposon
Plasmid-mediated Resistance on a Transposon:
Resistance genes on a transposon can move very efficiently to a chromosomal location or to a plasmid.
Affects multiple drugs classes and cross bacterial species
184
Plasmid-mediated high-level resistance to vancomycin
carried on a plasmid in Enterococcus (Tn1546)
Discuss how this occurs.
Plasmid-mediated high-level resistance to vancomycin
carried on a plasmid in Enterococcus (Tn1546)
H A X: Resistance genes-
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H = reductase (pyruvate lactate)
A = D-ala-D-lactate LIGASE
X = D-D-dipeptidase
```
185
Overuse of antibiotics facilitates emergence of
antibiotic resistance: Growth of bacteria in the presence of an antibiotic selectsfor resistant bacteria, through their ability to continue growing despite the antibiotic.
1954: 2 million pounds of antibiotics produced
2009: 7.3 million pounds for human use
28. 9 million pounds for livestock use
20-50% maybe unnecessary: New FDA guidelines, recommend limiting antibiotics use in farm animals and require a veterinarian’s prescription
Informational
186
Meningococcal disease is fatal how often (%)?
Who should be treated for it and wishing what time span?
Meningococcal disease is serious and is estimated to be fatal about 10% of the time.
Household members and close contacts of disease patients should receive antibiotic treatment.
Treatment should begin within 2 weeks.
187
What is a reflection of the probability of resistance in a specific organism?
When should antibiotics be used?
What do we need instead of antibiotics prophylaxis.
Clinicians need to be aware of resistance patterns where?
The probability of resistance in a specific microorganism is a reflection in part of antibiotic usage patterns.
It is crucial that antibiotics be used only
when there is likely to be a clear clinical benefit
need better (quicker) diagnostic tests to improve time sensitive treatments instead of antibiotical prophylaxis.
Clinicians need to be aware of resistance patterns not only in general but specifically in the area where they practice
188
CDC’s PROGRAM TO ENCOURAGE APPROPRIATE USE
OF ANTIBIOTICS:
Discuss physician education and hand disinfection programs.
1.Physician education:
discouraging bacterial antibiotic prescription when infection is not life threatening and/or likely to be viral
2. Hand disinfection programs:
reduction of healthcare associated cross infection of patients
189
Consequences of using more than one antibiotic
Compare additive to synergistic effects. Define antagonistic effects.
Consequences of using more than one antibiotic
1. Additive effects: If a dose of Drug-A that produces 50% of the maximum response is given concurrently with a dose of Drug-B that produces 50% of the maximum response, then the maximum response is produced. If a dose of Drug-A that produces 25% of the maximum response is combined with a dose of Drug-B that produces 50% of the maximum response, then 75% of the maximum response is produced.
2. Synergistic effects: combined effects of two agonists exceed that predicted by the individual actions of these compounds (i.e., the resulting effect is more than additive).
3. Antagonistic effects: When the combined effects of two drugs is smaller than the effect of each one alone.
190
Define selective toxicity. And distinguish between the two types and their corresponding nuances.
Selective Toxicity
•The ability of a compound to interfere with an essential life process of one organism that is in intimate association with another organism such that the host is not harmed.
•ABSOLUTE: the drug reacts with a target which is unique to the parasite. Does not mean the host cannot be harmed but does have a specific target.
•RELATIVE: the target is shared by both parasite and host.
Can be dose dependent: microbe can have better uptake or affinity for antimicrobial compared to host.
191
Which one is relative/absolute selectivity?
Inhibition of cell wall synthesis
Inhibition of cell membrane synthesis or function
Inhibition of protein synthesis
Inhibition of nucleic acid synthesis or function
Inhibition is absolute. The others are relative.
192
Discuss some issues that can come from chemotherapy relating to the host (5)
Problems in chemotherapy—Host
Sites of poor vascularization promotes an issue of drug getting to site.
* Poor host defenses (granulocytopenia, leukopenia, HIV, cancer chemotherapy, immunosuppressives
* Undrained pus
* Foreign body
* Dead tissue
* Site of infection - prostate is acidic, BBB, gonads
193
Problems in chemotherapy—Drug (6)
* Inappropriate drug --> doc responsibility
* Inadequate dose -> doc responsibility
* Improper route of administration --> mostly pt. not knowing
* Malabsorption --> some diseases affect the absorption of drug
* Accelerated drug excretion or inactivation --> usually is an increased rate of metabolism (inactivation)
* Poor penetration into privileged sites
194
Problems in chemotherapy—Pathogen
•Development of resistance --> already resistant microbe selected for by drug
•Superinfection:
- candidiasis - can occur with Rx, thus we treat the candidiasis as well.
- Clostridium treatment - can disrupt water reabsorption and lead to diarrhea.
* Complex life cycle
* Dual infection
195
Note: dose regimen is also dependent on the actual mechanism of action against organism
Informational
196
Pharmacokinetic-Pharmacodynamic Aspects
Discuss PAE and PALE
PAE (Post antibiotic affect) - there is a post phase in which it takes time for the microbes to recover and being to replicate at the rate when Tx first began.
PALE (Post antibiotic leukocyte effect) - With Tx exposure to leukocytes will have an enhanced effect on the microbe.
197
Determining Dosage of Antibiotics
•PAE:
PALE: Ratio of AUC/MIC or Peak/MIC
determine the percent of time a drug remains above the MIC. This will determine dose and interval of consumption.
198
Mechanisms of Resistance (8)
* Decreased permeability
* Increased metabolism by the parasite
* Increased concentration of critical metabolite
* Alternative metabolic pathway - e.g. parasites
* Increased concentration of inhibited enzyme
* Decreased lethal synthesis - some drugs need a modification to be activated e.g. phosphorylated
* Decreased affinity of target
* Increased efflux - up regulation of efflux pumps.
199
Minimizing Emergence of Resistance (4)
* Use most specific drug for the organism
* Do not underdose
* Use combination of drugs for specified infections (TB, HIV)
* Do not use antimicrobial drugs promiscuously
200
What is the starting compound in the drug activation of sulfanilamide and what is the enzyme that gets the job done!!!!!!!?
Prontonsil ----- azo reductase ---> sulfanilamide + phenyltriamine
201
MOA SULFANILAMIDE. Type of agent/selective toxicity. What is it's site of inhibition? What kind of inhibition is it and overall result on velocity of making folate?
Mechanism of Action
* Sulfas compete with p-aminobenzoicacid in the synthesis of folic acid
* Exhibit absolute selective toxicity
* Bacteriostatic
pteridine + p-aminobenzoic acid ---- *pteroate synthetase------> pteroic acid + glutamic acid ----dihydrofolate synthetase---> DHF ---- **DHFR ----> THF ----> nt (purines and purines), aa
** a step common to mammalian, bacteria, and some protozoans.
- Competitive inhibition, decreases the rate.
202
Mark the atomically differences/similarities of PABA and SULFA. Draw out the structure
both have two oxygen, same amino group, and sulf A has the S instead of C.
203
Structural requirements for Sulfonamide (5)
1. N4 nitrogen must be a free amino group
2. Amino group must be in paraposition
3. Other cyclic ring structures are inactive
4. Substitutions on ring reduce activity
5. Substitutions on N1 nitrogen are permissible, usually heterocyclic aromatic nuclei
heterocyclic aromatic nuclei (a cyclic compound that has atoms of at least two different elements as members of its ring)
Note: increase flux of electrons at the S-N1 increases activity of sulfonamide.
204
Resistance Mechanisms of Sulfonamide
Resistance Mechanisms
* Decreased uptake of sulfonamide (most common)
* Decreased affinity for pteroate synthetase
* Increased concentration of PABA
205
Representative Sulfonamides (1-3 of 8): Comment on properties (solubility, length of action, and corresponding nuances)
Triple Sulfa
Sulfisoxazole
Sulfamethoxazole
1. Triple Sulfa
- Rapidly absorbed,rapidly excreted,short acting
- Single dosage contain-ing equal amounts of each sulfa; - REDUCES INCIDENCE OF CRYSTALLURIA
2. Sulfisoxazole
- Highest urine solubility (pKa is most soluble in pH or urine), short acting
- Most commonly used single sulfa
3. Sulfamethoxazole
- Urine solubility less than sulfisoxazole, intermediate acting
Usually administered as fixed-ratio combination (SYNERGISTIC) with trimethoprim
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Representative Sulfonamides (4-6 of 8)
Discuss the properties and uses.
Sulfacetamide
Silver sulfadiazine
Sulfasalazine
4. Sulfacetamide
- Topical use for trachoma (granular conjunctivitis caused by Chlamydia trachomatis), 30% solutions have a pH of 7.4; at this pH does not irritate eye. Used for neonates to protect against clymydia.
5. Silver sulfadiazine
- Topical use only, elemental silver has antibacterial activity
- Prophylaxis of burn patients, little or no pain
6. Sulfasalazine
- Split by intestinal flora to yield 5-amino-salicylate and sulfapyridine
- Treatment of ulcerative colitis and other inflammatory bowel disease, salicylate has therapeutic value
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Representative Sulfonamides (7-8 of 8)
Discuss properties, nuances, and comments
Sulfadoxine
Sulfadiazine
7. Sulfadoxine + pyrimethamine
- Rapidly absorbed, ultra long half-life (~ 9 days)
- Chloroquine-resistant falciparum malaria. High incidence of dermatitis reactions
8. Sulfadiazine + pyrimethamine
- Rapidly absorbed, intermediate half-life: 18 hrs
- Treatment of toxoplasmosis
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Pharmacokinetics of Sulfonamides
* Well absorbed orally
* Distributed throughout body water, high concentrations in urine; penetrates CSF IN ABSENCE of inflammation; crosses the placenta
* Bound to plasma albumin
* N-acetylated (free amino group N4 is acetylated to be excreted); some metabolites are less water soluble than parent compound; glucuronidated
209
Adverse Effects of Sulfonamides (5).
```
1.Drug allergy
•Rashes
•Eosinophilia
•Drug fever
•Rare: Stevens-Johnson syndrome (necrotizing syndrome where epidermis is eroded, over 25% of body mortality is likely)
```
2.Renal toxicity —crystalluria
3.Kernicterus - perinatal or neonatal : (days - weeks old)
neonates don't have blood brain barrier. Thus bilirubin (highly toxic in brain) may penetrate into CNS attacking the sheaths and neurons. The severity of attack is proportional to time of exposure and amount of bilirubin.
•Displacement of bilirubin from albumin.
4.May displace drugs from albumin binding sites and/or decrease clearance
•Oral anticoagulants
•Uricosuric agents
•Methotrexate
5.Hemolytic anemia in individuals with G-6-PDH deficiency
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Therapeutic uses of Sulfonamides (3).
* Urinary tract infections (E. coli)
* Nocardiosis
* Chlamydial infections including trachoma
Note: Nocardiosis is an infectious disease affecting either the lungs (pulmonary nocardiosis) or the whole body (systemic nocardiosis). It is due to infection by bacterium of the genus Nocardia
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Trimethoprim serves as a competitive inhibitor of which enzyme? What is its selectivity? What population is TMP/Sulf A contraindicated? TMP/Sulf A are not additive they are?
Competitive inhibitor of dihydrofolate reductase.
- not absolutely selective (relatively selective). Affects both host and parasite. There exists a selectivity difference between human host and bacteria/protozoan.
- contraindication for pregnant women to take Sulf A and TMP during pregnancy for the increased demand of folate.
- synergistic (potentiation): due to the fact that you don't need 50/50 to get 100% effect. Instead you would need less than 50% both to get to MIC.
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Advantages of SMZ-TMP (3)
Sulfamethoxazole/Trimethoprim : their half amounts of MIC are equal. This is what you would like to get an effective combinatory synergistic effect.
* Increased potency
* Increased spectrum
* Decreased incidence of resistance
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Adverse effects of SMZ-TMP (5)
* Folate deficient patients may experience megaloblastosis, leukopenia or thrombocytopenia
* pregnancy
* very poor nutrition
* Nausea, vomiting
* Skin reactions (75%); may be serious
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Therapeutic Uses of SMZ-TMP (5)
* Urinary tract infections
* Respiratory and ear infectionsH. influenzaeand Strep. pneumoniae
* Shigellaand salmonella infections
* Pneumocystis jirovecipneumonia
* Toxoplasmosis and Plasmodialinfections
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MOA of Fluorquinolones (4)
Mechanism of Action
* Quinolones inhibit bacterial DNA gyrase(topoisomerase II)
* Inhibit topoisomerase IV in Gram + organisms
* Bactericidal
* Relatively selectively toxiceukaryotic topoisomerase II is 100–1000 times less sensitive
216
MOA of topoisomerase with stabilizes a positive node.
topoisomerase reduces the torsion. cuts and resealing occurs to change direction of segment.
Topoisomerase stabilizes the positive node, cuts the segment, and reseals the break after changing the direction of the DNA segment to get a negative node.
217
Ciprofloxacin, Levofloxacin, Trovafloxacin are examples of what drug type? What are the structural features of this drug (5)?
Fluoroquinolone
Structural Features
•Positions 2, 3 & 4 are inviolate
•F at position 6 confers resistance
•X = C, N
•Halogen on position 8 leads to phototoxicity
•R7 is Nitrogen containing saturated ring
218
Resistance Mechanisms against Fluoroquinolones?
* Point mutation of the A subunit of DNA gyrase decreasing affinity (chromosomal)
* Efflux pump identified in Staph. aureus, Pseudomonas aeruginosa & Mycobacteria
Note: better for host that it's chromosomal because only daughter cells will have the mutation.
219
Pharmacokinetics of Fluoroquinolones (4)
* Orally active
* Widely distributed into tissues including bone; but NOT CNS except for levofloxacin (90% of serum levels)
•Elimination:
–primarily renal; 20% metabolites
–Moxifloxacin: non-renal elimination (fecal elimination)
Half-lives permit once or twice a day dosing
–ciprofloxacin, 3–4 hrs
–levofloxacin, 5 hrs
–moxifloxacin, 10 hrs
*** don't need to know the exact half lives*****
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Adverse Effects of Fluorquinolones (8)
•Gastrointestinal (3–6%):
nausea > abdominal discomfort > vomiting > diarrhea
•CNS (1–4%): (blocks GABA receptor)
headache > dizziness > agitation > insomnia, rarely seizures
* Allergy (0.5–2%): rash or pruritus
* Photosensitivity; pefloxacin
* Arthropathy: damage to cartilage of weight bearing bones, joint pain no evidence of significant effect in children (>1,000 treated)
* Tendinitis (Tendon rupture particularly with concomitant steroid use)
* Crystalluria(particularly at alkaline pH) - acidify urine to avoid this
* QTC prolongation (increased risk of arrhythmia): levofloxacin, moxifloxacin
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Drug Interactions of Fluoroquinolones (2)
•Antacids and mineral supplements reduce oral absorption
Chelate with Mg++, Al+++, Zn+++, Fe+++
Sucralfate contains aluminum ions
•Ciprofloxacin and levofloxacin may interfere with metabolism of theophylline (used in some types of asthma) and warfarin
Note: (the chelate is not absorbable. Allow 2-4 hrs in taking the two substances)
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Therapeutic uses of Fluoroquinolones (
•Urinary tract
- E. coli, K. pneumoniae, Proteusand sensitive strains of Ps. aeruginosarespond well to quinolones (pH and Mg++influence)
* Prostatitis- penetration into the prostate tissue
* STDs- N. gonorrheainfections including PPNG (commonly resistant) Effective in pharyngeal and rectal gonococcal infections
* Gastrointestinal- Effective in Shigella, Salmonellaand coliform infections because of high concentrations in bowel
* Respiratory- Community acquired pneumonia particularly caused by Gram –organisms such as H. influenzaeTrovafloxacin for penicillin resistance Strep.pneumoniaeinfectionsActivity by the oral route is a distinct advantage when treating chronic reinfections in cystic fibrosis
M. tuberculosis: part of the regimen for MDR-TB
Prophylaxis and treatment of pulmonary form of anthrax. Exposed individuals are treated for 60 days for prophylaxis.
•Bone and joints
Effective alternative to parenteral agents (e.g. ahminoglycosides) against Gram –organisms
•Anaerobes
Moxifloxacin has activity
223
Methenamine: The rate of dissociation is slow
3 hrs to reach 90% of equilibrium
224
Decomposition of Methenamine is dependent on what? Comment on this further.
pH dependent.
```
% of theoretical amount
of HCHO released
7.4 0%
6.0 6%
5.0 20%
```
- Essential to maintain urinary pH at 5.5 or less
225
Methnamine Adverse Rxns (5):
1. Gastrointestinal upset is most common
2. High doses (4–8 g/day) may cause bladder irritation
3. Contraindicated in hepatic insufficiency because of ammonia production
4. Organic acids contraindicated in renal insufficiency because of crystalluria
5. Drug Interaction: Do not use with sulfas because formaldehyde reacts with sulfa producing insoluble product
226
Nitrofurantoin is used for what Tx. Discuss what type of agent, the rate of excretion, and the probability of resistance to develop.
Tx for simple uncomplicated UTI. No known teratogenic effects.
```
Bacteriostatic against E. coli
Rapidly excreted (half-life = 20–60 min)
Resistance rarely develops
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227
Nitrofurantoin Adverse Reactions (5)
1. Nausea, vomiting & diarrhea most common
2. Allergy: fever, chills, allergic pneumonitis particularly in the elderly
3. Neurological: vertigo, headache, nystagmusHigh dose—polyneuropathy of motor and sensory nerves
4. Hemolytic anemia in G–6–P DH deficient individuals
5. Colors urine brown
228
Phenazopyridine: In what Tx is it used for, what are its effects, and what's visible defining characteristic of it? When should we discontinue use of Phenazopyridine?
- Urinary tract analgesic; not antimicrobial
- Alleviates dysuria, frequency, urgency and burning
- Colors urine orange-red
Note: only give it for the first couple of days so that it does not interfere with the pain assessment of the infection and whether the antibiotic is working.
229
Fosftomycin MOA.
Mechanism of action:
- inhibitor of pyruval transferase which is important in the assembly of the muramic acid monomer of the peptidoglycan cell wall
230
Fosftomycin: what's the rate of absorption, where is it absorbed, comment on the exertion. What is the approved single dose, what is the most common side effect? In what special population has this been tested on?
* Rapidly absorbed from GI tract and excreted unchanged achieving high concentrations in the urine
* Approved for single dose (3 g) therapy of uncomplicated UTI
* Diarrhea is most common side effect
* Pregnancy category B (Category B- Animal reproduction studies have failed to demonstrate a risk to the fetus and there are no adequate and well-controlled studies in pregnant women.)
231
Cost of regimens for the treatment of UTI. Discuss the equieffectiveness of Fosftomycin compared to
Fosfomycinhas been shown to be equieffective to norfloxacin or nitrofurantoin in eradicating bacteriuria in some studies but not as effective as TMP/SMZ or ciprofloxacin in others.
RegimenCost:
fosfomycin (one dose) $25.25
TMP/SMZ (q.d x 3 d) $7.50
cotrimoxazole (q.d. x 3 d) $0.54
ciprofloxacin (b.i.d. x 3 d) $14.40
232
Beta-lactam antibiotics: Draw 6-amino penicillanic acid
The R group comes is located where and what does it determine? Where is the site of hydrolysis and what agent accomplishes this and what is the result of hydrolysis?
How many substitutions can cephalosporins make?
What is the only current monobactam?
List the Carbapenems available (4) and how many sites for substitution does it have?
R group is off an amino carboxyl from C6 of the beta lactic ring (6-amino penicillanic acid) and confers its stability to acid or b-lactamase hydrolysis
Hydrolysis by acid or b-lactamase inactivates the beta-lactam.
Cephalosporins has two regions for substitution.
-Aztreonam
- 3 posisitions for substitution
1. Imipenem which has formamidine in position R3.
2. Meropenem
3. Doripenem
4. Ertapenem
233
Against what type of culture are beta-lactams effective and what is the MOA?
beta-lactam bind irreversibly to transpeptidase. Has to be a growing culture for this to be effective.
234
Mechanism (applies to all ß-lactam antibiotics) - (5)
1. Growing cultures required
2. Peptidoglycan cell wall producing bacteria
3. Irreversibly inhibit the transpeptidation reaction
4. Osmotic pressure causes the bacterial cell to burst—bactericidal
5. Activate autolysins in some organisms
235
Mechanisms of Resistance (ß-lactam antibiotics) - (4)
* ß-lactamase breaks the ß-lactam ring rendering the ß-lactam inactive. May be chromosomal or plasmid
* Reduced binding to Penicillin Binding Proteins (PBPs); Methicillin Resistant Staph. aureus [MRSA]
* Decreased access to Gram neg. organisms via down-regulation of porins
* Increased efflux pumps found in some Gram neg. organisms
236
Types of Penicillins: (4) Discuss the route of administration, type of bacteria used for
Types of Penicillins
•Penicillin G (IV) (Pen V (GOOD ABSORPTION) is the oral prep)
- meningococci, penicillin-susceptible pneumococci, streptococci, non-ß-lactamase producing staphylococci, Treponema palladium (SYPHILLUS), clostridia, actinomyces
•Acid-stable (BETTER FOR ABSORPTION) or Antistaphylococcal (isoxazolyl) penicillins (methicillin*, ox-, clox-and dicloxacillin)
- Penicillinase producing Staphylococcus aureus
****penicillin G (pen V) and acid stables are MOSTLY FOR GRAM POSITIVE.******
•Extended-spectrum [covers GRAM NEGATIVE] (amoxicillin (ORAL), ampicillin (IV))
- Proteus mirabilis H. influenzae, E. coli, Listeria monocytogenes, salmonella, shigella, Enterococcus faecalis
•Antipseudomonal or ureido penicillins (piperacillin)
- Pseudomonas aeruginosa, Klebsiella spp., Serratia marcescens
237
Penicillin G spectrum of action (3)
1. GRAM + cocci : pneumococcal pneumonia (steptococcus pneumonia is the major cause)
2. Spirochetes: syphilis (single treatment of penicillin is very effective)
3. GRAM - cocci : gonorrhea (silver nitrate used for newborns)
238
Ampicillin spectrum of action (2):
Ampicillin is one of the drugs to use for _____.
- USE FOR meningitis (H. influenza) : GRAM (-) rod
- GRAM (+) bacilli
- GRAM (-) rods
239
Antiseudomonal penicillins spectrum of action or _____.
Spectrum of action (1).
- ureido penicillin
| - PIPERACILLIN, carbenicillin, ticarcillin
240
General Pharmacokinetics of Penicillins
```
route
distribution
____ - dependent
excretion
half life
site of penetration for bacterial meningitis
```
•Orally active: Pen V, amoxicillin, ampicillin, isoxozolyl penicillins (acid stable)
- Intravenous only: piperacillin, pen G, methicillin
•Distribute well - Doesn't cross membranes very well because of they all have acids on 'em
- Most distribution is in extracellular fluid thus not effective for intracellular pathogens e.g. legionella.
* Time-dependent killing (plasma levels should be >MIC for at least 70% of every 24 hrs) : Have long PAE.
* Excreted unchanged by glomerular filtration and active secretion into tubular fluid.
•Half-lives are rather short: between 0.5 to 1 hour
-- Active pump at nephron to get beta-lactams excreted thus have short half lives.
•Penetrate into CNS only in bacterial meningitis (inflammation)
Note: Time-dependent killing as opposed to dose dependent killing - Administer more mg to get maximum effect.
241
Effect of inflammation on penicillin distribution into CNS
There is rapid penetration of penicillin when meninges are inflamed. As the penicillin becomes effective against the bacteria there is less inflammation and thus less penicillin are able to get into CNS.
242
Adverse Effects of Penicillins
```
TI
Possible reactions (5)
Ampicillin specific reaction
Methicillin specific induction
Augmenting specific test result
```
•Remarkably safe drugs (TI are very high 900-1000)
1. Hypersensitivity: rashes, anaphylactic reactions, drug fever. (1-7% are sensitive)
* **Caution if have anaphylactoid reaction because of cross reactivity (when body defenses reacts with one antigen but also able react to another) with other ß-lactams
2. Neurotoxicity: may provoke seizures in high concentrations (interfere with GABA receptor)
3. Diarrhea due to disruption of normal balance of intestinal flora
4. •Platelet dysfunction: Decreased platelet aggregation with ticarcillin
5. Cation toxicity
–Ampicillin: 3.4 meq Na+/g
–Ticarcillin: 5.2 meq Na+/g
–Piperacillin: 1.88 meq Na+/g
* Ampicillin –maculopapular rash which is NOT allergic
* Methicillin induced nephritis
* False positive for urinary glucose (Augmenting®) - Clavulanic acid/ Amoxicillin
Note: Anaphylactoid reactions refer to an identical clinical pattern that is however non-IgE mediated. Certain allergens including drugs can trigger the mast cell cascade directly without involving IgE as the initial mediator.
243
Adjunct medications to Penicillins: Discuss (2).
Adjunct medications
•Probenecid: inhibits the renal tubular secretion of penicillins and can increase the half-life
•ß-lactamase inhibitors: clavulanic acid, sulbactam, tazobactamIn to extend the spectrum of amoxicillin and other penicillins to organisms producing ß-lactamase the inhibitor is added to the compound.
- They are NOT antibacterial; they only inhibit ß-lactamase.
244
General properties of cephalosporins
```
MOA
Mechanism of Resistance
Pharmacokinetics
Adverse effects
Describe the Disulfiram-like effect
```
* Mechanism of action: identical to penicillins
* Mechanism of resistance: same as penicillinsIn general, third generation are more resistant to ß-lactamases.
* Pharmacokinetics: similar to penicillins
•Adverse effects: similar to penicillins
- cefamandole or cefoperazone taken with alcohol produce a Disulfiram-like effect. This is due to inhibition of aldehyde dehydrogenase.
-In the body, alcohol is converted to acetaldehyde, which is then broken down by aldehyde dehydrogenase to get acetate. If the dehydrogenase enzyme is inhibited, acetaldehyde builds up and causes unpleasant effects (hang-over feeling almost immediately after drinking alcohol)
245
Cephalosporins
1st generation: For bone Pen G is substituted for _____.
2nd gen. : Range of effect
3rd gen. : effective against what type of bacteria? Give two examples and comment on the half lives
4th gen. : Range of spectrum for what organisms and give an example of Rx.
```
First Generation (gram (+) cocci, gram (-) rods)
Pen G substitutes Cafazolin (prophylaxis) for bone. Given to avoid infection.
```
```
Second Generation (Cef-Aclor) (gram (+/-) cocci, gram (-) rods)
Similar to first but also activity vs H. influenzae, E. coli, Neisseria spp and others
```
```
Third Generation (gram (-) cocci/rods)
Gram neg. bacilli: N. gonorrhea and N. meningitidis, H. influenzae meningitis,
e.g. ceftriaxone ; Antipseudomonal: ceftazidime
```
Fourth Generation
Wide spectrum vs Gram pos. & neg. organisms
Cefepime
246
Cephalosporins spectrum of action
What is the piece of knowledge when we are not sure of the organism's origin?
if not sure of organism origin do not give cephalosporin e.g. to try and cover Listeria monocytogenes. Give ampicillin instead.
Note: Listeria monocytogenes is a cause of meningitis especially in newborns. During pregnancy stay away from cheese.
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Adverse Effects (2)
CEFAMANDOLE
CEFOPERAZONE
Cefotetan
Cefonicid
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Give an example of synergism with a beta lactam.
- Risk of disulfiram effect (inhibition of acetaldehyde DH and get build up acetaldehyde)
- Hypoprothrombinemia
- aminoglycoside (Depending on their concentration, they act as bacteriostatic or bactericidal agents) with beta-lactam can be a good combination to treat serious infections.
Note: Normally a bacteriocidal/static don't work well together.
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Clinical Uses of 3rd Generation Cephalosporins (3)
* Gonorrhea: ceftriaxone; cefixime (oral)
* Meningitis: due to pneumococci, meningococci &H. influenzaebut not Listeria monocytogenes
* Empiric therapy of sepsis of unknown origin in both immunocompetent and immunocompromised hosts---> may use 3rd gen. cephalosporin or cefepime (4th gen. cephalosporin)
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Carbapenems- they are members of the beta lactam class of antibiotics, which kill bacteria by binding to penicillin-binding proteins and inhibiting cell wall synthesis.
Give examples of them (4)
Effective against what types of bacteria?
Administration/penetration
Adverse effects
* Imipenem, meropenem, doripenem, ertapenem
* Effective vs. gram-positive, gram-negative aerobic and anaerobic cocci and bacilli
* Parenteral only; penetrate fluids well including CSF.
* Adverse effects: NVD, skin rashes, infusion site reactions; imipenem is convulsant at high doses
Note: Carbapenems get into cells. Can penetrate CSF even without the presence of inflammation.
Note: parenteral - Taken into the body or administered in a manner other than through the digestive tract, as by intravenous or intramuscular injection.
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Carbapenem uses
Treatment of _____.
Monotherapy for _______ (7) for patients who can't tolerate?
Use which carbapenem for bacterial meningitis?
* Treatment of hospital-acquired resistant infections
* Monotherapy for septicemia, neutropenic fever, intra-abdominal, lower respiratory tract, genitourinary, gynecological, skin and soft tissue, and skin and bone infections in patients who cannot tolerate a cephalosporin
* Meropenem as an alternative treatment in bacterial meningitis only because you cannot tolerate other drugs for that Tx.
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Monobactam: Aztreonam
-Have beta-lactam ring
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Spectrum
Route
Excretion, penetration
Type of reaction to drug
Advantage to using it
Used as an alternative to _______ (3)
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* Spectrum is like aminoglycosides: gram-negative aerobic bacteria
* Give IM or IV (parenteral)
* Filtered and secreted by kidney; enters CNS without inflammation
* Injection site reactions; e.g. pain
* Advantage: little or no cross allergy with penicillins or cephalosporins
* Used as an alternative to aminoglycosides; penicillins and cephalosporins
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Vancomycin: compared to beta-lactam has a wide/limited spectrum? Comment on the sizeIt is effective against what type of bacteria and give examples (7).
- Limited
•Naturally derived complex tricyclic glycopeptide (MW = 1448 daltons)
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•Effective only against Gram pos. bacteria
–Staph. aureusincluding MRSA
–Staph. epidermidisincluding MRSE
–Strep pneumoniae
–Strep viridian's group
–Entercoccus
–Clostridium species
–Listeria monocytogenes
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Vancomycin Mechanism of Action
Note: the disaccharide unit for Gram (+) comes out of cytoplasm with the lipid carrier, penta-glycine, and pentapeptide. The crosslink is done by the penta-glycine. For gram (-) there is no penta-glycine just the penta-peptides making the connection from 4-3 (5 position peptide always pops off) and is still accompanied by the lipid carrier.
Binds to D-alanine-D-alanine of the pentapeptide of N-acetylmuramic acid. By sheer size and the 3D conformational nature of peptidoglycan Vancomycin disrupts transglycosylation (addition of disaccharide units NAM-NAG) and transpeptidation
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Vancomycin resistance, how?
Van A resistance is caused by an ensemble of 9 genes which are on a transposable element present on a plasmid.
The 5th position pentapeptide is converted from Dala to D lactate
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Vancomycin Pharmacokinetics
route
distribution (location)
What is required of administration if it needs to get to CNS?
excretion
* Must be administered parenterally
* Distributes well into pleural, pericardial, synovial and ascitic fluids.
* Does not enter CNS unless there is inflammation –may require intrathecal administration
* Excreted unchanged in the urine
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Vancomycin adverse effects (4)
* Administer slowly because it causes the release of histamine causing hypotension and erythema of face and upper trunk; histamine vasodilator arterioles and capillaries
* Phlebitis - causes irritation of vein
* Ototoxic - interaction with CN VIII
* Nephrotoxic - dangerous because causes damage to the organ that filters it so upstream accumulation of even more adverse effects!!!
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Vancomycin uses (4). What is used to treat Clostridium difficile pseudomembranous colitis?
* MRSA caused sepsis or endocarditis
* Vancomycin plus gentamicin for enterococcal endocarditis in patient with serious penicillin allergy
* Vancomycin plus cefotaxime or ceftriaxone or rifampin for highly penicillin-resistant pneumococcal meningitis
* Resistant strains of Enterobacter faecalisand faeciumhave emerged.
* No longer drug of choice for Clostridium difficile pseudomembranous colitis; Metronidazole is the drug of choice now
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Streptogramins are a class of antibiotics. Give examples of (2) and give two examples of oxazolidinone class.
Streptogramins are effective in the treatment of vancomycin-resistant Staphylococcus aureus (VRSA) and vancomycin-resistant Enterococcus (VRE), two of the most rapidly growing strains of multidrug-resistant bacteria.
Quinupristin/Dalfopristin - given together
Linezolid, which is a synthetic antibacterial agent of the oxazolidinone class, and cycloserine.
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Why were macrolide once thought to exhibit an irreversible nature?
What is the MOA of macrolide? What does it inhibit?
- Macrolides have a high avidity with a very slow dissociation.
* Binds reversibly to a site (P site) on the 50s ribosomal subunit and causes the dissociation of the peptidyl t-RNA from the ribosome.
* Inhibits the translocation step (movement of peptidyl t-RNA from the acceptor to the donor site).
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Bacterial Resistance to macrolides (3).
•Decreased affinity of binding site because of methylation of adenine nucleotide in ribosomal RNA (23S RNA of the 50s ribosome).
- The methylase may be constitutive or macrolide-inducible
* Decreased penetration or increased efflux
* Enzymatic inactivation of macrolide by an esterase
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What are macrolides advantageous for (spectrum)?
Comment specifically on the three macrolides.
Intracellular organisms. It has a lactone rings.
Erythromycin
–Gram positive streptococci, staphylococci and corynebacteria (diphtheria). Intracellular organisms such as, Mycoplasma, Legionella, Chlamydia spp. and certain atypical mycobacteria
•Clarithromycin
–Similar to erythromycin and active vs. H. influenza
•Azithromycin
–Less active vs streptococci and staphylococci than erythromycin. Preferred treatment for C. trachomatis–single dose of 1 gram
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In the treatment of syphilis if pt cannot tolerate penicillin what is given? What are some infections macrolides can treat (5)?
Erythromycin.
-Chlamydial infections, Mycoplasmal pneumonia, Syphilis, Corynebacterium Diphtheriae, Legionnaire's Disease.
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Pharmacokinetics of macrolides.
absorption
How does Erythromycin have to be prepared?
Distribution, which macrolides distributes the best
Where does it not penetrate?
* Well absorbed orally. Erythromycin is given as esters or in enteric coated tablet to protect against stomach acid
* Well distributed with high intracellular concentrations (azithro the highest); do not penetrate the CNS
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Metabolism & Excretion of macrolides
* Erythromycin: extensively metabolized by CYP 450 dependent enzymes
* Clarithromycin has an active metabolite, 14-hydroxyclarithromycin
* Excreted in urine and bile with biliary excretion being most important
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Adverse Effects of macrolides
Discuss ea. macrolide (3)
•Erythromycin
–Epigastric distress —stimulates the gastric motilin receptor
–Cholestatic jaundice (estate - not given anymore)
–Ototoxicity –transient at high doses
–Drug interactions –inhibits the metabolism of many drugs
including (warfarin, cyclosporine and theophylline; digoxin)***
*** These drugs all involved in CP450 metabolism and have very narrow TI, any change in the rate of metabolism the higher the risk of seeing adverse effects since Erythromycin also uses CP450 for metabolism***
* Clarithromycin –similar to erythromycin
* Azithromycin –does not inhibit P450 dependent drug metabolism
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Clindamycin
Class of antibiotic
MOA
Type of agent
* Lincosamidine antibiotic
* Binds to 50s ribosomal subunit and interferes with the peptidyl transferase reaction
* Bacteriostatic
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Resistance Mechanisms of Clindamycin (3)
* Decreased penetrability
* Decreased affinity to binding site of 50s subunit (like macrolides)
* Enzymatic inactivation by an O-nucleotidyl transferase (Staph. aureus)
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Clindamycin Spectrum (4)
* Streptococci
* Anaerobes including Bacteroides, Prevotella, Porphyromonasand other oral anaerobes
* Toxoplasmosis in combination with pyrimethamine
* Topically for rosacea and acne
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Clindamycin pharmacokinetics
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Bioavailability %
Distribution
Where does it accumulate (similar to another class of Rx)
Penetration specialty
Metabolism
Excretion
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* 90% bioavailability (great oral absorption)
* Distributed well into bone (used by dentists for teeth) and body fluids except CNS
* Accumulates (like macrolides) in macrophages and leukocytes
* Crosses the placenta
* >90% hepatic metabolism via oxidative demethylation to an ACTIVE METABOLITE - HOWEVER MOST DOESN'T GET BACK INTO CIRCULATION
* Biliary excretion
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Therapeutic Uses (4) Clindamycin
* Refractory bone infections - an infection of bone which has persisted or recurred after an intial course of treatment for osteomyelitis.
* Treatment of anaerobic infections of female genital tract, pelvic infections and abdominal penetrating wounds
* As a substitute for amoxicillin or pen V for acute orofacial infections in situations where the organisms are penicillinase producing
* As an alternative to penicillin-sensitive patients requiring prophylaxis to prevent endocarditis
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Adverse Effects of Clindamycin (4)
* NVD
* Hypersensitivity –rashes, fever
* Risk of Clostridium difficilesuperinfection (pseudomembranous colitis)
* Neuromuscular blockade at high IV doses
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Protein Synthesis Inhibitors (2). Just list.
- STREPTOGRAMINS
| - OXAZOLIDINONES
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Streptogramins : What is the name of the synergist duo?
- Synercid®
Quinupristin (Group B: hexadepsipeptides) / dalfopristin (Group A: macrolactones)
Quinupristin / Dalfopristin
30 : 70
Note : alone static, combined --> cidal
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Mechanism of Action of Streptogramins
•Bind to 50s ribosomal subunit
–Dalfopristin (Group A) inactivates both the donor and acceptor sites of peptidyltransferase, and Induces a conformational change to increase the binding of the Group B streptogramin
–Quinupristin (Group B) prevents the translocation step
•Individually they are bacteriostatic, together they are bactericidal
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Mechanisms of Resistance of Streptogramins (3)
* Methylation of 23S RNA binding site prevents binding of quinupristin
* Bacterial enzymatic inactivation; particularly dalfopristin
* Increased efflux
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Pharmacokinetics of Streptogramins
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route
penetration of cells
metabolism
excretion
half lives
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* Must be given IV
* Penetrates macrophages and PMNs
* Both undergo extensive non-enzymatic conversion to non-active products
* Excretion 80% bile; 20% urine
•Short half-lives:
Quinupristin 0.85 hr
Dalfopristin 0.7 hr
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Adverse Effects of Streptogramins (4). Careful to monitor which drug when given in combination?
* Injection related (40%): pain, inflammation, edema, injection reactions (incompatible with saline)
* NVD, headache
* Myalgia, arthralgia
* Marked drug interactions because of CYP3A4 inhibition, e.g. cyclosporine levels should be monitored
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Streptogramin Use (2)
1. Treatment of vancomycin resistant Enterococcus faecium(not Enterococcus faecalis)
2. Complicated infections of skin and skin structures caused by Staphylococcus aureus(methicillin sensitive)
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Linezolid (synthetic) to what?
Linezolid mechanism of action
Resistance - specific strain of bacteria.
Oxazolidinone
* Binds to site on the 50S subunit similar to chloramphenicol and prevents the formation of the 70S initiation complex
* Resistant strains of S. aureus bind less antibiotic but no cross-resistance with other antibiotics
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Linezolid spectrum of action
* Similar to vancomycin and gram-positive anaerobes
* Approved for nosocomial pneumonia, community-acquired pneumonia, skin infections, vancomycin resistant enterococcal infections, MRSA
Gram (+) cocci
Gram (-) bacilli
Clostridium perfringes
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Pharmacokinetics of Linezolid
route, bioavailability
Distribution
metabolism
excretion (% to specific organs)
•Orally active: 100% bioavailability
- also IV preparations
* Distributed to well-perfused tissues
* Metabolized to 2 inactive metabolites non-enzymatically
* 30% renal; 65% biliary
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Adverse Effects of Linezolid (5)
* GI disturbances (NVD), tongue discoloration
* Hematologic: anemia, thrombocytopenia, neutropenia—requires weekly monitoring
* Headache
* Rashes
* Pseudomembranous colitis
