Learning Objectives for Disease and Defense
Week 1
Absorption and Distribution
1. Identify the factors that determine a given drug’s ability to cross biological membranes.
a. Molecular size: smaller MW drugs will be absorbed more readily, size also affected by the drug binding
to plasma protein (increases size, decreasing absorption)
b. Lipid solubility: increased lipid solubility leads to increased absorption (drug can easily cross lipid bilayer
of membranes), estimated by oil:water partition coefficient
c. Degree of ionization: affected by pH, will influence lipid solubility (more unionized=more lipid
soluble=increased absorption), requires H-H equation
d. Concentration gradient: high concentration created at site of drug administration, drug will move from
[high] to [low].
2. Describe the mechanisms by which drugs cross biological membranes (diffusion, transport, etc.).
a. Passive diffusion
i. MOST IMPORTANT ROUTE, driven by concentration gradient
ii. Aqueous diffusion/filtration (drug flows through aqueous channel): limited capacity, channel
size varies (generally for drugs of MW less than 100-200)
iii. Lipid diffusion (drugs pass via hydrophobic bonding with membrane lipids): favored if drug has
high lipid:water partition coefficient, often pH dependent, unionized moiety crosses membrane
down concentration gradient, most important mechanism for drugs with MW of 500-800.
b. Carrier mediated diffusion
i. Facilitated diffusion: driven by concentration gradient (no energy required)
ii. Active transport: energy dependent, selective, saturable, unidirection, for durgs which resemble
endogenous compound. Many cells also contain less selective membrane transporters that are
specialized in expelling foreign molecules (ie: P-glycoprotein). Drugs are inhibitors of these
transporters can be involved in certain drug-drug interactions via alteration of substrate drug
levels in tissues.
c. Endocytosis
i. Of minor importance to drug passage, pinocytosis or phagocytosis
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3. Summarize the therapeutic advantages and disadvantages of the various routes of drug administration,
especially with regards to bioavailability and rate of onset of effect.
ROUTE
BIOAVAILABI RATE
OTHER FACTORS
LITY
Enteral (GI
Oral
0-100%:
Slow (15-30 Most common, drug absorption occurs via passive
tract)
depends on
min for
diffusion (favors lipophilic/nonionized drugs), rate of
survival in GI immediate
absorption of drug from intestine ˃stomach (because of
environment, release)
large SA of intestine), increased GI motility and empty
ability to
stomach=increased absorption, duration: min-hrs
cross GI
Slower
Enteric drug coat: protects stomach from irritation and
membrane,
(hours) for
protects drugs from low stomach pH
efficiency of
enteric/sust Controlled-release prep: rate of absorption is slowed by
drug
ained
slowing rate of product dissolution (allows for fewers
metabolism
release)
administrations, increased compliance, overnight
by gut wall or
therapy, elimination of peaks/troughts, BUT greater
st
liver (1
interpatient variability and formulation could fail giving
pass)
pt entire dose “dose dumping”
Enteral (GI
Rectal
Variable, but Not rapid
Useful with oral route is unavailable (vomiting,
tract)
generally ˃
unconscious, post-GI surg, uncooperative pt), 50% of
oral
dose will BYPASS liver (first pass metabolism is ˂ for oral,
absorption is irregular/incomplete
Parenteral
Sublingual
Generally
Within
Absorbed from mouthSuperior vena cava (protects
(Outside GI) Buccal
high
minutes (5- drugs from hepatic 1st metabolism + faster onset), useful
10 min)
for drugs that are lipid soluble and relatively potent
(˂1mg dose) as there is smaller SA for absorption
relative to GI tract
Parenteral
Intravenous
100%
MOST Rapid Most direct route, circumvent all factors related to
(Sec-min)
membrane passage/absorption, accurate and fast drug
delivery, used for drugs with narrow therapeutic
window, requires aseptic technique, most haxardous
route b/c easy to reach irreversible toxic levels quickly,
duration= t1/2-dep
Parenteral
Intramuscular
Approaches
Aqueous
Absorption/onset effected by blood flow/muscle activity
2
100%
soln-rapid
(5-10 min),
slower in
depot form
5-10 min,
Slower,
constant
rate for
depot
(hours)
Rapid (˂5
min for
gaseous)Ne
gligible
Parenteral
Subcutaneous
Approaches
100%
Parenteral
Inhalation
Gaseous:
Gas/volatile
liquids
OR
Suspension:
Aerosol or
microparticles
Variable100%
a. about
100%
b. variable
Parenteral
Transdermal
at site of injection, depot form in oil or suspended=
slower/sustained absorption, used if drug is too irritating
for subQ, absorption may be erratic/incomplete with
solubility is limited, disadvantage: pain, tissue necrosis
(if high pH), microbial contamination
Only for non-irritating drugs, volume of dose is limiting,
period of drug absorption can be altered via particle
size, protein complexation, pH (insulin), addition of
vasoconstrictor (local anesthetics), pellet implantation
(contraceptives)
Gas: used for rapid onset of systemic drug effects
(nicotine, crack, general anesthetics), rapid rate of
absorption due to large SA and high blood flow in
pulmonary tissue
Particle: applied at site of action in lung, increases local
topical effects (reduces systemic effects), exampleasthma, depends on particle size
< 0.5 M: exhaled from lungs  no effect
1-5 M: deposited in small airways  therapeutic
> 10 M: deposited in oropharynx  side effects
Irritant drug may induce bronchospasms
Slow (hours)
Topical
Apply patch to skin for tx of systemic conditions,
prolonged drug levels attained, 1st pass metabolism is
avoided, drug must be potent (dose ˂2 mg), must
permeate skin w/o irritation, examples: estrogens,
testosterone, fentanyl, nicotine, nitroglycerin
Localized application via skin/mucous membrane
(vaginal, nasal, eye) for tx of local conditions, minimal
systemic absorption, in children potential for 3-fold
greater system availability that in adult (body SA: weight
is greater)
4. Explain the influence of pH on the ionization of weak acid / weak base drugs.
a. Most drugs are either weak acids or weak bases, therefore they are present in biological fluids are
ionized or non-ionized species.
b. Non-ionized forms are more readily absorbed. Ionized forms DO NOT cross lipid membranes.
c. Weak Acids:
i. HA (or R-COOH) ↔ H+ + A- (R-COO-)
ii. R-COOH is the protonated/non-ionized form of the acid and can cross biological membranes (in
acidic environment)
iii. R-COO- is the un-protonated form of the acid and will be “ion-trapped”
d. Weak Bases:
i. BH+ (R-NH3+) ↔ H+ + B (R-NH2)
ii. R-NH3+ is the protonated form of the base and will be “ion-trapped”
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iii. R-NH2 is the un-protonated/non-ionized form of the base and will cross biological membranes
(in basic environment)
5. Be able to use the Henderson-Hasselbach equation to qualitatively predict the ratio of ionized to unionized
species of a weak acid or weak base drug in various body compartments.
a. HH equation: determines the extent of ionization of an acid or a base (dependent on the strength of the
acid or base (pka) and the pH of the body fluid). Allows quantitation of the fraction of the total amount
of drug that is ionized or unionized and allows predictions of a pH that at which the majority of the drug
will be non-ionized and thus will be absorbed.
b. pH-pKa= log (non-protonated: A- or B)/ (protonated: HA or BH+)
i. pH= pH of the biological compartment the drug is in
ii. pKa= pH of a solution at which concentrations of the protonated and unprotonated forms of the
drug are equal.
c. If pH is lower than pKa (lots of protons): protonated form of weak acid (unionized-lipophilic) or weak
base (ionized) will predominate
d. If pH is higher than pKa (fewer protons): unprotonated form of weak acid (ionized) or weak base
(unionized) will predominate.
e. “Ion Trapping”: lipid barriers may separate two aqueous solutions with different pH’s. Only non-ionized
drugs can diffuse through membrane and this form of the drug will equilibrate and be the same on both
sides of the membrane. At equilibrium, un-ionized concentration of drug is the same on both sides of
the membrane, but total concentration of drug is greater on the side where ionization is greater.
i. Acidic drugs will be trapped in BASIC solutions. Basic drugs will be trapped in ACIDIC solutions.
They are trapped where they are predominantly ionized.
ii. Clinical significance: altering urinary pH to ion trap weak acids or bases and hasten renal
excretion (in aspirin overdose situations), greater potential to concentrate basic drugs in more
acidic breast milk
6. Explain the therapeutic consequences of anatomic “barriers” to distribution and selective accumulation of
drugs.
a. Tissues with tight junctions between cells (GI mucosa, BBB, placenta, renal tubules), require that drugs
pass through lipid membranes to or from this compartment and into or out of the blood. Drugs that
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can’t pass through membranes (large size, protein bound, highly charged, high water solubility) will be
UNABLE to move between these compartments and blood.
i. GI mucosa: negligible absorption of drug into blood in administered orally
ii. BBB/placenta: limited distribution of drug from blood into brain or into fetal circulation, due to
structural differences between brain and non-brain capillaries
i. Renal tubules: following filtration at
glomerulus (note that large or protein
bound drugs are NOT filtered), reduced
reabsorption of drug back into blood,
thus enhancing excretion via urine.
ii. Selective accumulation of certain drugs
may occurs in specific tissues and can be
harmful or beneficial (kidney, eye, lung,
bone, ear)
7. Describe how drug binding to plasma proteins can effect drug distribution and elimination as well as be a
potential source of drug-drug interactions.
a. Will influence distribution as only FREE DRUG is diffusible.
b. Acidic dugs bind to albumin and basic drugs bind to alpha-1 acid glycoprotein
c. As drug binding to protein increases:
i. Decreased concentration of free drug (can limit fetal exposure to drug)
ii. Decreased metabolic degradation and rate of excretion (will decrease elimination rate and
increase half-life), acis as circulating drug reservoir that can prolong drug action
iii. Decreased volume of distribution by enhancing apparent solubility in blood (because only free
drug can get out)
iv. Decreased ability to enter CNS across BBB (because only free drug enter brain more readily)
d. Mediates protein binding/displacement drug-drug interactions
i. Displacement of 1st drug from protein binding site by 2nd drug results in increased levels of
unbound 1st drug, but levels of total drug are unchanged because administration is unchanged.
e. Unlikely to be of clinical significance unless the displaced drug has narrow therapeutic index, displacing
drug is started in high doses, Vd of displaced drug is small, or response to drug occurs more rapidly than
redistribution.
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8. Explain the derivation and clinical relevance of the following pharmacokinetic parameters. Describe their use in
designing dosage regimens:
a. Bioavailability (F): Adjustment of dose for oral vs parenteral administration.
i. Bioavailability (F or f[%]): fraction of unchanged drug reaching the systemic circulation,
determined by comparing AUC following single dose of drug given by any route to the AUC
following single dose by IV route.
ii. F = Fraction Bioavailable = AUCORAL / AUCIVA
iii. Information on extent of
absorption (bioavailability) by
ORAL route is available for most
drugs. You can use this for
dosage adjustments when drug
is given by a different route
(common to switch from oral to
IV).
iv. F= 100% for IV, F=0-100% for
ORAL, F= about 100% for other
system drug action routes
b. Volume of distribution (Vd): Converting drug dose to plasma concentration, selecting loading dose,
implications of high or low values.
i. Vd: size of compartment necessary to account for total amount of drug in the body if it were
present throughout body at same concentrations found in plasma. It is the volume of the body
fluids into which the drug distributes following administration. Gives indication of the extent to
which a drug passes from plasma to extravascular tissues
ii. High values of Vd indicate drugs located mostly outside of plasma (increased tissue binding, high
lipid solubility)
iii. Low values of Vd indicate drugs located mostly inside the plasma or ECF (extensive binding to
plasma proteins or large size)
iv. Vd varies between patients due to: Body Weight, Fat vs. Lean and Changes in Protein Binding
v. Vd allows determination of the necessary single dose of drug (loading dose) to fill the
distribution volume with enough drug to achieve desired steady state level (Cp)
vi. Vd also allows prescriber to determine effect of any given dose (D) will have on the plasma
concentration
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Examples:
a.
b.
c.
d.
1. Loading Dose (LD)= Cp (desired) x Vd
2. Cp= Dose/Vd
3. Vd (L)= DOSE (mg)/concentration of drug in plasma (mg/L)
4. DOSE (mg)= concentration of drug in plasma (mg/L) x Vd (L)
5. Concentration of drug in plasma (mg/L)= DOSE (mg)/Vd (L)
Drug A, Dose (mg)= 1000, Cp0 (mg/L)= 333
i. Vd (L)=3, most drug stays in plasma
Drug B, Dose (mg)=1000, Cp0 (mg/L)= 66
i. Vd (L)=15
Drug C, Dose (mg)=1000, Cp0 (mg/L)=25
i. Vd(L)= 40
Drug D, Dose (mg)=1000, Cp0 (mg/L)= 2
i. Vd(L)= 500, most drug leaves plasma
Metabolism and Excretion
1. Describe the general principles and consequences of drug metabolism.
a. Drug metabolism: drugs undergo enzyme-catalyzed chemical structure transformation after
administration to the patient (if only terminated only by renal excretion, the duration of action would be
prolonged)
b. Drug metabolizing enzymes have endogenous substrates and play a role in normal metabolism
c. Liver is primary site of drug metabolism, but lungs (30%), intestines (6%), kidney (8%), skin (1%),
placenta (5%) and bacteria have enzymes capable of drug metabolism
d. Oxidation is most common pathway, but other types of chemical transofmration can occur. Many
transformation are catalyzed by membrane-bound enzymes of the SER called CYP450
e. Lipid-soluble compounds are generally converted to more H20-soluble (more polar) compounds that are
more readily excreted
f. Generally, metabolism is detoxifying process (active drug to inactive or less active compound), but can
also metabolize active drug into MORE active compound (ie: codeine morphine), metabolize inactive
compound into active ingredient, metabolize to toxic metabolite
2. Describe the general characteristics of Phase I (oxidation [CYP450 and non-CYPP450 (non-microsomal)],
reduction, hydrolysis) and Phase II reactions (conjugations: glucuronide, sulfate, glycine, glutathione) as related to:
Qualitative and quantitative role in drug metabolism
Classifications of reactions [ex., O-dealkylation is phase I oxidation (P450)]
PHASE I: inserts or unmasks a
functional group on the drug that
renders molecule more water-soluble
and the molecule can then undergo
conjugation in Phase II rxn.
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PHASE II: endogenous substrate
combines with pre-existing or
metabolically inserted functional
group on the drug forming a highly
polar (water soluble) conjugate that is
Reactions
Enzymes involved
Genetic Polymorphisms
General developmental patterns of
activity and age-related changes in
activity
Inhibitory/Inducibility
Relative ease of saturability at high
drug substrate levels
1. Oxidation—P450 dependent or
P450 independent (most common)
2. Reductions (azo, nitro, carbonyl
reductions)
3. Hydrolysis
1. CYP450 (includes NADPH,
flavoprotein NADPH-cytochrome P450
reductase, and O2) or non-CYP450
2. Reductase
3. Esterases or amidases
YES
Examples: Amplichip test available to
detect polymorphisms in CYP2D6 /
2C19
Lab tests available to detect genetic
variation in anticoagulant response.
Warfarin metabolizing enzyme:
CYP2C9. Warfarin target enzyme:
vitamin K reductase [VKORC1]
YES (decreases with age in 1/3 of pts)
YES/YES
Minimal
excreted via the urine.
Conjugations:
1. Glucuronidation
2. N-acetylation
3. Glutathione conjugation
4. Sulfate conjugation
1. Transferases (ie: glucuronyl
transferases, N-acetyltransferases)
Yes (less)
Yes (especially UGT)
YES (less)
Substantial Limited supply of
reactants renders Phase II reactions
more easily saturable (become zero
order elimination kinetics) than phase
I reaction
3. Explain the therapeutic consequences of induction and inhibition of metabolism. List the clinically relevant
inhibitors and inducers on page 8 of the drug metabolism notes.
a. Induction: increased drug metabolizing activity (increased clearance) via stimulation of the CYP450 system,
compound that causes induction is an “inducer”, mechanism is often increased synthesis of enzyme protein
accompanied with increase in liver weight, proliferation of SER, increases in NADPH and cytochrome P450.
Requires 48-72 hours to see onset of effect (slow compared to inhibition).
a. Therapeutic consequences: maximal effects seen in 7-10 days, production of pharmacokinetic
tolerance (induction by a drug of its own metabolism), induction by 1 agent may increase clearance
of another drug reduced therapeutic effect (via increased elimination) or increased toxicity (via
toxic metabolite)
b. Inhibition: decrease clearance of drug by inhibiting drug metabolizing activity, phase I enzymes are more
prone to inhibition compared to Phase II enzymes. Mechanisms: inhibit enzyme synthesis, inhibitor can act
as a substrate competing for the enzyme, inhibitor can be an ihbitor without being a substrate, inhibition
results from formation of metabolite that destroys enzyme via covalent bonding or form tight complex with
enzymes inhibiting its activity.
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a. Therapeutic consequences: inhibition of metabolism can occur as soon as sufficient hepatic
concentration is reached (within hours), inhibition by 1 agent of the metabolism of another can
result in decreased clearance of the inhibited drug increased toxicity.
INDUCERS (increased clearance)
INHIBITORS (decreased clearance)
Phenobarbital [1A2, 2C9, 2C19, 3A4]
Cimetidine [2D6, 3A4, 1A2]
Phenytoin [2C9, 2C19, 3A4]
Erythromycin / Clarithromycin [3A4]
Carbamazepine [2C9, 2C19, 3A4]
Azole antifungals [3A4]
Rifampin [1A2, 2C9, 2C18, 3A4]
Fluoxetine (other SSRIs) [2D6,3A4]
Ethanol [2E1]
Grapefruit juice [3A4]
St. John’s Wort [3A4]
HIV protease inhibitors [3A4]
Tobacco smoke (not nicotine) [1A2]
Omeprazole [2C19]
4. Describe the general characteristics of drug excretion by the kidney (filtration, secretion, reabsorption and the
influence of pH and protein-binding on these processes).
A. Excretion: loss of chemically UNCHANGED drug from the body. Kidneys are MOST important organ for
excretion (Especially for water-soluble and non-volatile compounds).
B. Filtration: glomerular, rate of 120ml/min, all drugs smaller than albumin (MW=69000) will be filtered, only
free drug is filtered (NOT protein bound), renal excretion affected by renal blood flow and renal function, drugs
cleared by this route have t1/2= 1-4 hours (but high protein binding can extend half-life)
C. Secretion: active tubule secretion, drugs transported from blood to urine, rate of 120-600 ml/min, occurs with
drugs that are stronger acids and bases in proximal tubule via secretory mechanism that are saturable (examples
of drug substrates for transporter= acids: penicillins, salicylate, diuretics, bases: morphine, catecholamines,
histamine), plasma protein binding (reversible) does NOT appreciably affect rate of secretion (t1/2= 1-2 hours),
poorly developed process in neonates (therefore prolonged half- lives).
D. Tubular Reabsorption: drugs that are lipid-soluble and uncharged WOULD be cleared at rate of urine
formation (1 ml/min) but the primary function of drug metabolism is to produce more water soluble metabolite
that is less likely to be reabsorbed. In order to be reabsorbed, drugs must pass through membranes.
i. Passive diffusion occurs with lipid soluble molecules in proximal and distal tubules, as water is
reabsorbed, lumen to blood back-diffusion is favored as drug is concentrated in luminal fluid.
a. Diffusion of weak acid/bases dependent upon urine pH (non-ionized form only will diffuse
across membrane), can change urinary pH with NH4Cl (acidify) or NaHCO3 (alkalinize)
ii. Active reabsorption is particularly important for endogenous compounds (glucose, aa’s), most drugs
REDUCE this active transport.
5. Describe the therapeutic implications of enterohepatic recirculation of drugs.
a. Enterohepatic Recycling: Drug metabolites in liver (usually conjugates that increase MW to ˃300)
are secreted into bile, stored in gallbladder, delivered to intestine via bile duct, hydrolyzed by
bacterial enzymes back to the parent drug (more lipid soluble) and undergo reabsorption from the
gut
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b. Reduces the elimination of the drug and prolongs its half-life and duration of action in the body.
c. Some drugs have “reservoir” of recirculating drug that accounts for 20% of total drug present in the
body.
d. Antibiotics that reduce gut glora can decrease enteroheptaic recycling and decrease plasma drug
levels and is a potential mechanism for drug-drug interaction
6. Describe the factors influencing drug passage from plasma to breast milk.
a. Most drugs do cross (unchanged) into breast milk, but at LOW levels. Resulting infant plasma levels is
substantially lower than therapeutic levels. To prevent infant exposure:
i. Desynchronize breastfeeding and peak milk/drug concentration
1. Breastfeed at end of dosing interval or administer after nursing
2. Administer a dose prior to infant’s longest sleep time
3. Fat (and thus drug) content of milk: increases during feeding period
ii. Choose Medication for breastfeeding mother carefully
1. Select drugs with little-no passage into breast milk
2. Drugs with rapid clearance
3. Milk is MORE acidic than plasma (5.6 vs 7.4 pH) and has tendency to accumulate basic
compounds by ion trapping
4. Lipid soluble compounds= increased milk concentration
5. High protein binding= decreased milk concentration
6. Never use drugs contraindicated by American Academy of PEDS
7. Drugs that can affect milk production/secretion/ejection (through prolactin, oxytocin)
Pharmacokinetics of Elimination
1. Explain the derivation and clinical relevance of the following pharmacokinetic parameters and be able to use
them in designing dosage regimens and predicting changes in drug plasma levels and drug response:
a. Clearance (CL): Selecting maintenance dose, dosage adjustments necessitated by alteration of kidney or
liver function.
i. Clearance: the volume of plasma which is completely cleared of drug in a given period of time by
the processes of kidney excretion and drug metabolism (with some contribution from other
tissues).
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b.
c.
d.
e.
1. Clearance = volume of distribution (Vd) x Ke
a. Ke: fraction of drug eliminated per unit time
ii. Clearance: proportionality constant that makes the average plasma concentration at steady
state equal to the rate of administration
1. Maintenance dose/tau= CL x Cp(ss)
2. CL= (MD/tau)/ Cp(ss)
Half-life (t1/2): Time to steady state or removal from body, selecting dosage intervals, relation to
fluctuations in plasma drug levels between drug doses (difference between Cp max and Cp min).
i. The time required to eliminate ½ of the drug amount present in the body
1. Drug with T1/2 of 3 hours, takes 3 hours for drug concentration to go from 1000 mg/ml to
500 mg/ml and another 3 hours from drug to go from 500 mg/ml to 250 mg/ml.
ii. Time it takes for drug to be essentially eliminated (4-5 half-lives)
iii. Time it takes to reach steady state when drugs administered continuously (4-5 half-lives)
iv. Degree of fluctuation between doses= 2x, where x=# of t1/2 in T
v. t1/2 = 0.693/ke
Elimination rate constant (ke)
i. ke is the fraction of drug leaving body per unit time via all elimination processes. ke is best
thought of simply as a number or constant that allows us to calculate the amount of drug
remaining at any time during the elimination process.
First-order: Implications for chronic dosing regimens
i. Virtually all drugs eliminated via first-order kinetics which means the rate of elimination (mg/hr)
is proportional to the concentration of drug in the plasma (mg/L)
1. If concentration of drug is doubled the rate of elimination is doubled
ii. As drug is eliminated from the body, its concentration is CONSTANTLY CHANGING, therefore
rate of elimination also changes constantly
iii. CONSTANT FRACTION of drug is eliminated per time and this is INDEPENDENT OF THE TOTAL
AMOUNT OF DRUG PRESENT
iv. Most clinically used drugs are eliminated by first-order kinetics when given in doses within the
therapeutic dosage range because the major biological processes responsible for drug
elimination, hepatic metabolism and renal excretion, are first-order processes.
Zero-order kinetics: Implications for chronic dosing regimens.
i. Process in which the rate of elimination of drug from the body is INDEPENDENT of the amount
of drug in the body. The amount of drug removed per unit time is constant.
ii. Most often occurs due to saturation of hepatic metabolic enzyme systems by drug
administration. This enzyme saturation occurs with therapeutic doses for only a FEW drugs
(aspirin, phenytoin, EtOH) and with toxic doses for most hepatically eliminated drugs. Drugs
eliminated by zero-order kinetics don’t have half-lives, can present dose adjustment challenges
especially at the upper end of the therapeutic range of narrow therapeutic index drugs, where a
small change in dose can produce large changes in plasma concentrations and subsequent
toxicity.
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12
Principles of Pharmacodynamics
1. Describe the drug-receptor concept and its consequences for pharmacotherapy.
a. Receptor: component of the biological system to which a drug binds to bring about a change in the
function of the system, specificity of the fit of drug to receptor (recognition) induces conformational
change in receptor protein.
b. Transduction: conformational change in the receptor leads to the transduction that alters cellular
function via effector molecules. Effectors accomplish the biologic effect after being activated by the
receptor (they translate the drug-receptor interaction into a change in cellular activity). Examples:
ligand-gated ion channels (fast response that changes membrane potential), G-protein-coupled
receptors (fast response that produces 2ndary messengers, IP3, cAMP, cGMP), kinase linked or hormone
(nuclear) receptors (slow response that changes gene expression/protein synthesis.
c. Types of Receptor Molecules:
i. Specialized: membrane proteins or ion channels designed to detect chemical signals and initiate
a response via signal transduction pathways
ii. General: biological molecules with any function including enzymes, lipids or nucleic acids.
iii. Proteins: binding site for majority of drugs, great specificity (due to secondary and tertiary
structure), example: hormone and neurotransmitter receptors, receptor or voltage gated ion
channels, enzymes, transport proteins, structural proteins
iv. Nucleic acids and membrane lipids: lower specificity
d. Consequences of Drug Receptor Therapy
i. Receptor mediate the actions of pharmacologic agonists and antagonists
1. Agonists: bind to and regulate function or receptor macromolecules in the same
manner as endogenous ligand promoting that receptor function
2. Antagonist: binding to receptors, but unable to generate characteristic response, effect
results from preventing the binding of endogenous agonist and blocking their action,
will have NO EFFECT in absence of the agonist for that receptor (extent of effect
depends on normal “tone”)
ii. Receptors are responsible for selectivity of drug action
1. Size, shape, electrical charge, etc. determine binding affinity of drug to particular
receptor relative to many other binding sites on patient
iii. Theory allows determination of quantitative relation between dose or concentration of drug
and its pharmacologic effects via use of dose-response curve
1. Quantify relationship between drug dose and effect helps to understand the drugreceptor interactions. Knowing potency and therapeutic efficacy is important when
choosing a drug.
e.
2. Explain the theoretical aspects and therapeutic consequences of the hyperbolic shape of the dose-response
curve.
a. Drug receptor theory assumes that interaction follows simple mass action relationships, binding is
reversible and that response is proportional to receptors [R] occupied by drug [D]
b. R+ D ↔ R-D  R-D is proportional to response
c. Dose response curves generated by giving increasing doses of drug and measuring the specified
response to each dose
d. Hyperbolic shape of curve confirms the mathematical relationship between dose and response as show
by this equation:
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i. e/Emax =
[D]/ EC50 + [D]
=
[D]/ED50 + [D]
e. Therapeutic consequences:
i. Curve is relatively linear (straight) at low doses meaning that at low doses the response
increases in direct proportion to the dose, this is consistent with receptor theory that states that
the great the number of receptors occupied by drug, the greater the response provided.
ii. Curve levels off at high drug doses meaning that there is a limit to the increase in response that
can be achieved by increasing the drug dose, consistent with theory that says the response by
administration of drug is proportional to amount of receptors occupied by drug (HIGH dose=all
receptors occupied, no further increase can be achieved and Emax achieved)
3. Describe the advantages of the log dose-response curve versus the dose-response curve.
a. Advantages of log dose-response:
i. Allows for wide range of doses to be plotted allowing easy comparison of different drugs
ii. Dose-response relationship is nearly a straight line over large range of doses (corresponds to
therapeutic range)
4. Explain the terminology of log dose-response curves:
a. Potency: affinity / Kd / EC50
i. Potency: concentration (EC50) or dose (ED50) required to produce 50% of that drug’s individual
maximal effect, depends on affinity (Kd) of receptors for binding the drug and in part of the
efficiency of this drug-receptor complex to generate a response and is designated by EC50.
Provides information on how much drug (dose) will be required to produce a given effect (more
potent, less drug needed). EC50 values used to compare potencies of different drugs.
b. Efficacy: power / Emax
i. Maximal effect or maximal efficacy (Emax): limit of the dose-response relationship on the
response axis (y-axis), indicates the relationship between binding to the receptor and the ability
to initiate a response. Most important determinant of drugs clinical utility. Power is often used
interchangeably with efficacy to describe the ability to initiate a response.
c. Agonist - Partial agonist - Antagonist
i. Agonist: drug that activates its receptor upon binding and brings about the characteristic tissue
response
ii. Partial agonist: occupy the same receptor as full agonist, but bring about less than maximum
response even at full dosage levels. These are less efficacious.
iii. Full agonist: occupy receptors and bring about a full or maximal response (max response being
defined as that produced by the most powerful agonist in that tissue)
14
5. Explain the use of log dose-response curves to compare potency and efficacy of different drugs.
a. Figure A: EC50x (half maximal effective concentration of drug x) occurs at a concentration that is 1/10
the half-maximal effective concentration of drug y, EC50y. Therefore, drug x is MORE potent than drug
y.
b. Figure B: Emax for x is 100%, Emax for y is 50%. Therefore, drug x is more efficacious than drug y. Drug
y is a partial agonist.
15
c. Potency: Based on position of curve along the dose [x] axis. Drugs A and B are more potent than Drugs C
and D. Drug A is less potent than Drug B (partial agonist) because Drug A has a larger EC50 compared to
Drug B. Potency: B ˃A˃C˃D. For therapeutic purposes, the potency of a drug is expressed in dosage
units for a particular therapeutic endpoint (not as EC50s)
i. 20 mg of lisinopril will lower BP by 10-15 mm Hg, as will 50 mg of captopril
ii. 200 mg of ibuprofen will alleviate headache pain, as will 650 mg of aspirin
d. Maximal Efficacy: this is the limit of the dose-response relation on the response [y] axis. Drugs A, C and
D are full agonists with greater maximal efficacy than drug B, a partial agonist. Efficacy refers to the
extent a given clinical effect can be achieved in an intact patient (not action at specific target). Efficacyemax: A=C=D˃B. Efficacies of different drugs are compared even though they act at different receptors or
targets:
i. Furosemide (inhibits Na+-K+-2Cl- cotransporter) is a more efficacious diuretic than
hydrochlorothiazide (inhibits Na+-Cl- cotransporter)
ii. Morphine (mu opioid receptor agonist) is a more efficacious analgesic than aspirin (inhibits
cyclooxygenase)
6. Distinguish between characteristics of the different types of antagonism (pharmacological [competitive
reversible and noncompetitive irreversible], physiological, chemical) and provide examples.
a. Antagonism: antagonist is a drug that inhibits the action of an agonist but has no effect in the absence of
an agonist, divided into receptor and non-receptor antagonists.
b. Pharmacological antagonists (aka: receptor anagonists): bind to the same receptor as the agonist.
i. Competitive reversible antagonist: binds reversible to the active site of receptor, but does not
stabilize the conformation change required for receptor activation. Antagonist blocks agonist
from binding to its receptor and maintains receptor in inactive conformation. EC50 increases
(potency decreases) - Emax unchanged (because agonist concentration can be increased to
outcompete the antagonist). Example: metoprolol is competitive reversible antagonist of NE in
Beta1 receptors in heart that produces a reduction in heart rate. But increase in endogenous NE
during exercise will still increased heart rate
16
ii. Noncompetitive irreversible antagonist: binds irreversibly (covalently) or pseudo irreversibly
(with very high affinity-slow dissociation) to the active site of the receptor. Removes functional
receptors from the system, limiting the number of available receptors that can contribute to the
response. Noncompetitive because an irreversibly bound active site cannot be outcompeted
even at high concentrations). The curve shifts downwards and the maximal efficacy (Emax) of
agonist is reduced. No shift on the x-axis.
iii. Noncompetitive Allosteric Antagonist
1. Drug binds to different site on the receptor than agonist drug. They do no compete with
agonist for receptor binding, but inhibit receptor from responding to agonist, so curve
shifts downward, result in a dose-dependent decrease in maximal efficacy (Emax). High
concentrations of agonist cannot active the receptor. May see a shift to the right
(decrease in apparent potency) if spare receptors available.
17
c. Non-receptor antagonists: include physiological antagonists that bind to a different receptor and
chemical antagonists that bind the agonist molecule directly (do not involve any receptor binding).
i. Physiological: activates or blocks a distinct receptor that mediates a physiologic response that is
opposite to that of activation of the receptor for agonist.
ii. Chemical: does not involve receptor binding, antagonism occurs via inactivation of agonist itself
by modifying it or sequestering it so it is no longer capable of binding to and activating the
receptor. Examples: EDTA chelator, antacid neutralizes stomach acid, osmotic diuretics.
Adverse Drug Reactions (Toxicology) and Poisoning
1. Compare and contrast graded dose-response curves and population dose-response curves and explain the use
of population dose-response curves to evaluate drug safety (Therapeutic Index and Standard Safety Margin).
a. Dose-response curve: a number of increasing doses of a drug are given to the same subject and the
increase in response for each dose is measured (graded in increments) allowing determination of the
maximal effect of the drug (Emax). ED50 is the dose that produces 50% of the maximal response
possible in an individual.
b. Population dose-response curve (quantal): characterize pharmacologic responses that are all-or-nothing
events (not graded) in a population of subjects (not an individual), generated by arbitrarily defining
18
some specific therapeutic effect (ie: relief of headache) and then determining the minimum dose to
produce this response in each member of the population. Single given dose of drug in an individual test
subject will either bring about the response or not (all-or-nothing). Data is plotted as fraction of
population that response at each dose of drug vs. the log of dose administered. Quantal curves are not
used to determine Emax (like dose-response). ED50 is the dose that initates the response in 50% of the
test population.
c. Degree of risk evaluated by comparing the quantal dose-response curves for the desirable and toxic
effects. Generated in the same way except for that the all-or-nothing effect is a toxic effect (side effect,
death) and TD50 is the dose that produces an undesirable side effect in 50% of subjects and LD50 is the
lethal dose that causes death in 50% of subjects.
i. To compare the dosage necessary for desired effect vs. dose with undesirable effect use:
1. Therapeutic index: compares midpoint in the population (ED50 and LD50)
a. TI= LD50/ED50
b. The higher the TI, the safer the drug, clinically used drug are ˃10-20
2. Standard safety margin: looks at the extremes in the population (ED50 and LD1)
a. SSM= [(LD1/ED99)-1] x 100
b. More conservative measure that TI, more reliable if pt response to therapy to
specific drug varies, takes into account the extremes, SSM can be negative.
2. Describe the general FDA categories for drug use in pregnancy and the implications for drug prescribing.
a. Used to classify risk to fetus of using drugs during pregnancy.
i. A: controlled studies show no risk, possibility of harm to fetus is remote, KCl
ii. B: no evidence of risk in humans (opioids, acetaminophen, ondanstron, thiazide diuretics)
iii. C: risk cannot be ruled out (pseudoephedrine, antidepressants)
iv. D: positive evidence of human fetal risk, but potential benefits may outweigh the risks (in a lifethreatening situation or serious disease) (oral anticoags, ACE inhibitors, diazepam-lorazepan,
alprazolam, paroxetine)
v. X: contraindicated in pregnancy, risks involved in the use of drug clearly outweighs the benefits,
(HMG CoA reductase inhibitor-statins, isotretinoin)
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3. Describe the major pharmacokinetic (via effects on absorption, distribution, metabolism, and excretion) and
pharmacodynamic (via pharmacologic [receptor] or physiologic effects) mechanisms underlying drug-drug and
food-drug interactions and the potential for clinical significance of each.
a. Pharmacokinetic
i. Absorption: decreased motility=decreased absorption rate=lower peak plasma drug levels
(doesn’t change bioavailability), increases in the rate of absorption less important clinically.
Physiochemical inactivation via change in pH or formation of insoluble complexes reduced
bioavailability.
ii. Distribution: protein binding, displacement interactions, competitive binding may increase
amount of free drug, cellular distribution interactions. Displacement of 1st drug from protein by
2nd drug results in increased levels of unbound-free 1st drug (total drug unchanged)
a. Can be of clinical consequence if:
i. Displaced drug has narrow therapeutic index
ii. Displacing drug is started in high doses
iii. Vd of the displaced drug is small
iv. Response to drug occurs more rapidly than redistribution
iii. Metabolism: metabolic rate increased by inducers reduced +/- subtherapeutic levels, rate
decreased by inhibitorsincreased and possibly toxic levels. Most interactions occur via effects
on cytochrome P450 (oxidation Phase 1 reactions)
iv. Excretion: most excretion interactions occur in the kidneys and they include:
1. glomerular filtration rate
a. Decreased by nephrotoxic drugs (e.g., aminoglycosides)   Cp
b. Increased by displacement from plasma proteins   Cp
2. tubular secretion
a. Decreased by competition for active transport (penicillins)   Cp
3. Change in tubular reabsorption via increase in urine pH
a. Decreased for weak acid drugs (e.g., aspirin)   Cp
b. Increased for weak base drugs (e.g., amphetamine)   Cp
4.
 pH 
i. Weak Acid:
R-COOH   R-COO− + H+
ii. Weak Base:
R-NH3+   R-NH2 + H+
5. Change in tubular reabsorption via decrease in urine pH
a. Increased for weak acid drugs   Cp
b. Decreased for weak base drugs   Cp
6.
  pH
i. Weak Acid:
R-COOH   R-COO− + H+
ii. Weak Base:
R-NH3+   R-NH2 + H
b. Pharmacodynamic
i. Antagonistic effects: Two drugs with opposite pharmacologic effects given together
1. -blocker (hypertension) + -agonist (asthma)  bronchospasm
ii. Synergistic or additive therapeutic effects: Two drugs with similar therapeutic effects given
together
1. -blocker + diuretic  enhanced blood pressure lowering
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iii. Synergistic of additive side effects: Similar to above, but involves side effects
1. Ethanol + benzodiazepine  enhanced CNS sedation
iv. Indirect pharmacodynamics effect: Pharmacologic effect of one drug indirectly affects second
drug
1. Diuretic (hypokalemia) + digoxin  enhanced digoxin toxicity
4. List the pharmacokinetic (decreased absorption or enhanced elimination) interventions that are available for
treatment of drug overdoses and poisoning and the limitations and contraindications for each.
a. Prevention of Absorption
i. Emesis: empties stomach contents rapidly
1. Ipecac: emesis after 15-30 minute lag, may repeat once in 20 minutes, local irritation
and CNS stimulation of chemoreceptor trigger zone (CTZ), effective orally, must be
given BEFORE activated charcoal, Ipecac should no longer be used in HOME treatment
2. Apomorphine: dopamine agonist, produces emesis by stimulation of CTZ, rapid action
parenterally, respiratory depressant, toxic in children, rarely used today
3. Contraindications of emetic agent: comatose pt (lacks gag reflex risk of aspiration),
ingestion of corrosive poisons (strong acid/base), ingestion of CNS stimulant (risk of
seizures), ingestion of petroleum distillate (risk of pneumonitis, pregnancy category C
(weigh the benefit: risk, unknown if drug will cause harm).
ii. Gastric lavage: most rapid and complete method to empty the stomach, but lavage + emesis
only empties 30% of oral poisons. Washing of stomach contents with saline and removal via
nasogastric tube, best within 60 minutes of poison ingestion.
iii. Chemical adsorption Activated charcoal: binds drug in gut to limit absorption (but also binds
Ipecac), effective without prior gastric emptying and can reduce elimination half-lives of drugs
that have been given IV (back-diffusion of drug from blood with ion-trapping in stomach),
underutilized or used in insufficient doses (best to give 10:1 ratio to toxin), serial admin may be
helpful (every 4 hours), difficult to administer and poorly accepted in children, home treatment
is NOT recommended.
iv. Osmotic cathartics: decrease time of toxin in GI tract (osmotic laxative effect), indicated if toxin
was ingested ˃60 minutes, if toxin is in enteric coated tablet or if toxin is hydrocarbon.
1. Sorbitol 70%: recommended, given with charcoal to prevent “charcoal briquet”
formation
2. Magnesium citrate or sulfate: avoid in renal disease or poisonings with nephrotoxic
agents
3. Sodium sulfate: avoid in CHF or HTN (system absorption fluid overload)
4. Polyethylene glycol: whole bowel irrigation that promotes elimination of entire
contents of intestines, for poisonings with sustained-release drugs, metal ions, drug
packets.
b. Enhancement of Elimination
i. Extracorporeal removal: lots of complications, pt must REALLY need this and treatment must
have significantly increased rates of toxin elimination compared to normal hepatic metabolism
or renal excretion
1. Hemodialysis/peritoneal dialysis: blood pumped through filter, most effective for drugs
with small Vd (if large Vd, poorly removed by this method as most of drug is outside
21
plasma), toxin should have low protein binding capacity (if bound to protein, toxin
won’t cross dialysis membrane), assists in correcting fluid and electrolyte imbalance
2. Hemoperfusion: blood pumped through column of adsorbent material, useful for high
MW toxins with poor water solubility, risks: bleeding (removal of platelets) and
electrolyte disturbances.
ii. Enhanced metabolism: induction of cytochrome P450 metabolism is NOT realistic (due to 1-3
day onset of action), enhancement of detox metabolism pathways with N-acetylcysteine in
acetaminophen toxicity and thiosulfate in cyanide poisoning, inhibition of metabolism to block
formation of toxic metabolites (inhibition of alcohol dehydrogenase in methanol or ethylene
glycol toxicity)
iii. Enhanced renal excretion: previously popular but unproved value
1. Forced diuresis (fluids [normal saline] plus high efficacy diuretics [furosemide]), small
effect, with danger of fluid overload, protects kidney (benefit)
2. Block reabsorption from kidney: prevention of passive reabsorption via alteration of
urinary pH and ion trapping, alkalinize urine with NaHCO3 (trap weak acids pKa=3-7.5
like aspirin and barbiturates), acidify urine with NH4Cl or ascorbic acid (trap weak bases
pKa=7.5-10.5 like phencyclidine or amphetamine)
iv. Chelation of heavy metals: combines aspects of enhancing the elimination of the toxin
(increases renal excretion) and inactivating the toxin (decreases ability to interact with and
damage target tissue)
1. Heavy metal ions: ability of form coordinate covalent bonds with protein side chain
nucleophiles, interact with macromolecules that are essential for normal physio
function, toxin effects are due to enzyme inhibition and alteration of membrane
structure. Treatment: admin of chelating agents that complex with free metal ions in
body fluids reducing their concentration and promoting the dissociation of metals from
these functional intracellular macromolecules, metal ion-chelator complex is excreted
in the kidneys.
5. Compare and contrast the concepts of toxicokinetics (seen with toxic amounts of drugs) to “normal”
pharmacokinetics (seen with therapeutic doses and therapeutic plasma levels).
a. Toxicokinetics: the study of the absorption, distribution and elimination of toxic parent compounds and
metabolic products that aids in prediction of amount of toxin that reaches site of injury and the
resulting damage. A toxic dose of drug may result in alterations of “normal” pharmacokinetics.
i. Absorption: large amount of ingested drug may slow tablet dissolution, alter GI emptying, injure
GI tract altered absorption delayed peak effect
ii. Volume of distribution: useful in predicting which drugs will be removed by dialysis/exchange
transfution (low Vd values)
iii. Clearance: important to know contribution of each organ to elimination of the toxin or drug in
planning treatment strategy
iv. Half-life: published values are for therapeutic doses, may be prolonged in toxic overdoses due
to saturation of the elimination mechanisms
6. Describe the mechanism of acetaminophen overdose toxicity and its treatment (role of hepatic bioactivation to
toxic metabolite and depleted hepatic glutathione in hepatocellular injury).
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a. 70-80% of acetaminophen is conjugated with glucuronic acid (developed in adults) or sulfate (in
children) (phase II reaction). 5-10% proceeds through a Phase I cytochrome P450 oxidation via CYP2E1
and this metabolite is the chemically reactive N–acetyl-p-benzoquinonimine (NAPQI or Ac*), a strong
electrophile detoxified by phase II GSH-transferase and excreted as mercapturate.
b. Hepatocellular injury occurs when there is saturation of the phase II sulfate and glucuronide
conjugation pathways by toxic doses. Results in excessive formation of Ac* by the unsaturated phase I
P450 pathway, eventual depletion of cellular glutathione and the binding of Ac* to critical protein or
cellular constituents. Predisposing factors: increased CYP2E1 activity and decreased haptic glutathione
content (occurs with excess EtOH consumption).
c. Toxicity is divided into 4 stages: 1. Initial 24 hours—symptoms do NOT reflect potential seriousness
(nausea, vomiting, abd pain), 2. 24-48 hours—clinical indications of hepatic damage apparent (elevated
plasma aminotransferases, prothrombin time prolonged), 3. 72-96 hours—peak hepatotoxicity, 4. 7-8
days—recovery if timely treatment OR severe liver damage in 10% with 10-20% dying of liver failure.
Treatment: activated charcoal and gastric lavage to
remove residual drug, best within 4 hours, vigorous
supportive therapy needed when intox is severe. Nacetylcysteine recommended within 12-36 hours of
ingestion as this drug is through to serve as a precursor
for glutathione synthesis (provides source of cysteine),
administer orally (LD=140mg/kg) + 70 mg/kg every 4
hours for 17 doses, IV formulation available and decreases
nausea and vomiting and has NO interference with action
if emetic agent of charcoal used.
7. Describe the basic pharmacodynamic parameters of methanol and ethylene glycol that underlie their toxicities:
rapid oral absorption, metabolism by common hepatic enzyme systems, these metabolic products selectively
damage different tissues or organs, and toxicities of both can be treated through similar interventions.
a. Methanol and ethylene glycol are well absorbed via the oral route with subsequent extensive
metabolism to organic acids, have minimal toxicity until metabolized to formic acid (retinal damage and
blindness) and oxalic acid (acute renal failure). The rate-limiting enzyme in the following pathway is
alcohol dehydrogenase and inhibition of this enzyme (with fomepizole) is the cornerstone of treatment.
Symptoms: delayed onset because need time to metabolize, serve metabolic acidosis in 4-12 hours,
methanol: visual disturbances (snowstorm), ethylene glycol: deposition of calcium oxalate crystals 
acute renal failure. Treatment: 1. Suppress production of toxic metabolites by inhibiting alcohol
dehydrogenase (infusion of EtOH to maintain a blood level of 0.1% b/c EtOH is competitive inhibitor of
alcohol dehydrogenase saturates the enzyme reduces formic acid and oxalic acid) OR fomepizole,
inhibitor of alcohol dehydrogenase that doesn’t produce CNS depression (more effective, more more
expensive), 2. Hemodialysis, correction of metabolic acidosis
23
DD - Bacterial Structure, Function and Growth
1.
Describe the major structural features of bacteria and explain the principal function(s) of each feature.
b. Cell wall and cell surface structures: rigid cell wall external to plasma membrane that contains
peptidoglycan. Rigidity of cell wall is essential for resisting osmotic lysis and maintaining cell shape.
i. Each bacterial isolate has a characteristic, rigid shape
ii. Bacterial shape is determined by both intracellular elements and by rigid components of the cell
wall. Peptidoglycan layer forms a rigid mesh that surrounds the cytoplasmic membrane. It
consists of a polymer with repeating units of 2 hexose sugars: N-acetylglucosamine and Nacetylmuramic acid. MurNAc residues are linked to tetrapeptide chains that contain amino
acids only found in bacterial cell walls (ie: meso-diaminopimelic acid (DAP), D-glutamic acid and
D-alanine). The tetrapeptides are cross-linked from one chain (via DAP in gram-negative and via
L-lys in gram-positive) to D-ala on another chain. Lysozyme in body secretions contributes to
innate hose defenses because it hydrolyzes the peptidoglycan by cleaving the bond between
MurNAc and GlcNAc (gram-positive is MORE susceptible due to due to exposed peptidoglycan
layer)
Intracellular localization of each protein type
iii. Other cell surface structures:
24
1. Capsules: loose, gelatinous outer surface layers and consists of complex
polysaccharides. Enhance virulence by enabling the encapsulated bacteriato resist
phagocytosis. Most capsular polysaccharides are antigenic, some are used as vaccines
to prevent specific bacterial infections.
2. Flagella: appendages originating in cytoplasmic membrane that function as organs of
motility. Peritrichous: flagella distributed all over surface. Polar: 1+ flagella at 1 end of
the cell. Chemotaxis: movement toward attractive nutrients and away from toxic
substances, uses flagella. Counterclockwise= swimming, clockwise= tumbling. Most
flagella are antigenic.
3. Pili: aka fimbriae are long slender, proteinaceous antigenic, hair-like structures on
surface of bacteria. Play a role in adherence to surfaces and tissues. Antibodies against
pili may block adhernace. Sex pili: role in bacterial conjugation for ssDNA transfers.
c. Cytoplasmic membrane: called the inner membrane in gram-negative, anatomical and physiological
barrier between the inside and outside of the bacterial cell. Lipid bilayer made of phospholipids and
proteins, but doesn’t contain sterols and has higher protein content 60-70% (compared to animal cells).
i. Exhibits selective permeability: impermeable to charged substances, only hydrophobic or
unchanged molecules no larger than glycerol can pass
ii. Electron transport system is located on cytoplasmic membrane. Generates proton motive force
during respiration.
iii. Other functions: metabolite transport, biosynthesis of lipids, DNA replication and flagellar
rotation.
d. Cytoplasm: aqueous solution of proteins and metabolites, site of metabolism
i. Ribosomes: 70S ribosomes, important for protein synthesis, Polycistronic: encode more than 1
protein product on mRNA.
ii. Nucleoid: where the DNA of bacteria is located, DNA is packed and supercoiled, no nuclear
membrane around the nucleoid therefore transcription and translation occur as coupled
processes. Bacterial genome consists of:
1. Chromosome: single, double-stranded, circular DNA molecule, cytoskeleton as a
primitive mitotic apparatus
2. Plasmids: extra-chromosomal, self-replicating DNA molecules (smaller than
chromosomes), usually not essential for bacterial viability, often encode virulence
factors, plasmids called “R factors” carry genes that determine resistance to antibiotics
3. Bacteriophages: viruses that infect bacteria. Temperate bacteriophages: intergrate into
chromosomes and replicated as part of chromosome, usually encode for bacterial
toxins, virulence factors or resistance of antibiotics. Phage conversion: change in
phenotype of host bacterium as a consequence of gene expression that is encoded by a
bacteriophage within the host bacterium.
2. Explain the importance of differences in cell wall structure among bacteria.
a. Gram-positive: the extent of cross-linking of peptidoglycan chains is typically much greater in grampositive. Thick, extensively cross-linked peptidoglycan layer that also contains teichoic acids. Teichoic
acids have a repeating polyglycerol-P or polyribitol-P backbone substituted with other molecules and
25
they are covalently attached to the peptidoglycan layer. Lipoteichoic acids are attached to underlying
cytoplasmic membrane and help anchor the cell wall to membrane.
b. Gram-negative: thin, sparsely cross-linked peptidoglycan layer and other major components that are
located exterior to the peptidoglycan. Outer membrane is a lipid bilayer that contains
lipopolysaccharide (LPS), lipoproteins and porins (transmembrane channels for diffusion)—acts as
barrier to entry of some antibiotics and protects cell against detergents and toxic compounds. LPS is
located exclusively in outer leaflet of outer membrane (inner leaflet consists of phospholipids). LPS
contains Lipid A (toxic component of endotoxin), core polysaccharide, and O side chain oligosaccharides
that function as somatic antigens (O antigen)
3. Draw a typical bacterial growth curve and explain the characteristics of each growth phase.
a. Lag phase: physiologic adjustment for starting cells or inoculum. Induction of new enzymes and
establishment of proper intracellular environment for optimal growth.
b. Exponential (logarithmic) phase: rate of increase in cell number/cell mass is proportional to cell
number/cell mass already present. A constant interval of time is required for doubling of cell
number/cell mass—called the generation time (doubling time).
c. Stationary phase: as essential nutrients are consumed and toxic products of metabolism accumulate,
growth slows or ceases, growth that does occur is balanced with cell death. Leads to marked increase in
resistance to antibiotics because antibiotics act on growing cells. In nature, bacteria are most often
found at this phase.
26
d. Death phase: OPTIONAL. The number of viable bacteria decreases over time. If cell lysis (autolysis)
occurs, the mass of intact bacteria in culture will decrease.
4. Describe how bacteria are classified according to their nutritional requirements
a. Nutrition: provision of proper environmental conditions for promoting bacterial growth including
nutrients, pH, temperature, aeration, salt concentration osmotic pressure. Under adverse nutritional
conditions sporulation occurs.
b. Heterotrophic: obtain carbon from an organic source
c. Autotrophic: obtain carbon exclusively from CO2
d. Fastidious bacterial pathogens that are deficient in 1+ biosynthetic pathway, they require in addition to
carbon and energy, a number of essential growth factors like amino acids, vitamins, purines, pyrimidines
and inorganic ions.
e. Obligate intracellular bacteria: grow within eukaryotic cells but cannot be cultivated on artificial media.
f. Aerobe: requires oxygen to grow, produces toxic metabolites such as H2O2 and superoxide, but use
catalase and SOD to protect them against ROS.
g. Anaerobes: killed by oxygen, although anaerobes that are frequently associated with disease are more
aeroterant than strict anaerobes.
Growth Response
Aerobic
Anaerobic
Comment
Example
Aerobe (strict
aerobe)
+
-
Requires oxygen;
cannot ferment
Mycobacterim
tuberculosis
Anaerob e (strict
anaerobe) *
-
+
Killed by oxygen;
fermentative
metabolism
Clostridium sp
Bacteroides sp
Indifferen t
(aerotolerant
anaerobe)
+
+
Ferments in presence
or absence of O 2
Streptococcus
pyogenes
Facultative
(facultative
anaerobe)
+
+
Respires with O 2;
ferments in absence
of O 2
Escherichia coli
Staphylococcus
aureus
(+)
+
Grows best at low O 2
concentrations; can
grow withou t O2
Campylobacter jejuni
Type o f Bacteria
Microaerophilic
( + ) indicates small amoun t of growth
27
5. Define respiration and fermentation and explain how metabolic “energy currency” is generated.
a. “Energy currency” in bacteria: ATP and electrochemical gradients (proton motive force), these 2 types of
potential energy are interconvertible by the membrane ATPase.
b. Fermentation: organic compounds serve as both electro donors are electron acceptors (no net oxidation
of substrates occurs). Anaerobic and facultative bacteria grown under anaerobic conditions obtain
energy by fermenting organic substrates. Indifferent organisms obtain energy by fermentation under
anaerobic and aerobic conditions because they are incapable of respiration.
c. Respiration: generate ATP through electron transport and use molecule oxygen as final electron
acceptor, anaerobic respiration—certain bacteria use inorganic substrates such as nitrate as terminal
electron acceptors instead of O2.
6. Explain why unique bacterial components are important as potential targets for antimicrobial therapy.
a. Selective toxicity: selective inhibition of microbial growth at drug concentrations tolerated by the hose.
Some components of bacteria that are NOT present in eukaryotes or are sufficiently different from their
counterparts to be effective targets for antimicrobial agents.
7. Identify the principal targets for the major groups of antibiotics used in human medicine.
a. Cell wall-active microbials: selective toxicity due to lack of peptidoglycan in mammalian cells
i. Beta-lactams, vancomycin, cycloserine
b. Outer and cytoplasmic membrane-active microbials: polymyxins are cationic surfactants that disrupt
bacterial outer and cytoplasmic membranes, they are less active on mammalian cell membranes
c. Inhibitors of protein synthesis at the ribosomal level due to the differences between bacterial and
mammalian ribosomes
i. Aminoglycosides, tetracyclines, chloramphenicol, macrolides/lincomycins
d. Inhibitors of nucleic acid synthesis
i. Quinolones, rifampicin
e. Metabolic inhibitory antimicrobials
i. Sulfonamides, trimethoprime, isoniazid, metronidazole
28
DD LAB- Principles of Bacterial Isolation and Identification
1. To isolate the individual pathogenic organism from a mixed culture and identify the individual bacteria by applying
basic microbiological techniques such as Gram stain, biochemical properties and serological testing
2. To identify a pathogenic organism from a simulated urine sample, to illustrate how many bacteria must be present in
samples to detect them by microscopy.
3. Understand differential and selective media including identifying which media is differential or selective and how
these media are used in microbiology.
DD - Host-microbe interactions
a. Define and describe
a. Infection: process whereby a microbe enters into a relationship with the host. It may or may not cause
disease.
b. infectious disease: disease caused by an infection with a microbe. Some infections are communicable
(transmitted patient to patient) and others are not.
c. Pathogenicity: ability (usually of a species) to cause disease
i. Frank pathogens: causes disease readily in normal hosts
ii. Opportunistic pathogens: cause disease in compromised hosts, many normal flora are
opportunistic pathogens
d. Virulence: denotes degree of pathogenicity. If a strain is highly virulent, then it is likely to cause disase
when introduced to a host in small numbers
2. Explain how a microbe is shown to be the cause of a specific disease
a. Use Koch’s Postulates:
i. Specific microbes are present regularly in characteristic lesions of the disease
ii. Specific microbes can be isolated and grown in vitro
iii. Injection of cultured microbes into animals reproduces the disease seen in humans
iv. Specific microbes can be re-isolated from lesions of the disease in animals
v. LIMITATIONS:
1. Some infectious diseases do not have a characteristic (pathognomonic) lesion
2. Some microbes cause specific infectious diseases but cannot be grown in vitro
3. Traditional concepts of pathogenicity focus primarily on properties of microbes vs.
hosts
4. The characteristics of infectious diseases usually reflect complex interactions between
microbes and their hosts.
3. Describe typical stages in pathogenesis of an infectious disease and explain their importance
a. Encounter: how the agent meets the host
i. Endogenously (normal flora) or exogenously (from environment), route of infection, what is
infectious dose
b. Entry: how the agent enters the host
i. Did microbe cross epithelial barrier (by invasion or passively?)
ii. Colonization of body surfaces
iii. Adherence
c. Spread: how the agent spreads from the site of entry
29
i. Spreading factors: hyaluronidase, elastase, collagenase
ii. “wall off” infection: coagulase
d. Multiplication: how the agent multiples in the host
i. Must replicate at levels that exceed their clearance by the host.
e. Damage: how tissue damage is caused by the agent and/or the host response
i. Aggressins: microbial products that damage the host
ii. Impedins: microbial products that block host defenses
f. Outcome: dose the agent or host win the battle or do they learn to coexist
g. Mechanisms of host response to infection
4. Compare mechanisms of innate and acquired host defense against infections
5. Describe the composition and importance of the normal flora of the body
a. Factors that influence normal flora
i. Diet (breast-feeding, bottle feeding, solid food)
ii. Suppression of flora with antibiotics
iii. Anatomic abnormalities (blind loop increase flora)
iv. Genetic differences between individuals
b. Physiologic importance of normal flora
i. Effects on tissue/organ differentiation (normal vs. germfree animals)
ii. Production of vitamins by gut flora
iii. Biochemical conversions
iv. Competition with pathogens for colonization of body surface
30
6. Compare several disease paradigms that illustrate selected mechanisms of pathogenesis
a. Cholera: toxin mediated disease, organism is non-invasive
b. Pneumococcal pneumonia: acute inflammation caused by invasive extracellular bacteria, invades and
replicates
c. Tuberculosis: infection by a facultative intracellular bacterium, grows in phagocytes
d. Rheumatic fever: pathology mediated by an immune response, from streptococcal infections
DD - Microbial Toxins
1.
Define and describe the term “microbial toxin”.
a.
Macromolecular products of microbes that cause harm to susceptible animals by altering cellular
structure or function, they are very potent, the clostridian neurotoxins are the most toxic biological
substances known. Importance: some toxins cause the major manifestations of specific diseases, other
toxins contribute to pathogenesis without causing unique signs or symptoms, toxin-mediated diseases
cause significant morbidity and mortality
2.
Explain how a microbial toxin is implicated in pathogenesis of an infectious disease
e. Show that purified toxin causes the same symptoms or signs as infection by the toxin-producing microbe
f. Show that antitoxin prevents disease caused by the toxin producing microbe
g. Show that virulence of individual bacterial strains correlates with the amount of toxin that they produce
h. Show the nontoxinogenic mutants are avirulant and that virulence is restored if the microbe regains the
ability to produce toxin.
31
3. Explain the mechanisms of action of the microbial toxins described here and compare the properties of microbial
toxins that have different mechanisms of action
a. toxins that facilitate spread of microbes through tissues:
toxic enzymes break down ECM or degrade debris in necrotic tissue
b. toxins that damage cellular membranes:
called hemolysins (easy to see action of RBC), but act on other cells too, therefore cytolysins is accurate
name. These toxins insert into membrane, assemble into complexes that form forms and lyse the target
cell.
c. toxins that stimulate cytokine production:
pyrogenic exotoxins are from a larger class of molecules called superantigens—the most potent known T
cell activators (bind to both MHC class II molecules and to V-beta chains on T cells and they activate a
larger number of T cells than any specific antigen does.
d. toxins that inhibit protein synthesis:
diphtheria toxin and Pseudomonas aeruginosa exotoxin A inactivate elongation factor 2 (required for
peptide chain elongation)
Shiga toxins and ricin (plant toxin) remove one adenine residue from RNA of ribosome to inactivate it.
e. toxins that modify intracellular signaling pathways:
Heat-labile enterotoxins of Vibrio cholera and E. coli increase cell membranes associated cAMP, activate
alpha subunit of stimulatory Gs regulatory protein active chloride secretion and secretory diarrhea.
Pertussis toxins: increase cAMP and inactivate the alpha subunit on inhibitory Gi regulatory protein.
f. toxins that inhibit release of neurotransmitters
4. Explain the principles of immunization against toxin-mediated diseases.
a. Antitoxin antibodies (antitoxins) bind to toxins and prevent their toxicity (neutralization). Antitoxins don’t
prevent infection by toxin-producting bacteria or reverse toxic effects after toxin has entered host cells.
b. Toxoids are derivatives of toxins that retain immunogenicity but lack toxicity, they are used as vaccines for
long term protection
c. passive immunization is the administration of antibodies to a patient to provide immediate but temporary
protection against a toxin or infectious disease
d. active immunization involves the administration of toxoid to a patient in order to elicit production of specific
anti-toxic antibodies. Primary series of immunizations and periodic boosters required, active immunity can
persist for years because of immunologic memory.
5. Explain the principles for developing novel therapeutic agents based on toxins.
 Immunotoxins are hybrid molecules which contain the active (A) fragment of a toxin (e.g., diphtheria
toxin, exotoxin A, ricin, etc.) chemically conjugated to, or expressed as a fusion protein with, ligands (e.g.
monoclonal antibodies, "single-chain" antibodies, or the receptor-binding domains of hormones) for
specific receptors that differ from the receptors for the native toxins. The rationale is to eliminate the
receptor-binding component of the native toxin and provide a new receptor-binding moiety that will
redirect the toxic component to target cells that express the alternative receptor.
 Many immunotoxins are designed to kill tumor cells that display a tumor-specific receptor but not
normal cells that lack that receptor. Immunotoxins are being tested as potentially valuable therapeutic
agents for treatment of specific cancers, autoimmune diseases, and other disorders.
32
DD - Genetic Variation, Gene Transfer and Evolution of Virulence
a. Describe the mechanisms that generate genetic diversity within a bacterial species and how these contribute to
the evolution of virulence
a. Spontaneous mutation: single base changes, deletions and insertions. Mutation rates are very low: 10-610-10 per cell-generation. Examples: increased resistance to antimicrobials in Pseudomonas and
Mycobacterium tuberculosis.
b. Recombination: either site-specific or homologous recombination within a particular organism or
genetic exchange and recombination between closely related organisms
i. Example: recombination between variant pilin genes produces hybrid genes that encode pilin
with new unique antigenic properties
c. Acquisition of new DNA segments: lateral transfer from other bacteria, even from unrelated species.
New genes may alter virulence potential, survival characteristics, antimicrobial resistance
i. Acquisition of transposable elements—a discrete segement of DNA which is capable of moving
itself from one chromosomal location to a new location within the cell. Typically encode 1+
proteins that mediates transposition (transposase).
1. Insertion sequences: transposons that only encode transposase
2. Complex transposons: carry additional genes such as those encoding antibiotic
resistance, toxins, adhesions, etc.
ii. Bacteriophage conversion: certain virulence genes are carried on bacteriophage and are not a
“normal” component of the respective bacterial genome.
iii. Acquisition of plasmids: can be transferred from one bacteria to another by conjugation or
transduction. They can carry virulence genes or genes conferring resistance to antibiotics
iv. Acquisition of pathogenicity islands: Pathogenicity islands are large segments of DNA present in
the chromosome of some, but not all strains of bacteria. The encode genes that contribute to
the virulence of these isolates. Lacking PI may make bacteria avirulent.
b. Discuss how spontaneous mutation and selection can interact to determine the genetic composition of bacterial
populations
a. Errors occur that result in base pair changes, deletions, duplications, etc.
b. Typically changes are deleterious or neutral
c. In rare instances, a mutation may confer a selective advantage
d. Spontaneous mutation to antibiotic resistance occurs once in approx 108 – 1010 organisms
c. Distinguish between transformation, transduction and conjugation as mechanisms of gene transfer. Identify the
salient features of each mechanism
a. Transformation: first to be discovered, active component in transformation is naked DNA (chromosome
fragment or plasmids), many transformable species only become competent for DNA uptake at certain
points in their growth cycle, occurs most frequently between members in same species.
b. Transduction: gene transfer mediated by a bacteriophage, bacterial virus (bacteriophage) transfers
segments of DNA from 1 cell to another.
c. Conjugation: genetic transfer that is dependent upon physical contact between the donor and recipient,
mediated by bacterial plasmids
33
d.
e.
d. Discuss the properties of bacterial viruses. Distinguish between the lytic and lysogenic state.
a. Lytic: phage multiples and host cell is lysed.
b. Lysogenic: host cell remains viable and infecting phage DNA is maintained by the host cell in a noninfectious state called prophage.
e.
34
f.
Describe how errors in bacteriophage development can lead to phage-mediated gene transfer
a. During phage assembly, if there is any error in DNA packaging and the packaging system inserts a
“headful” sized piece of bacterial DNA into a maturing phage capsid in place of a normal phage DNA
molecule then the transducing particles contain no viral genetic information, but they are still able to
attach to other host cells and inject the bacterial DNA which they contain. Injected DNA may then
recombine with homologous segement in recipient to produce a genetic recombinant, transductant.
g. Define lysogenic conversion. Distinguish between lysogenic conversion and generalized transduction
a. Lysogenic conversion: certain temperate bacteriophage encode genes which may be expressed during
the lysogenic state and cause the appearance of a new phenotypic trait in the lysogenic host. This is
called bacteriophage conversion or lysogenic conversion. The genes controlling the new phenotypic
trait are found only as a component of the phage genome (the converting genes are NOT found alone as
normal constituents of the bacterial genome). Examples: diphtheria toxin, scarlet fever toxin, cholera
toxin.
b. Generalized transduction: process in which any segment of the donor cell genome (chromosome or
resident plasmids) may be passed into another cell.
35
DD - Common Bacterial Pathogens
Genus
Bacteria
Staphyl
ococcus
Staphylo
coccus
aureus
Appearanc
e (micro)
blue/purpl
e cocci in
clusters,
GRAPES
Staphyl
ococcus
Staphylo
coccus
epidermi
dis
Blue cocci
Streptoc
occus
Streptoc
occus
pyogenes
Blue cocci
in chains of
pairs
Gram Positive (+) Cocci
Disease
General
Primary pathogenic species
in genus. Asymptomatic in
30% of healthy ppl. Site of
carriage: anterior nares,
perineum. Endogenous
flora can lead to infection of
self or others (ie: healthcare
worker transmits to
patient). Catalase +.
Prototype of SSNA’s (staph
species, not aureus) or CNS
(coagulase – staph).
Considered normal skin
flora/non-pathogenic, but
can cause localized infection
usually associated with
foreign body (catherter,
shunts, prostheses).
Catalase +.
Catalase (-). “Group A
Strep” of which there are 80
serotypes (antigenic
differences of M protein).
36
Treatment/Drugs
Typical disease:
1. Cutaneous infection (boils,
folliculitis, wound infection),
characteristic lesion is localized
abscess. Bacteria + host help form
fibrinous capsule that walls off
infection and limits spread, but
limits access of phagocytic cells,
antibodies, etc. “coagulase” is
virulence factor that forms fibrin
capsule and deposits fibrin on cell
surface. Cutaneous staph
associated with foreign body
(suture/splinter) that interferes
with bacterial clearance by
phagocytes + provides surface for
bacteria to grow.
2. toxin-mediated disease, certain
strains have genes for 1+ protein
toxins. Scalded skin syndrome
(exfoliatins) Local infection, toxin
production (epidermolytic toxins A
and B), systemic effects.
Widespread desquamation in
infants; localized in older. Toxic
shock syndrome (toxic shock
syndrome toxins produced and
circulate leading to systemic
effects) and staph food poisoning
(staph enterotoxins via ingestion
of contaminated food).
3. pneumonia: pts with impaired
host defenses at risk
“slime” production, an
extracellular glycocalyz that allows
organisms to adhere very
tenaciously to implanted device
and allows them to grow in a
protected biofilm on surface.
Drain abscess.
1. Pharyngeal Infection: Cause of
“strep throat”. Virulence factor
“M” protein is a surface exposed
protein that inhibits phagocytosis
Untreated is self-limiting
weeks. Production of Mantibody by host makes b
killing and leads to recove
DRUGS:
Pen G/Pen V
if PCNase-producing (MSS
DICLOX, 1st CEPH
if CA-MRSA: TMP-SMX, TC
If HA-MRSA: VANC, LIN, D
Difficult to treat, require
Antibiotic resistance (incl
limited access of drug to
Transmission: nasal
secretions or by droplets
from coughing.
and killing by PMN’s and increases
adherence to epithelial cells.
2. Skin and wound infection:
typical lesion is that of a spreading
infection of cutaneous/subQ
tissue. Bacteremia/sepsis
possible. Infection uses hydrolytic
enzymes to break down tissue and
damage/kill phagocytic cells
(facilitates spread of organism).
Streptoc
occus
Streptoc
occus
pneumon
iae
Streptoc
occu
Enteroco
ccus
faecalis
Blue cocci
in pairs,
diplococcic,
“pneumoc
occus”
Normal flora in UR tract of
40% of healthy people.
Predisposing factors:
young/old, alcoholism,
respiratory viral infection
Primary cause of
enterococcal infections.
Infection sites: urinary tract,
surgical wounds, biliary
37
3. Post-streptococcal diseases:
a) Glomerulonephritis: immune
complex disease that may follow
skin or pharyngeal infection by
Group A strep. Streptococcal
antigen-antibody complexes
deposited in kidney and
accumulate. Self-limiting.
Complete-mediated damage to
kidney.
b) Rheumatic fever: autoimmune
inflammatory disease that may
follow group A strep pharyngitis
(this is why we treat). Symptoms:
fever, inflamm heart, joints.
Antibodies produced during
response to pharyngeal infection
self-react (only from
rheumatogenic strains) are
recognize and bind specific host
antigens in myocardium and heart
valves. NOT like endocarditis (a
true bacterial infection of heart
values) because there are no
bacterial colonies .
1. Pneumonia
2. Meningitis
3. Bacteremia
4. Sinusitis, otitis media, bronchitis
Pathogenesis: related to ability ot
grow and evade host defenses,
antiphagocytic polysaccharide
CAPSULE (91 distinct antigenic
capsules), recovery/immunity due
to anticapsular antibody
1. Frequent cause of nosocomial
(acquired IN-hospital) infections,
transferred on hands of hospital
staff. Bacteremia, meningitis, UTI.
antigenically related strai
DRUGS:
PEN G/PEN V
AMOX
MAC
1st CEPH
VANC
Vaccination: 23-valent va
13- valent in children. Eff
"serotype replacement”
DRUGS:
PEN G/PEN V
MAC
FQ
If resistant, 3rd CEPH
DRUGS:
VANC
CARB
PEN G/AMP +/- AG
Gram Positive (+) Rods
Genus
Bacteria
Appearanc
e
Clostridi
um
Clostridiu
m
difficile
Blue rods
tract. Normal flora in GI
tract of some people.
Often seen as mixed infection of
several different organisms,
including anaerobes (important in
cases of perforated colon and
release of contents)
General
Diseases
Treat
Diseases associated with antibiotic treatment for
unrelated conditions (first described with use of
clindamycin). Caused by depletion of intestinal flora
by antibioticovergrowth of C. difficile (self bacteria
or transmitted from another patient). Bacteria
produces 2 discrete toxins that damage mucosa of
intestines.
1. Diarrhea
2. Pseudomembranous colitis
1. Tetanus, toxin-mediated. Local infection
(anaerobic) and toxin production
Retrograde transport of toxin to CNS
Blocks inhibitory interneurons in CNS
Vaccine vs antitoxin
Resis
antib
DRUG
METR
VANC
Fidax
Strict anaerobes—killed by
O2
Endospore formers (Critical
to persistence of these
bacteria)
Clostridia most likely to be
encountered in
hospitalnosocomial
infections. Normal flora of
GI tract in 10% of healthy
ppl.
Clostridiu
m tetani
Soil and animals
(importance of spores)
Clostridiu
m
botulinu
m
Soil and animals
(importance of spores)
Clostribiu
m
perfringe
ns
Gram Negative (-) Rods
Escherich “typical”
ia coli
gram (-)
rod
If VRE—LIN/SG
DRUG
CLIN
1. Botulism, toxin-mediated
Preformed toxin in food
Toxin blocks acetylcholine transmission at neuromuscular junctions
Part of normal flora in
human GI tract (large
intestine)
38
1. Gangrene/tissue infections
DRUG
CLIN
Disease caused by endogenous organisms or
acquisition (ingestion)
1. GI disease: drinking contaminated H2O, ETEC
(enterotoxigenic E. coli, traveler’s diarrhea). Bacterial
properties: adherence to intestinal mucosa (pili) and
toxins that disrupt electrolyte balance in gut. Selflimiting.
2. UTI: typically endogenous from GI tract, access the
UT via urethra—bladder—kidney. “special” strains get
into “wrong” place. Bacteria properties: adherence to
bladder epithelium, specific interactions with bladder
epithelial cells, hemolytic.
3. ABD infection: escape of contents of colon to
1. Flu
DRUG
FQ
TMPAMO
1st CE
AG
NF
Pseudom
onas
aerugino
sa
Gram Negative (-) (Diplo)cocci
Neisseria
gonorrho
eae
Very common
environmental bacterium,
most ppl highly resistant to
infection
Causative agent of
gonorrhea and
conjunctivitis blindness in
infants born to infected
mothers
peritoneal cavity, surgical/trauma wounds, colon
cancer. Bacteriologically mixed cultures. Associated
with anaerobic bacteria to form anaerobic abscess.
Opportunistic pathogen, infection of burns, wounds.
Seen in immune compromised patients.
1. infections of traumatic injury, surgical wounds and
especially BURNS
2. Chronic lung infection of patients with CF: S. aureus
infections are common early in life (viscous
secretions statsis in lungs infection), but treated
with antimicrobials. By age 15-20 CF pts become
chronically infected with P. aeruginosa (intrinstic
resistance to many anti-staph drugs and protected
from phagocytosis by viscous lung secretions/mucois
exopolysaccharide made by bacteria) and progressive
lung damage due to action of toxins and host immune
response begins frequent cause of death in CF pts
Key to infectivity is pilus: required for virulence,
adherence and interferes with bacterial killing by
neutrophils. Different strains have antigenically
distinct pili and bacterial can undergo process of
antigenic variation during infection (cells switch to
distinct type of pili). Means pt can have repeated
infection.
Growth on mucosal surfaceinflamm
responsepurulent discharge/local tissue invasion.
Prolonged infection=scarring and fibrosis.
Males: asymptomatic to urethritis
Females: ˃rate of asymptomatic is higher in females,
infection of cervix, urethra, ascends to uterine
tubesfibrosis and infertility.
Diffic
patie
resist
to ma
diffic
drugs
replic
DRUG
FQ, C
Antib
prote
Asym
easily
becau
they
DRUG
CEFT
FQ
MAC
Anaerobic Bacteria
Most common anaerobic bacteria (besides Clostridia) are members of normal flora that inhabit colon, mouth (gums/teeth), female
Killed in presence of molecular O2. Diverse gram staining/morphology. Common properties: generally of endogenous origin (norm
typical lesion: abscess (just like Staph aureus). Hallmark: mixed infection containing aerobic and anaerobic bacteria (aerobes metab
conducive to anaerobe growth). Because of anaerobic metabolism some drugs are more effective (metronidazole) and some less (a
Obligate intracellular bacteria: grow only in infected eukaryotic cell (Cannot be Cultured)
Rickettsia
1. Rocky Mountain spotted fever, lost capacity to
Drug
synthesize own ATP (rely on host cell)
cell e
DRUG
DOXY
CHLO
Chlamydi
Obligate intracellular
1. Trachoma: chronic conjunctiva infection
Drug
a
bacteria
scarring/blindness, endemic in Asia and Africa where
cell e
poor hygiene prevalent
2. Genital: common STD, found with coinfections of N. DRUG
gonorrhoeae. Causes “non-gonococcal urethritis” in
TCN
men and urethritis, cervicitis and PID in women.
MAC
3. Neonatal: if mom has disease, baby can be infected
at birthconjunctivitis and pneumonia.
39
Bacteria without cell walls and containing sterols in plasma membrane
Mycopla Mycoplas
Person-to-person (infected respiratory
sma
ma
secretions). Organism grows in specialized
pneumon
cell-free bacteriologic medium, but is
iae
difficult/slow. DX:serological test, gram
stain rules out other bacterial causes.
40
1. Common cause of pneumonia in ages 5-20.
Generally mild w/fever, HA, sore throat, nonproductive cough, aches, fatigue. Recovery in 14 weeks. Adheres to respiratory epithelial cells,
growth is extracellular, bacteria produce H2O2
and superoxide radicals damages host tissue.
Becau
shape
pleom
are n
DRUG
MAC
Antibacterial Agents I-III
Define and / or give examples for:
1. Selective Toxicity
a. Effects of antimicrobial agents should be exerted selectively on the MICROBE, NOT the host.
Fundamental feature of antibiotic therapy. No perfect antibiotics exist.
b. Biochemical differences between pathogen TARGET and host must be discovered and exploited.
i. Inhibition of metabolic pathway found in bacteria, NOT humans
1. Folate metabolism: bacteria make folate intracellularly, humans take up folate from
environment
ii. Pathway exists in bacteria and humans, but differences in enzyme structure
1. Protein synthesis: bacterial ribosome is 30S and 50S, mammals use 40S and 60S
2. Nucleic acid synthesis: DNA gyrate (bacteria) vs. topoisomerase (humans). RNA
polymerase is structurally distinct in bacteria too.
iii. Macromolecular structure does NOT exist in humans
1. Cell wall synthesis: peptidoglycan component does not occur in eukaryotes.
iv. Macromolecular structure differs between microbes and humans
1. Fungal cell membrane: ergosterol is major constituent f fungal membranes vs.
cholesterol in mammals.
2. Antibacterial Spectrum: Narrow vs extended vs broad
a. Narrow spectrum: effective against either gram positive OR gram negative), most effective on
susceptible organisms, less disturbance on host flora.
i. Animoglycosides, bacitracin, clindamycin, vancomycin, metronidazole, Pen G, Pen V,
Penicillinase-resistant penicillins, monobactams
b. Extended spectrum: effective against gram positive AND gram negative
i. Extended-spectrum penicillins, cephalosporins, fluoroquinolones, carbapenems
c. Broad spectrum: effective against gram positive, gram negative and atypical, sacrifice efficacy for
greater scope of activity for initial empiric coverage, less likely to cause superinfections, acute severe
infections should be treated with broad spectrum (target empiric therapy to likely bacteria) with a
switch to narrow spectrum ASAP (target definitive therapy to bacteria based on lab results).
i. Macrolides, chloramphenicol, sulfonamides, tetracyclines, trimethoprim
3. Resistance: Natural vs escape vs acquired (chromosomal vs plasmid-mediated)
a. Natural (intrinsic) therapy: when a bacteria naturally doesn’t have a susceptible target for drug action.
Example: fungal cell walls don’t have peptidoglycans and mycoplasma don’t have cell walls at all,
therefore they are naturally resistant to penicillins which interfere will cell wall production
b. Escape: microbe is sensitive and antibiotic reaches target but organism escapes the consequences dueto
availability of purines, thymidine, serine, methionine released from purulent infections (sulfonamide
resistance) or failure to lyse due to lack of osmotic pressure difference (penicillin resistance),
emphasizes important role of surgical drainage if needed.
c. Acquired resistance: selective pressure (ie: antibiotic administration) successive generations of
organisms with biochemical traits that minimize drug action
i. Mutational (chromosomal) resistance: rate is 1 in 10^7-10^12. Each generation becomes more
resistant if allow to survive, thus proper dosing and duration of antibiotic therapy prevents
survival of slightly resistant strains.
41
ii. Plasmid mediated resistance: clinically important source of multiple drug resistance that can
emerge during a SINGLE course of treatment. Nonpathogenic coliform bacteria can code for
resistance to multiple drugs via a protein that moves antibiotics out of cell. Exchange of genetic
information occurs via:
1. Conjugation: 2 physically attached bacteria, exchange of plasmid w/ resistant
determinant
2. Transformation: ability of bacteria to pick up free DNA from environment
3. Transduction: with virus (bacteriophage) carrying resistance determinant R to bacteria.
4. Mechanisms of resistance and implications for therapy
a. Altered targets or receptors
i. Penicillin-binding proteins [MRSA, S. pneumoniae, Enterococci)
1. -lactam antibiotics (penicillins, cephalosporins, carbapenems)
ii. DNA gyrase [S. aureus, Pseudomonas species]
1. Fluoroquinolones
iii. Peptidoglycan side chain [Enterococci (VRE), Staphylococci (VRSA)]
1. Vancomycin
iv. 50S ribosome methylation [Streptococci, Staphylococci, Enterococci]
1. Erythromycin, Clindamycin
b. Enzymatic destruction or inactivation of antibiotic
i. -lactamase [S. aureus, P. aeruginosa, Bacteroides, Enterococci]
1. -lactams (penicillins, cephalosporins)
ii. Acetyl-/phospho-/adenylyltransferases [Enterococci]
1. Aminoglycosides
iii. Acetyltransferase [Staphylococci, Streptococci, Neisseria]
1. Chloramphenicol
c. Alternative resistant metabolic pathway
i. Overproduction of PABA or thymidine nucleotides [Streptococci]
1. Sulfonamides
d. Decreased entry (natural resistance)
i. Pseudomonas aeruginosa
1. -lactam antibiotics
ii. Pseudomonas species
1. Fluoroquinolones
iii. E. coli, Pseudomonas
1. Aminoglycosides
e. Increased efflux (MDR may be encoded by single gene)
i. Streptococci, Staphylococci, Enterococci
1. Tetracyclines, Macrolides
ii. Pseudomonas species
1. Fluoroquinolones
f. Resistance can be minimized by only using antibiotics when need is established, selecting antibiotic on
basis of susceptibility tests, using adequate concentration and duration to prevent first and second step
mutants.
42
5. Describe:
a. Classifications of antimicrobial mechanisms of action
i. Inhibitions of synthesis or damage to cell wall
ii. Inhibition of synthesis or damage to cell membrane
iii. Modification of synthesis or metabolism of nucleic acids
iv. Inhibition or modification of protein synthesis
v. Modification of intermediary metabolism (folate metabolism)
b. Which mechanisms generally result in bacteriostatic or bactericidal effects
i. Whether antibiotic has –cidal or –static action is determined by its mechanim of action
(TARGET), concentration achieved in vivo and the specific microorganism.
ii. Bacteriostatic: organisms that are prevented from growing
1. Mechanism: inhibition of protein synthesis (exception are aminoglycosides, which are
–cidal), inhibition of intermediary metabolic pathways.
iii. Bactericidal: organisms are killed
43
1. Mechanism: inhibition of cell wall synthesis, disruption of cell membrane function,
interference with DNA function or synthesis
c. Advantages of bactericidal agents
i. Preferred in severe infection
ii. Act more quickly and their action is often irreversible
iii. Compensate for patients with an impaired host defense
iv. Required for treatment of infections in locations that are not accessible to host immune system
responses
d. Importance of pharmacokinetic and host factors in selection of antimicrobial therapy
i. Absorption: necessary to achieve adequate concentration in systemic circulation, accomplished
via oral or IV route, some infections can be treated with topicals (to skin or mucous membranes)
or non-absorbable drugs for tx of GI tract infections.
1. Oral route advantages: easy to administer, patient acceptance, lower cost.
2. Oral route disadvantages: GI upset, diarrhea, alter natural flora, incomplete absorption,
unsuitable for NPO pts.
a. Take on empty stomach: when antibiotic is unstable to increased acidity that
occurs when food is in the stomach
b. Take with food or meal: when drug is stable but may be irritating to stomach
3. IV route advantages: most rapid, predictable plasma levels
4. IV route disadvantages: greater training needed, greater expense, aseptic technique
required
5. Typically, patients are switched to oral antibiotics ASAP from IV (to reduce $ and
complications)
ii. Distribution:
1. CNS: most antibiotics distribute well to tissues outside of CNS, but vary in ability to cross
BBB.
a. Readily enter CSF: chloramphenicol, sulfonamides-trimethoprim, cephalosporins
(3rd/4th), rifampin-metronidazole
b. Enter with inflammation: penicillins, vancomycin, ciprofloxacin, tetracycline
44
c. Enter CSF poorly: aminoglycosides, cephalosporins (1st/2nd), erythromycin,
clindamycin
2. Selective distribution (accumulation):
a. Beneficial accumulation: clindamycin into bone (tx osteomyelitis), macrolides
into pulmonary cells (tx URI), tetracyclines into gingical crevicular fluid and
sebum (tx peridntitis and acne), excretion of nitrofurantoin into urine (tx UTI)
b. Increased potential for toxicity accumulation: aminoglycoside binding to cells of
inner ear and renal brush border ototoxicity and nephrotoxicity, tetracyclines
binding to Ca++ in developing bone and teeth abnormal bone growth and
brown tooth discoloration in children.
3. Fetus: antibiotics can have adverse effects on fetus if they cross placenta. Drugs to use
with caution during pregnancy: aminoglycosides, metronidazole, chloramphenicol,
tetracyclines, fluoroquinolones, voriconazole
iii. Elimination
1. Renal excretion: renal dosing (dose +/- frequency of antibiotic is adjusted based on renal
function) may be necessary for pt with kidney dysfunction. Renal status monitored with
serum creatinine (Scr) and creatinine clearance (CrCl)
2. Hepatic metabolism: metabolic drug-drug interactions, interpatient differences in
metabolic rate (genetic polymorphisms), hepatotoxic antibiotic actions. No lab value to
give estimate of liver’s ability to metabolize antibiotics. Generally, heptatically
metabolized antibiotics are avoided in pt with liver dysfunction
3. Duration of antimicrobial activity: half life information helps to guide dosage regimen
a. Too short or dose too low: resistance can develop, reccurance of infection
b. Too long: superinfection more likely
c. Too high: dose-related toxicities
d. Post-antibiotic effect: some anitbiotics continue to kill/inhibit bacterial growth
for several hours after [drug] falls below MIC, can be given less frequently
e. Concentration-dependent killing: antibiotics that kill bacteria faster when given
in doses that result in higher plasma concentrations
6. For the following drugs and drug categories (listed below) describe their: General mechanism of action (include
discussion of -cidal vs -static, mechanisms of resistance) Pharmacokinetics: Absorption (oral vs parenteral) /
Distribution (esp., CNS penetration) / Elimination (renal vs hepatic) Spectrum with respect to the following
broad bacterial classifications (bolded) and Clinical Uses (underlined) as related to common causative organisms
(italics) COCCI
a. Penicillin:
i. Substitutions on R group to increase acid stability (in GI), decrease renal excretion, increase
metabolic stability, minimize bacterial resistance, increase antibacterial spectrum by increasing
bacterial penetration
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ii. Mode of Action
1. Inhibit Bacterial Cell Wall Synthesis at Stage 3:
a. Stage 1: Synthesis-assembly of cell wall subunits occurring in the cytosol
(inhibited by fosfomycin and cycloserine.
b. Stage 2: Linear polymerization of subunits occurring at cell membrane (inhibited
by bacitracin & vancomycin). Target  transglycosylase.
c. Stage 3: Cross-linking of peptidoglycan polymers occurring at the cell wall
(inhibited by penicillins, cephalosporins. Target  transpeptidase,
carboxypeptidase (PBPs)
2. Penicillins are bactericidal to growing organisms, Pen G confined to gram + organisms
and gram – cocci.
3. Penicillin Binding Proteins: Beta-lactam antibiotics acylate several bacterial proteins
termed penicillin binding proteins (PBP’s). PBPs include (but aren’t limited to)
transpeptidase enzymes. Penicillin’s inhibit these enzymes by irreversible covalent
interaction. Certain beta-lactam antibiotics bind to distinct PBPs. Binding is not
uniform. Beta-lactams trigger autolytic activity via presence of endogenous autolytic
enzymes by depressing the natural inhibitory action of autolysins. Effect persists when
drug is gone due to penicillin’s covalent binding to bacterial proteins. Maximal killing is
function of growth rate of organism.
4. Resistance:
a. Production of penicillinase enzyme via plasmid. Production of beta-lactamase
induced in presence of penicillin. Transmitted to sensitive organisms by
bacteriophages. Major problem with staphylococcus.
b. Alterations in penicillin binding proteins, responsible for methicillin resistance in
staph (MRSA) and penicillin resistance in pneumococci.
c. Inability to penetrate bacterial cell
d. Metabolically inactive organisms or “L” forms can survive in hypertonic
environment like kidney.
iii. Pharmacokinetics
1. Absorption: moderately strong acids, highly water-soluble, acid-lability impairs oral
absorption of many penicillins and optimal absorption is on empty stomach. Chemical
modification of R group improves absorption by increasing acid stability. Rapidly
absorbed from IM parenteral sites.
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2. Distribution: throughout body, penetrates tissues poorly (ionized at physiological pH),
high concentration in liver, kidney, skin. Can enter inflamed tissues or membranes (CSF,
joint, eye) more readily than normal.
3. Metabolism-Excretion: most excreted as active drug via kidney, 1 hour half-life, 90% by
tubular secretion, blocked by probenecid, metabolism increases in cases of renal failure,
excreted in break milk (consider risk:benefit)
iv. Toxicity/Adverse reactions
1. Virtually non-toxic, except for hypersensitivity reactions
a. Type I reaction: very rare (0.05%) but life threatening, mediated by IgE (mast
cells) antibodies, onset: few minutes- 30 minutes. Symptoms: urticarial,
angioedema, respiratory obstruction, vascular collapse.
b. Type II reaction: rare, due to cytotoxic antibodies of IgG or IgM class,
complement-dependent cell destruction (hemolytic anemia).
c. Type III reaction delayed (˃72 hours), formation of IgG or IgM immune
complexes with penicillin that act as antigens and can activate complement and
lodge in tissue skin rash, serum sickness, arthralgias, allergic vasculitis
d. Type IV reaction: cell mediated allergy, delayed reactions, mediated by Tlymphocytes, skin eruptions and thrombocytopenia.
e. Other: idiopathic, most common reaction (1-4%) is maculopapular or
morbilliform rash (mild and reversible)
v. Individual Penicillins
1. Prototype Penicillin (narrow spectrum of antimicrobial activity)
a. Penicillin G: prototypical, powerful, inexpensive, penicillin of choice in most
cases, disadvantages: hydrolyzed by acid and penicillinase enzyme. 30-50%
bound to plasma protein. Most common use is via parenteral route to pts with
serious infections.
b. Penicillin V: acid resistant, better absorbed than Pen G, but still incompletely.
Pen V is preferred for oral therapy due to higher reliability of absorption.
Antimicrobial efficacy ˂ that of Pen G, but suitable for mild-mod infections.
2. Penicillinase-resistant penicillin
a. In order of efficacy (greatest first): Methicillin (obsolete) ˃ Nafcillin (parenteral,
erratic oral) ˃ Oxacillin (oral) ˃ Cloxacillin (oral) and Dicloxacillin (oral).
b. These antibiotics are ˂ potent against Pen G-sensitive organisms and are not
subsitutes for Pen G except when penicillinase-producing organisms are
present.
c. Acid resistance varies among group members, these penicillins are less
susceptibe to beta-lactamase than cephalosporins.
d. Eliminated by both renal and hepatic routes
e. All are narrow spectrum: gran +, gram – cocci.
3. Extended spectrum penicillin
a. Increased hydrophilicity allowing penetration through porins of outer
membrane of gram – organisms
b. Ampicillin and amoxicillin have additional activity against gram – bacilli. Acid
resistant, but not resistant to penicillinase, but can be given with beta47
lactamase inhibitors to extend their microbial spectrum. Amox is more
completely absorbed after oral administration and food interferes less with its
absorption, therefore LESS diarrhea.
c. Anti-pseudomonal penicillins (not resistant to penicillinase)
i. Ticarcillin and Piperacillin: given parenterally, effective against
Pseudomonas aeruginosa and enterococci and Bacterides fragilis.
4. Beta-lactamase inhibitors: Clavulanic Avid, Sulbactam, Tazobactam (combat penicillin
resistance)
a. Resemble beta-lactam molecules but have weak or no antibiotic activity. They
act as potent, irreversible inhibitors of beta-lactamase. Beta-lactamase
inhibitors extend antimicrobial spectrum of accompanying penicillin if bacterial
resistance is due to beta-lactamase destruction AND if inhibitor is active against
the particular beta-lactamase.
b. Clavulanic Acid + amoxicillin and ticarcillin (oral). Sulbactam + ampicillin
(parenteral). Taxobactam + piperacillin (parenteral).
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1. Vancomycin
a. Tricyclic glycopeptide acts by inhibiting cell wall synthesis at site different from penicillin (blocks linear
polymerization, Stage 2)
b. Poor oral absorption, administed IV (except for GI tract indications), excreted through kidneys (in renal
failure half-life extended 6-10 days)
c. Adverse reactions: chills, fever, rash (infusion related), ototoxicity is most SEVERE (pretreat with
acetaminophen and diphenhydramine)
d. Antimicrobial Spectrum/Clinical uses: use reserved from situations when less toxic agents are ineffective
or not tolerate (Pen allergy)
2. Daptomycin
a. Cyclic lipopeptide, parenteal, once dailym more rapidly bactericidal alternative to vancomycin.
Mechanism involves action at bacterial membrane and loss of intracellular ions leading to cell death.
Active against vancomycin resistant strains of staph (VRSA) and enterococci (VRE). Side effects: GI
disorders, fever, HA, dizziness).
3. Cephalosporins
a. Structure
b. Relative to Pen G and V, cephalosporins have broader spectrum of action against gram – bacteria, less
susceptibility to penicillinase, but cephalosporinases are emerging, less cross-reactivity in penicillin
sensitive patients (1-5%), classified into 3 or 4 generations, breadth of activity against gram – bacteria is
basis for classification.
c. Absorption: acid-stable, orally given include: cephalexin, cephradine, cefadroxil (1st gen), cefaclor,
cefuroxime (2nd gen), cefdinir, cefpodoxime, cefprozil (3rd). ALL OTHERS: IV/IM only.
d. Distribution: penetrate tissues and fluids (including placenta) except CSF and brain. Major feature of 3rd
gen CEPH is ability to penetrate into CSF.
e. Metabolism-Excretion: primarily excreted through kidneys. Cefotaxime is the only to undergo
metabolism.
f. Classifications
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i. First Generation: Cefaxolin, Cephradine, Cephalexin, effective against gram + cocci, gram – cocci
and some gram – bacilli. Rarely drugs of first choice, antibacterial spectrum like amoxicillin,
more stable than penicillins to many beta-lactams. Cefaxolin is prototype because low cost, low
toxicity, good penetration into tissues an 90 min half-life (longer)
ii. Second Generation: Cefuroxime, Ceftin, Cefotetan, Cefoxitin, Cefaclor, greater activity against
gram – bacteria than 1st gen. Little to no activity against Pseudomonas. Increased spectrum of
activity is due to increased penetration of gram – envelope and increased affinity for penicillinbinding proteins. Active against anaerobes like Bacteroides.
iii. Third Generation: Cefdinir, Cefotaxime, Ceftraixone, Ceftazidime, Cefixime, Cefpodoxime,
Cefprozil. Expanded gram – coverage compared to 2nd gen. More active against enteric gram –
bacilli. Ceftazidime has moderate antipseudomonal activity.
iv. Fourth Generation: Cefepime, similar to 3rd gen, but more resistant to chromosomal and
extended spectrum beta-lactamases, good against Pseudomonas species
g. Toxicity: well tolerated due to high selective toxicity
i. Allergy/hypersensitivity: anaphylaxis, skin rash, nephritis, hemolytic anemia, rxns not as severe
as with penicillins. Cross-hypersensitivity with penicillins (1-5%). Should not be given to pt with
HX of immediate sensitivity to penicillin.
ii. Other: nausea, vomiting, diarrhea. Super infection: with 2nd and 3rd gen (broader spectrum).
Action to suppress intestinal flora can intensify effort of oral anticoagulants.
h. Antimicrobial Spectrum/Clinical Uses
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WEEK 4
Antimicrobials III
Carbapenems: Imipenem-Cilastatin, Meropenem, Ertapenem
Protein synthesis inhibitors
Macrolides: Erythromycin, Clarithromycin, Azithromycin
Tetracyclines: Tetracycline, Doxycycline, Minocycline
Lincosamides: Clindamycin
Synthetic: Chloramphenicol
Aminoglycosides: Tobramycin, Gentamicin, Neomycin, Streptomycin
Streptogramins: Quinupristin / Dalfopristin
Oxazolidinones: Linezolid
Miscellaneous Antibiotics
Fluoroquinolones: Ciprofloxacin, Levofloxacin, Moxifloxacin
Nitrofurantoin, Metronidazole
Antimicrobial Resistance Mechanisms
1. Discuss the issues associated with an increased use of antimicrobial agents in the past several decades both in
terms of human medicine and agriculture.
a. World-wide problem, 90,000 people become ill with infections caused by MRSA and 15,000 of them
die/year.
b. Affects clinical outcome - associated with  death rates
c. Results in higher healthcare costs, Estimated >$5 billion, More expensive drugs
d. Leads to prolonged hospitalization
e. Increased challenges for appropriate management: Empiric therapy and Directed therapy
2. What are the major selective pressures that drive the emergence of antimicrobial resistance?
a. Antibiotic use for growth promotion, prophylaxis and therapy (FOOD/ANIMALS)
b. Antibiotic use for therapy and prophylaxis (HOSPITALIZED PATIENTS)
3. What are the major reservoirs of resistant organisms?
a. Food/Animals, Humans in community, Hospitalized patients
4. What are the indicators that antimicrobial resistance is a global issue and not merely related to the increased
usage of antibiotics in doctors’ offices and hospitals in developed countries?
a. Substaintially more antibiotics are used in feedlots that in medicine. In some third-world countries, the
antibiotic chloramphenicol is still used in animal feed lots to increase beef productivity.
b. Resistant organisms can arise in a single location and be transmitted to other parts of the world. Even in
seemingly isolated locations the identical resistance mechanisms exist that emerged only a few years
earlier in distant countries. Human movement in today’s world makes the spread of resistance very
easy and fast.
5. Describe the general mechanisms, for example intrinsic and acquired, associated with antimicrobial resistance.
a. Intrinsic: Resistance that is already present in particular types of bacteria, Gram-negative bacteria are
intrinsically resistant to some unmodified penicillins by the nature of their outer membrane
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i. Biofilms: highly resistant structure which is composed of a matrix (usually polysaccharide)
surrounding a group of bacteria. Bacteria in the biofilm are in a quiescent state of growth and
they are highly resistant to antibiotics because the compounds require actively growing cells.
Also, some antibiotics may not be able to diffuse through the matrix and would not reach a
clinically useful concentration.
ii. Efflux Pumps: mechanism for pumping out toxic substances from their cytoplasma through their
cell membranes. A single efflux pump can pump out completely different classes of antibiotics.
b. Acquired: Organisms acquire resistance from chromosomal mutations, or by the exchange of genetic
material with resistant bacteria, “infectious antibiotic resistance” because is can be transmitted from
one type of bacteria to another
i. Resistance Transfer Factors (RTF): plasmids that carry genes for their own replication (oriR) and
genes (tra) from their own transfer from 1 bacteria to another. Does not have to be of the same
species or genera. RTF’s may or may not have antibiotic resistance genes. If a bacteria has a
resistance factor plasmd (one that has genes for antimicrobial resistance)the RTF can mobilize
that resistance factor to another cell wither by integrating the resistance factor into its DNA or it
can provide transfer functions (sex pili) for the resistance factor that cannot transfer itself.
ii. IS elements: DNA pieces that carry genes encoding proteins from their own transmission from 1
replicon (plasmid, bacteriophage, chromosome) to another. They cannot transmit themselves
from 1 bacteria to another except by hopping into a piece of DNA that already has the ability to
transfer itself. They cannot replicate autonomously.
iii. Transposons: DNA fragments that are composed of 2 ID elements with extra baggage DNA
(usually antibiotic resistance genes, or virulence factor genes or metabolic factors) between the
IS elements. They can hop from 1 replicon to another and some can leave a copy of themselves
behind when they leave (replicative transposition). Other cannot leave a copy behind. Cannot
exist separately from another replicon (like IS elements)
1. Conjugative transposons: transposons that can mobilize themselves from 1 cell to
another, they have acquired transfer genes like the RTF’s have, but still need another
replicon for survival.
6. Discuss the several major biochemical and physiological mechanisms by which bacteria become resistant to
antimicrobial agents. Describe how the alteration in the regulation of a gene can lead to resistance. Which of
these mechanisms are responsible for clinically significant resistance?
a. Mutational alteration of the target of the antibiotic: the resistant organisms would be less of a “fit” than
the susceptible organisms because of altered target
i. Examples: mutation in a ribosomal protein resistance to AG, mutation in cell wall structure
beta-lactam resistance
b. Enzymatic alteration of the antibiotic target
i. Examples: vancomycin resistance, series of enzymes that alter the peptidoglycan precursor
target of vancomycin. Erythromycin, methylation of antibiotic target, a ribosomal protein, the
gene encoding this enzyme is regulated by erythromycin.
c. Enzymatic alteration of the antibiotic: genes for this resistance usually are associated with mobile
genetic elements (plasmid or transposon), but not always.
i. Beta-lactams
ii. AG’s modifying enzymes—all types leads to clinically significant resistance to AG’s
d. Transport of the antibiotic out of the cell
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i. Efflux pumps that transport different classes of antimicrobials OUT
e. Alteration in the expression of endogenous genes that lead to antimicrobial resistance
i. A mutation in promoter of gene encoding a porin that is required for antibiotic uptake
ii. Mutation in promoter of inducible gene results in constitutive expression of enzyme that
inactivates an antibiotic.
f. Routing of metabolic pathway around the antimicrobial target
i. Sulfonamides inhibit enzyme dihydrofolate reductaseinterference in folic acid and subsequent
pyrimidine synthesis in bacteria.
7. What is meant by
significant?
“inducible resistance", and can it be clinically
a. If an isolate of staphylococcus tests resistant to erythromycin and susceptible to clindamycin (a
lincosamide), it could still possess the inducible (iMLSB) resistance mechanism.
b. Macrolide-Lincosamide-Streptogramin B [MLSB] resistance is usually encoded by ermA or ermC genes.
c. Induction tests to determine clindamycin resistance utilize erythromycin and clindamycin disks located
on an agar plate in close proximity.
d. This can be performed by the standard disk-diffusion procedure. After overnight incubation, if there
is a flattening of the clindamycin zone adjacent to the erythromycin disk ("D-zone"); this indicates that
the organism has iMLSB resistance.
e. Organism is induced to turn on resistance genes to clindamycin in the presence of erythromycin (a
strong inducer)
Clinical Antibiotic Use: Bacterial Resistance and Antibiotic Resistance
1. Describe the laboratory tests used to determine measures of antimicrobial susceptibility.
a. Tube Dilution (Broth Dilution) Susceptibility Test
i. Serial dilutions of an antibiotic are made in liquid medium which is inoculated wth standardized
# of organisms and incubated a certain time. The tubes are examined for bacterial growth
(turbidity). The tube with the lowest concentration of antibiotic that has NO visible growth is
the MIC. (in example, MIC is 3.1)
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b. Disk diffusion or Kirby-Bauer Test
i. Suspension of isolate at specific concentration is spread evenly onto agar in petri dish. Disks
with defined concentrations of different antibiotics are placed onto agar surface. After
incubation the diameter of a clear circular zone of inhibition of growth is measured. The
diameter is proportional to the sensitivity of the organisms to the antibiotic. Using reference
tables the size of the zone can be linked to the MIC and the organism can be classified as
Susceptible, intermediate or resistant.
c. E test
i. Use a strip with a gradient of different concentrations of antibiotic. Strip is placed onto agar
that has been prepared with isolate of interest. After incubation, an ellipse of inhibition is
created and where the ellipse meets the strip, the MIC can be read. No tables are needed.
2. Define
a. antimicrobial susceptibility: MIC is less than antibiotic levels (maximum dose) that can be achieved in
the blood (exceptions: H. influenzae, S. pneumoniae)
b. MIC: minimum inhibitory concentration is the lowest concentration of an antimicrobial that will inhibit
the visible growth of a microorganism after overnight incubation. Variables for MIC: growth media, pH
of growth media (pH can affect drug activity, antibiotic specific. Low pH ↓ aminoglycoside effect, pH
effect are controlled in lab but in vivo pH is different), concentration of drug you need to inhibit bacteria
(organism concentration, higher=higher drug concentration).
c. MBC: minimum bactericidal concentration is the lowest concentration of the antibiotic that kiss 99.9%
of the original inoculum in a given time.
d. Bacteriostatic: inhibits bacterial growth, MBC=MIC
e. Bactericidal: kills bacteria, MBC ˃˃˃MIC
3. Discuss the variables that affect clinical outcome of antimicrobial use
a. HOST
i. Organism concentration at site of infection, pH in the site of infection, antibiotic concentration
and its penetration to the site of infection (urine= 100X % [plasma concentration of drug, soft
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tissue=50-100%, lung= 50-100%, joint=50-100%, abscess= 5-10% and CSF= 5-10%), host
defenses (immunocompromised hosts), some antibiotics depend on immune system to kill the
bacteria (they just inhibit growth).
4. Discuss clinical and economic considerations for parenteral vs oral administration of antimicrobials
a. CLINICAL
i. Determine diagnosis, Consider Age and Preexisting Dx, Define common organsims, Determine
likely resistance, Obtain proper cultures*, Initiate logical empiric therapy, Modify parenteral
therapy based on cultures and patient response, Consider home IV or PO therapy (* Serious
infections, immune compromised or poor response to prior therapy)
ii. Switch to PO from IV: Define infection, Determine cause of infection & antimicrobial
susceptibility, Achieve favorable response to IV, Determine if comparable levels can be achieved
with PO doses, Assure compliance, Measure levels, Follow clinical response
b. ECONOMIC
i. 100 Pts, 6 wk treatment course with IV or PO after 5 hospital days, Hosp Day = $1000
IV Atb = $100/day, PO Atb = $10/day, Home Care = $300/day IVC complication rate 3/1000 pt
days
5. Discuss the patterns and sources of the development of antimicrobial resistance
a. Bacteria evolve as very fast rates and can develop mechanisms of resistance to antibiotics faster than we
can develop new antibiotics. The more an antibiotic is used, the faster the resistance develops.
b. Where antibiotics are used: human use= 50%, 20% of that is hospital and 80% is community (20-50% is
unnecessary), agricultural use=50%, 20% of that is therapeutic and 80% is growth promotion and
prophylactic (40-80% highly questionable).
c.
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6. Discuss the strategies to deal with emerging antimicrobial resistance
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