Transcript PlayingWithMicrobesLabPart3L6

Drugs,
Microbes, Host
– The Elements
of
Chemotherapy
Chapter 12
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12.1 Principles of Antimicrobial Therapy
• Goal of antimicrobial chemotherapy: administer
a drug to an infected person, which destroys the
infective agent without harming the host’s cells
• Rather difficult to achieve this goal
• Chemotherapeutic agents described with regard
to their origin, range of effectiveness, and
whether they are naturally produced or
chemically synthesized
The Origins of Antimicrobial Drugs
• Antibiotics are common metabolic products of
aerobic bacteria and fungi
• Bacteria: Streptomyces and Bacillus
• Molds: Penicillium and Cephalosporium
• Chemists have created new drugs by altering the
structure of naturally occurring antibiotics
• Also Searching for metabolic compounds with
antimicrobial effects in species other than
bacteria and fungi
12.2 Interactions Between Drug and
Microbe
• Goal of antimicrobial drugs
• Disrupt the cell processes or structures of
bacteria, fungi, and protozoa
• Or inhibit virus replication
• Most interfere with the function of enzymes
required to synthesize or assemble
macromolecules or destroy structures already
formed in the cell
• Drugs should be selectively toxic- they kill or
inhibit microbial cells without damaging host
tissues
Targets of Antimicrobials
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4. Protein synthes is inhibitors acting
on ribosomes
1. Cell wall inhibitors
Block synthesis and repair
Penicillins
Cephalosporins
Carbapenems
Vancomycin
Bacitracin
Fosfomycin
Isoniazid
Ribosome
Erythromycin
Clindamycin
Synercid
Pleuromutilins
Substrate
2. Cell membrane
Causelossofselective permeability
Polymyxins
Daptomycin
Enzyme
Product
Site of action
30S subunit
Aminoglycosides
Gentamicin
Streptomycin
Tetracyclines
Glycylcyclines
Both 30S
and 50S
3. DNA/RNA
Inhibit replication and transcription
Inhibit gyrase(unwinding enzyme)
Quinolones
Inhibit RNA polymerase
Rifampin
Site of action
50S subunit
Blocks initiation of protein
synthesis
Linezolid
mRNA
DNA
5. Folic acid synthesis
Block pathways and inhibit
metabolism
Sulfonamides (sulfa drugs)
Trimethoprim
Mechanisms of Drug Action
• Inhibition of cell wall synthesis
• Inhibition of nucleic acid structure and function
• Inhibition of protein synthesis
• Interference with cell membrane structure or
function
• Inhibition of folic acid synthesis
Antimicrobial Drugs that Affect the
Bacterial Cell Wall
• Active cells must constantly synthesize new
peptidoglycan and transport it to the proper
place in the cell envelope
• Penicillins and cephalosporins react with one
or more of the enzymes required to complete
this process
• Bactericidal antibiotics
Penicillin and Bacterial Cell Wall
Figure 12.2
Penicillin and Bacterial Cell Wall
Figure 12.3
Other Drugs Targeting Cell Wall
• Bacitracin – from Bacillus subtilis,
Neosporin
• Isoniazid – TB
• Vancomycin- MRSA, penicillin allergies
Antimicrobial Drugs that Affect Nucleic
Acid Synthesis
• Block synthesis of nucleotides
• Inhibit replication
• Stop transcription
• Inhibit DNA synthesis
Transcription of DNA
• This is a difficult target because the process is
well conserved
• Rifampin – Binds to RNA polymerase
• Specific for bacteria
• Prevents elongation of transcript after initiation
• Useful drug for tuberculosis
DNA Replication
• Few clinical drugs affect polymerization
• Mechanisms are conserved: lead to toxicity
• DNA gyrase inhibitors
• Nalidixic acid inhibits DNA gyrase
• Fluouroquinolones
• Ciprofloxacin – used in the 2001 anthrax attack
Antimicrobial Drugs that Block Protein
Synthesis
• Inhibit translation by reacting with the ribosomemRNA complex
• Prokaryotic ribosomes are different from
eukaryotic ribosomes- selective
Drugs Affecting Protein Synthesis
Figure 12.4
Antimicrobials and Protein Synthesis
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Antimicrobial Drugs that Disrupt Cell
Membrane Function
• Damaged membrane invariably results in
death from disruption in metabolism or lysis
• Specificity for particular microbial groups
based on differences in the types of lipids in
their cell membranes
Membrane-Active Drugs
• Detergents
• Bind to phospholipid and lipid A
• Disrupt membranes
• Poor selective toxicity
• Polymyxins in Triple Antibiotic Creams
• Must be used topically
Antimicrobial Drugs that Inhibit Folic
Acid Synthesis
• Sulfonamides and trimethoprimcompetitive inhibition
• Supplied to cells in high concentrations to
make sure enzyme is constantly occupied
with the metabolic analog rather than
the true substrate
Inhibition of Folic Acid Synthesis
Figure 12.5
Concept Check
Which molecule do sulfanomides disrupt
formation of?
A. Peptidoglycan
B. RNA
C. Glycogen
D. Folic acid
12.3 Survey of Major Antimicrobial Drug
Groups
• About 260 different antimicrobial drugs
• Classified in 20 drug families
• Largest number of antimicrobial drugs are for
bacterial infections
Spectrum of Activity
Penicillins
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Nucleus
(Aminopenicillanicacid)
• Discovered by Fleming
• Block cross-linking of
peptidoglycan
• All have beta-lactam
ring
R Group
Betalactam
Thiazolidine
S
N
CO
CH3
CH3
N
Nafcillin
O
COOH
S
CH
N
CO
COONa
CH3
CH3
N
O
COOH
S
Ticarcillin
• Different spectra of
action
Cl
S
CO
N
CH3
CH3
N
• Penicillinases; betalactamases
N
O
O
COOH
CH3
Cloxacillin
S
CH
CO
N
CH3
COONa
Carbenicillin
CH3
N
O
COOH
Cephalosporins
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R Group1
O
• Different ring
structure
• Block crosslinking of
peptidoglycan
Basic Nucleus
C
CH2
O
Cephalothin
(first generation*)
S or O
5
6
4
3
N1
2
COOH
O
CH2
N
OC
CH3
CH2
N
S
NH2
N
CH3
CH2
CH2
N
CH3
Moxalactam
N (thirdgeneration)
O
N
OH
CH
C
CH2
S
N
N
CH3
NH2
S
Cefepime
(fourthgeneration)
O
N
C
C
NH
CH2
• Different spectra
of action
Cefotiam
(secondgeneration)
N
N
S
COONa
• All have betalactam ring
N7
S
R Group2
N
N
CH3
OCH3
OH
Ceftobiprole
(fifth generation)
N
N
NH2
S
N
N
O
O
*New improved versions of drugs are referred to as new “generations.”
NH
Subgroups and Uses of
Cephalosporins
• Broad-spectrum
• Resistant to most penicillinases
• Cause fewer allergic reactions than
penicillins
• Four generations of cephalosporins exist
based on their antibacterial activity
Concept Check
What is the mechanism of action of penicillin?
A. Disrupting protein synthesis
B. Block peptidoglycan cross-linking
C. Disrupt the cell membrane
D. Inactivate DNA transcription
Aminoglycosides
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50S
aa
aa
• Binds 30S
30S
• Distorts the
ribosome
50S
aa
aa
30S
• Causes
translation errors
• Streptomycin
• Neomycin
Triple antibiotic
cream
30S
50S
30S
30S
aa 50S aa
30S
30S
mRNA
Aminoglycoside Drugs
• Products of various species of soil actinomycetes
in the genera Streptomyces and Micromonospora
• Relatively broad spectrum because they inhibit
protein synthesis
• Subgroups and uses
• Aerobic gram-negative rods and certain gram-positive
bacteria
• Streptomycin: Bubonic plague and tularemia and good
antituberculosis agent
• Gentamicin: Less toxic and used for gram-negative
rods
Tetracyclines
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50S
aa
aa
Aminoglycosides
mRNA
is misread,
protein is
incorrect
mRNA
30S
• Blocks the A site
Chloramphenicol
• Prevents tRNA entry
• Reversible reaction
50S
aa
aa
30S
mRNA
Prevent
initiationand
blockribosome
assembly
Oxazolidinones
• Bacteriostatic
Formation
of peptide
bonds is
blocked
30S
50S
Tetracyclines
30S
tRNA is
blocked,
no protein is
synthesized
mRNA
30S
aa 50S aa
Erythromycin
30S
30S
Ribosome
is prevented
From
translocating
mRNA
Tetracycline Antibiotics
• Broad-spectrum
• GI problems in some
• Subgroups and uses
• Gram –positive and gram-negative rods and cocci
• Aerobic and anaerobic bacteria
• Mycoplasmas, rickettsias, and spirochetes
• Doxycycline and minocycline for sexually
transmitted diseases, Rocky Mountain spotted
fever, Lyme disease, typhus, Mycoplasma
pneumonia, cholera, leptospirosis, acne, even
some protozoan
Chloramphenicol
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50S
aa
aa
• Binds 50S
• Prevents peptide
bond formation
• Toxic
Aminoglycosides
mRNA
is misread,
protein is
incorrect
mRNA
30S
50S
aa
aa
Chloramphenicol
30S
mRNA
Prevent
initiationand
blockribosome
assembly
Oxazolidinones
• Aplastic anemias
Formation
of peptide
bonds is
blocked
30S
50S
Tetracyclines
30S
tRNA is
blocked,
no protein is
synthesized
mRNA
30S
aa 50S aa
Erythromycin
30S
30S
Ribosome
is prevented
From
translocating
mRNA
Erythromycin and Clindamycin
• Erythromycin
• Large lactone ring with sugars attached
• Relatively broad-spectrum
• Fairly low toxicity
• Blocks protein synthesis by attaching to the ribosome
• Mycoplasma pneumonia, legionellosis, Chlamydia
infections, pertussis, diphtheria
• Clindamycin
• Broad-spectrum
• Derived from lincomycin
• Causes adverse reactions in the gastrointestinal tract,
so applications are limited
Tetracylcines and Erythromycin
Figure 12.10
Oxazolidinones
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50S
aa
aa
Aminoglycosides
mRNA
is misread,
protein is
incorrect
mRNA
30S
50S
aa
aa
• Bind to the ribosome
• Prevent initiation
Chloramphenicol
30S
mRNA
Prevent
initiationand
blockribosome
assembly
Oxazolidinones
• Linezolid
Formation
of peptide
bonds is
blocked
30S
50S
Tetracyclines
30S
tRNA is
blocked,
no protein is
synthesized
mRNA
30S
aa 50S aa
Erythromycin
30S
30S
Ribosome
is prevented
From
translocating
mRNA
Antibacterial Drugs Targeting Folic Acid
Synthesis
• Sulfonamides, Trimethoprim, and Sulfones
• Sulfonamides
• Sulfa drugs
• Very first modern antimicrobial drug
• Synthetic
• Shigellosis, acute urinary tract infections, certain protozoan infections
• Trimethoprim
• Inhibits the enzymatic step immediately following the step inhibited by
sulfonamides in the synthesis of folic acid
• Often given in combination with sulfamethoxazole
• One of the primary treatments for Pneumocystis (carinii) jiroveci pneumonia
(PCP) in AIDS patients
• Sulfones
• Chemically related to sulfonamides
• Lack their broad-spectrum effects
• Key drugs in treating Hansen’s disease (leprosy)
Sulfa drugs
• Tetrahydrofolate is an important carbon and
hydrogen carries
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Sulfonamides
inhibit enzyme
Trimethoprim
inhibits enzyme
Folic acid is coenzyme
required to synthesize
Purines
PABA
(a)
Dihydrofolic
acid
Tetrahydrofolic
acid (folic acid)
Pyrimidines
Aminoacids
Only certain bacteria
perform this step
PABA
(substrate)
Structural differences
H
Dihydropteroic
acid
H
N
HO
O
S
O
N
H
Sulfa drug
(inhibitor)
Sulfa drug
(inhibitor)
N
H
Sulfanilamide
(b)
O
C
H
H
PABA
Enzyme
(c)
Enzyme
Enzyme
Higherlevelsof
sulfadrug
morelikelyto
bindtoenzyme
Agents to Treat Fungal Infections
• Fungal cells are eukaryotic, so present special
problems
• Majority of chemotherapeutic drugs are
designed to act on bacteria and are ineffective
for fungal infections
• Similarities between fungal and human cellstoxicity to humans
• Four main groups
• Macrolide polyene antibiotics, Griseofulvin,
Synthetic azoles, Flucystosine
Anti-Fungal Drugs
Polyenes bind membrane
Nystatin
Amphotericin B
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OH
OH
OH
OH
OH
O
O
OH
O
OH
(a)
OH
Anti-Fungal Drugs
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• Azoles
• Inhibit ergosterol
synthesis
N
N
• Griseofulvin
• effective against
ringworm
• inhibits
microtubules
• prevents cell
division
(b)
C
Cl
Synthetic Azoles
• Broad-spectrum antifungal agents
• Ketoconazole, fluconazole, clotrimazole, and
miconazole
• Ketoconazole: orally and topically for
cutaneous mycoses, vaginal and oral
candidiasis, and some systemic mycoses
• Fluconazole: used in selected patients for
AIDS-related mycoses
• Clotrimazole and miconazole: mainly topical
ointments for infections in the skin, mouth,
and vagina
Flucystosine
• Analog of the nucleotide cytosine
• Can be used to treat certain cutaneous
mycoses
• Usually combined with amphotericin B for
systemic mycoses
Antihelminthic Drug Therapy
• Flukes, tapeworms, and roundworms have
greater similarities to human physiology
• Using drugs to block their reproduction is usually
not successful in eradicating adult worms
• Most effective drugs immobilize, disintegrate, or
inhibit the metabolism of all stages of the life
cycle
Mebendazole and Thiabendazole
• Broad-spectrum
• Used in several roundworm intestinal infestations
• Inhibit the function of microtubules of worms,
eggs, and larvae
Antiviral Chemotherapeutic Agents
• Selective toxicity is almost impossible to
achieve because a single metabolic system is
responsible for the well-being of both virus
and host
• Several antiviral drugs have been developed
that target specific points in the infectious
cycle of viruses
• Three major modes of action:
• Barring penetration of the virus into the host cell
• Blocking the transcription and translation of viral
molecules
• Preventing the maturation of viral particles
Anti-Virals
• Amantadine
• Inhibits the replication of Influenza A virus
• Acyclovir
• Disrupts Herpesviruses replication
• Nucleoside analog
• Ribavirin
• Blocks RNA synthesis
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Table 12.5 Actions of Antiviral Drugs
Mode of Action
Antiviral
Agents
Inhibition of Virus
Entry: Receptor/
Fusion/Uncoating
Inhibitors
Examples
1
Effects of Drug
HIV
Inhibition of Virus
Entry: Receptor/
Fusion/Uncoating
Inhibitors
Blocks HIV infection by
preventing the binding of
viral GP-41 receptors to cell
receptor 1 , thereby preventing
fusion of virus with cell
Amantadine and its
relatives, zanamivir
(Relenza), oseltamivir
(Tamiflu)
Block entry of influenza
virus by interfering with
2 3
Influenza
fusion of virus with cell
virus
membrane (also release);
stop the action of influenza
neuraminidase, required
Drug molecules
for entry of virus into cell (also
No infection
assembly) 2 3
Drug
molecule
Nucleus
Inhibition of Nucleic Acyclovir (Zovirax), other Purine analogs that
“cyclovirs,” vidarabine
terminate DNA replication in
Acid Synthesis
herpesviruses 4
Ribavirin
Herpesvirus
Purine analog, used for
respiratory syncytial
virus (RSV) and some
hemorrhagic fever viruses
4
Drug
molecule
Noviral
DNA synthesis
HIV
Zidovudine (AZT),
lamivudine (3T3),
didanosine (ddI),
zalcitabine (ddC), and
stavudine (d4T)
Nevirapine, efavirenz,
delavirdine
Inhibition of Viral
Assembly/Release
Indinavir, saquinavir
Drug molecules
Nucleotide analog reverse
transcriptase (RT) inhibitors;
stop the action of RT in HIV,
blocking viral DNA
production 5
Nonnucleotide analog reverse
transcriptase inhibitors;
attach to HIV RT binding site,
stopping its action 6
5
RT
Drug
molecule
Protease inhibitors; insert into
HIV protease, stopping its
action and resulting in inactive
noninfectious viruses 7
6
No reverse
transcription
HIV
7
Drug
molecule
Interferon (IFN): An Alternative to
Artificial Drugs
• Glycoprotein produced by fibroblasts and leukocytes in
response to various immune stimuli
• Produced by recombinant DNA technologies
• Known therapeutic benefits:
• Reducing the time of healing and some of the
complications in certain infections
• Preventing or reducing some symptoms of cold and
papillomaviruses
• Slowing the progress of certain cancers
• Treating a rare cancer called hairy-cell leukemia,
hepatitis C, genital warts, and Kaposi’s sarcoma in
AIDS patients
• Often results in serious side effects
Concept Check
Which of these antimicrobial compounds would
be best to treat a severe genital herpes
outbreak?
A. Amantadine
B. Acyclovir
C. Azidothymidine
D. Rifampin
12.4 Interactions Between Microbes and
Drugs: The Acquisition of Drug Resistance
• Drug resistance: an adaptive response in which
microorganisms begin to tolerate an amount of drug that
would ordinarily be inhibitory
• Can be intrinsic or acquired
• Microbes become newly resistant to a drug after
• Spontaneous mutations in critical chromosomal genes
• Acquisition of entire new genes or sets of genes via
transfer from another species (plasmids called
resistance (R) factors)
Resistance Mechanisms
• Antibiotics are present in nature
• Microbes are capable of adapting quickly to
selective pressures
• Drug resistance has arisen for all antibiotics
• Two main strategies employed by microbes
• Prevent access of the drug to the target site
• Alter the nature of the target site
Mechanism of Resistance
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1. Druginactivation
Result
S
R
1
O
S
R
Penicillinase
N
COOH
O
C
OH
Activepenicillin
N
H
1. Inactivation of a drug like penicillin by
penicillinase, an enzyme that cleaves
a portion of the molecule and renders
it inactive.
CH3
CH3
COOH
Inactivepenicillin
2. Decreasedpermeability
2. The receptor that transports the
drug is altered, so that the drug
cannot enter the cell.
Normal
receptor
Drug
2
Cell surface
of microbe
Cell surface
of microbe
Different
receptor
3. Activationofdrugpumps
Drug
Inactive
drug
pump
3
Cell surface
of microbe
Active
drug
pump
3. Specialized membrane proteins
are activated and continually
pump the drug out of the cell.
Cell surface
of microbe
4. Change in drug binding site
4. Binding site on target (ribosome)
is altered so drug has no effect.
4
5. Use of alternate metabolic pathway
Drug acts
5
A
B
C
X
C1
D
Product
D1
5. The drug has blocked the usual
metabolic pathway (green), so the
microbe circumvents it by using an
alternate, unblocked pathway that
achieves the required outcome (pink).
Transferring Drug Resistance
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Antibiotic Resistance
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Not drug-resistant
Drug-resistant mutant
Exposure
to drug
(a) Population of microbial cells
Remaining
population
grows
overtime
(b) Sensitive cells ( ) eliminated by drug;
resistant mutants survive
(c) All cells are now resistant
Preventing Drug Resistance
• Limit drug use - less selective pressure
• Proper drug use - viruses are not affected, use
full dose to ensure elimination of pathogens
• Narrow range antibiotics - kill only the targeted
microbes; less likely complications
• Multiple drug treatments - drugs can work
synergistically; much less likely to get drug
resistance.
Interaction Between Drug and Host
• Toxicity to organs
• Allergies
• Disruption of
normal flora
• Other adverse
effects
12.5 Selecting and Antimicrobial
• Antimicrobial susceptibility testing
• Therapeutic index considerations
Disk Diffusion Assays
• Kirby-Bauer
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Kirby-Bauer Disc Diffusion Test*
• Standardized
conditions
Enrofloxacin 5 g
(R < 17 mm;S 22 mm)
Oxytetracycline 30g
(R<17 mm;S 22mm)
0
mm
ENR
1 5
2
3
Gentamicin 10 g
(R < 17 mm; S 21 mm)
4
S
• Zones of
inhibition
• Larger zone
indicates more
susceptible
• Smaller zone
indicates more
resistant
OT
R
30
CTX
30
GN
I 10
AMP
I 10
S
R
C
30
Cefotaxime 30 g
(R < 14 mm; S 23 mm)
Ampicillin10g
(R<14mm;S22mm)
Chloramphenicol 30 g
(R < 21 mm; S 21 mm)
ENR = Antibiotic carrier (disc)
= Region of
5
imprinted with abbreviation
bacterial growth
and concentration
Disc Diffusion Test (schematic).
Example and evaluation of asensitivity test, agar diffusion method.
R = resistant, I = intermediate, S = sensitive
= Zone of Inhibition
(a) *R and S values differ from table 12.7 due to differing concentrations of the antimicrobials.
b: © Kathy Park Talaro
(b)
E-Test Strips
• Drug gradient used
• Can determine MIC
• Read where the zone
touches the strip
Tube Dilution Assay
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Same inoculum size of test bacteria added
Control
• Drug diluted in
series
• Inoculate and
incubate
0
Negative
control
0.2
0.4
0.8
1.6
3.2
6.4
12.8
m g/ml
Increasing concentration of drug
(a)
Growth
No growth
• Look for growth
(MIC)
(b)
Image courtesy David Ellis.
Therapeutic Index
• What is the best drug to use?
• Lowest risk of side effects versus
• Highest probability of killing the pathogen
• 50 µg is toxic and 5 µg is effective; T.I. = 10
• 50 µg is toxic and 1 µg is effective; T.I. = 50
• Higher T.I. are better
Concept Check
What does a large zone of inhibition indicate on a
Kirby Bauer disk diffusion assay?
A. High toxicity to the patient
B. The bacterium is resistant to the antibiotic
C. Low toxicity to the patient
D. The bacterium is susceptible to the antibiotic