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Antimicrobial agents
Antibiotics
Molecular targets
Principles and Definitions
• Selectivity
– Selectivity
vs
toxicity
• Therapeutic index
– Toxic dose/ Effective dose
• Categories of antibiotics
– Bacteriostatic
• Reversibly inhibit growth
• Duration of treatment sufficient for host defenses to
eradicate infection
– Bactericidal• Kill bacteria
• Usually antibiotic of choice for infections in sites such as
endocardium or the meninges where host defenses are
ineffective.
Principles and Definitions
• Selectivity
• Therapeutic index
• Categories of antibiotics
– Use of bacteriostatic vs bactericidal
antibiotic
• Therapeutic index better for bacteriostatic
antibiotic
• Resistance to bactericidal antibiotic
• Protein toxin mediates disease – use
bacteriostatic protein synthesis inhibitor to
immediately block synthesis of toxin.
Principles and Definitions
• Antibiotic susceptibility testing (in vitro)
– Bacteriostatic Antibiotics
• Minimum inhibitory concentration (MIC)
• Lowest concentration that results in inhibition of visible growth
(colonies on a plate or turbidity of liquid culture)
– Bactericidal Antibiotics
• Minimum bactericidal concentration (MBC)
• Lowest concentration that kills 99.9% of the original inoculum
Antibiotic Susceptibility Testing-MIC
Disk Diffusion Test
Determination of MIC
Str
Tet
8
4
2
1
Tetracycline (mg/ml)
MIC = 2 mg/ml
0
Ery
Chl
Amp
Size of zone of inhibition depends on sensitivity, solubility, rate of diffusion.
Compare results to MIC tables generated using standards.
Zone Diameter Standards for Disk Diffusion Tests
Zone diameter (mm)
Antimicrobial agent
(amt. per disk)
and organism
R
I
Enerobacteriacae
<11
12-13
Haemophilus spp.
<19
Enterococci
<16
Tetracycline (30 g)
<14
MS
Approx. MIC
(ug/ml) for:
S
R
S
>14
>32
<8
>20
>4
<2
Ampicillin (10ug/disk)
>17
15-18
>16
>19
>16
<4
Principles and Definitions
• Combination therapy
– Prevent emergence of resistant strains
– Temporary treatment until diagnosis is made
– Take advantage of antibiotic synergism
• Penicillins and aminoglycosides inhibit cell wall synthesis
and allow aminoglycosides to enter the bacterium and
inhibit protein synthesis.
• CAUTION: Antibiotic antagonism
– Penicillins and bacteriostatic antibiotics. Cell wall synthesis
is not occurring in cells that are not growing.
• Antibiotics vs chemotherapeutic agents vs
antimicrobials
– Antibiotics-naturally occurring materials
– Chemotherapeutic-synthesized in the lab (most
antibiotics are now synthesized and are therefore
actually chemotherapeutic agents.
Antibiotics that Inhibit Protein
Synthesis
•Inhibitors of INITATION
•30S Ribosomal Subunit (Aminoglycosides, Tetracyclines, Spectinomycin)
•50S Ribosomal Subunit (Chloramphenicol, Macrolides)
•Inhibitors of ELONGATION
•Elongation Factor G (Fusidic acid)
Initiation of Protein Synthesis
1 3
2 GTP
30S
1
2
3 GTP
Initiation Factors
f-met-tRNA
mRNA
Spectinomycin
3
GDP + Pi
P A
70S
Initiation
Complex
2
50S
1
Aminoglycosides
1
2 GTP
30S
Initiation
Complex
Elongation of Protein Synthesis
Tetracycline
P A
Tu GTP
Tu GDP +
GTP
Ts
Fusidic Acid
G GDP +
Pi
Ts
Tu
Ts
GDP
+
G
Pi
P A
GDP
Chloramphenicol
GTP
G GTP
P A
P A
Erythromycin
Survey of Antibiotics
Discuss one prototype for each category:
•Mode of Action
•Spectrum of Activity
•Resistance
•Synergy or Adverse Effects
Protein Synthesis Inhibitors
• Mostly bacteriostatic
• Selectivity due to differences in prokaryotic
and eukaryotic ribosomes
• Some toxicity - 70S ribosomes eukaryotic
in mitochondria
Antimicrobials that Bind to the
30S Ribosomal Subunit
Aminoglycosides
(only bactericidal protein synthesis
inhibitor)
streptomycin, kanamycin, gentamicin, tobramycin,
amikacin, netilmicin, neomycin (topical)
• Modes of action – Irreversibly bind to the 16S ribosomal RNA and freeze
the 30S initiation complex (30S-mRNA-tRNA) and
prevents initiation of translation.
– Increase the affinity of the A site for t-RNA regardless of
the anticodon specificity. Induces misreading of the
mRNA for proteins already being synthesized.
– Destabilize microbial membranes
– Multiple modes of action is the reason this protein
synthesis inhibitor is bactericidal.
Aminoglycosides (bactericidal)
streptomycin, kanamycin, gentamicin, tobramycin,
amikacin, netilmicin, neomycin (topical)
• Spectrum of Activity -Many gram-negative and
some gram-positive bacteria; Not useful for anaerobic
(oxygen required for uptake of antibiotic) or
intracellular bacteria.
• Resistance - Common
• Synergy - The aminoglycosides synergize with betalactam antibiotics. The beta - lactams inhibit cell wall
synthesis and thereby increase the permeability of
the membrane to aminoglycosides.
Tetracyclines
(bacteriostatic)
tetracycline, minocycline and doxycycline
• Mode of action - The tetracyclines reversibly bind to the 30S
ribosome and inhibit binding of aminoacyl-t-RNA to the acceptor
site on the 70S ribosome.
• Spectrum of activity - Broad spectrum; Useful against
intracellular bacteria
• Resistance - Common
• Adverse effects - Destruction of normal intestinal flora resulting
in increased secondary infections; staining and impairment of
the structure of bone and teeth. Not used in children.
Spectinomycin
(bacteriostatic)
• Mode of action - Spectinomycin reversibly interferes with m-RNA
interaction with the 30S ribosome. It is structurally similar to the
aminoglycosides but does not cause misreading of mRNA. Does not
destabilize membranes, and is therefore bacteriostatic
• Spectrum of activity - Used in the treatment of penicillin-resistant
Neisseria gonorrhoeae
• Resistance - Rare in Neisseria gonorrhoeae
Antimicrobials that Bind to the
50S Ribosomal Subunit
Chloramphenicol,
Lincomycin, Clindamycin
(bacteriostatic)
• Mode of action - These antimicrobials bind to the 50S ribosome
and inhibit peptidyl transferase activity. No new peptide bonds
formed.
• Spectrum of activity - Chloramphenicol - Broad range;
Lincomycin and clindamycin Restricted range
• Resistance - Common
• Adverse effects - Chloramphenicol is toxic (bone marrow
suppression) but is used in life threatening situations such as
the treatment of bacterial meningitis.
Macrolides (bacteriostatic)
erythromycin, clarithromycin, azithromycin, spiramycin
• Mode of action - The macrolides inhibit translocation of
the ribosome.
• Spectrum of activity - Gram-positive bacteria,
Mycoplasma, Legionella
• Resistance - Common
Antimicrobials that Interfere with
Elongation Factors
Selectivity due to differences in prokaryotic and eukaryotic
elongation factors
Fusidic acid
(bacteriostatic)
• Mode of action - Fusidic acid binds to elongation factor G (EF-G)
and inhibits release of EF-GDP from the EF-G/GDP complex. Can’t
reload EF-G with GTP.
• Spectrum of activity - Gram-positive cocci
Inhibitors of Nucleic Acid
Synthesis
Inhibitors of RNA Synthesis
Selectivity due to differences between prokaryotic and eukaryotic
RNA polymerase
Rifampin, Rifamycin, Rifampicin, Rifabutin
(bactericidal)
• Mode of action - These antimicrobials bind to DNA-dependent RNA
polymerase and inhibit initiation of mRNA synthesis.
• Spectrum of activity - Broad spectrum but is used most commonly
in the treatment of tuberculosis.
• Resistance - Common. Develops rapidly (RNA polymerase
mutations)
• Combination therapy - Since resistance is common, rifampin is
usually used in combination therapy to treat tuberculosis.
Inhibitors of DNA Synthesis
Selectivity due to differences between prokaryotic and eukaryotic
enzymes
Quinolones
(bactericidal)
nalidixic acid, ciprofloxacin, ofloxacin, norfloxacin,
levofloxacin, lomefloxacin, sparfloxacin
• Mode of action - These antimicrobials bind to the alpha subunit
of DNA gyrase (topoisomerase) and prevent supercoiling of
DNA, thereby inhibiting DNA synthesis.
• Spectrum of activity - Gram-positive cocci and urinary tract
infections
• Resistance - Common for nalidixic acid; developing for
ciprofloxacin
Antimetabolite Antimicrobials
Inhibitors of Folic Acid Synthesis
p-aminobenzoic acid + Pteridine
Sulfonamides
Tetrahydrofolate
required for the methyl
group on methionine,
and for thymidine and
purine synthesis.
Pteridine
synthetase
Dihydropteroic acid
Dihydrofolate
synthetase
Dihydrofolic acid
Trimethoprim
Dihydrofolate
reductase
Tetrahydrofolic acid
Methionine
Thymidine
Purines
Sulfonamides, Sulfones
(bacteriostatic)
• Mode of action - These antimicrobials are analogues of paraaminobenzoic acid and competitively inhibit pteridine synthetase,
block the formation of dihydropteroic acid.
• Spectrum of activity - Broad range activity against gram-positive
and gram-negative bacteria; used primarily in urinary tract and
Nocardia infections.
• Resistance - Common
• Combination therapy - The sulfonamides are used in
combination with trimethoprim; this combination blocks two distinct
steps in folic acid metabolism and prevents the emergence of
resistant strains.
Trimethoprim, Methotrexate,
Pyrimethamine (bacteriostatic)
• Mode of action - These antimicrobials binds to dihydrofolate
reductase and inhibit formation of tetrahydrofolic acid.
• Spectrum of activity - Broad range activity against grampositive and gram-negative bacteria; used primarily in urinary
tract and Nocardia infections.
• Resistance - Common
• Combination therapy - These antimicrobials are used in
combination with the sulfonamides; this combination blocks two
distinct steps in folic acid metabolism and prevents the
emergence of resistant strains.
Anti-Mycobacterial Antibiotics
Para-aminosalicylic acid (PSA)
(bacteriostatic)
• Mode of action - Similar to sulfonamides- competitively inhibit
pteridine synthetase, block the formation of dihydropteroic acid
• Spectrum of activity - Specific for Mycobacterium tuberculosis
Dapsone
(bacteriostatic)
• Mode of action - Similar to sulfonamides- competitively inhibit
pteridine synthetase, block the formation of dihydropteroic acid
• Spectrum of activity - Used in treatment of leprosy (Mycobacterium
leprae)
Antimicrobial Drug Resistance
Principles and Definitions
• Clinical resistance vs actual resistance
• Resistance can arise by new mutation or by gene
transfer (e.g. acquisition of a plasmid)
• Resistance provides a selective advantage.
• Resistance can result from single or multiple steps
• Cross resistance vs multiple resistance
– Cross resistance -- Single mechanism-- closely related
antibiotics are rendered ineffective
– Multiple resistance -- Multiple mechanisms -- unrelated
antibiotics. Acquire multiple plasmids. Big clinical
problem.
Antimicrobial Drug Resistance
Mechanisms
• Altered permeability
– Altered influx
• Mutation in a transporter necessary to import antibiotic can lead
to resistance.
– Altered efflux
• Acquire transporter gene that will pump the antibiotic out
(Tetracycline)
Antimicrobial Drug Resistance
Mechanisms
• Inactivation of the antibiotic
b-lactamase
Chloramphenicol Acetyl Transferase
Antimicrobial Drug Resistance
Mechanisms
• Mutation in the target site.
– Penicillin binding proteins (penicillins)
– RNA polymerase (rifampin)
– 30S ribosome (streptomycin)
Antimicrobial Drug Resistance
Mechanisms
• Replacement of a sensitive enzyme with a
resistant enzyme
– Plasmid mediated acquisition of a resistant
enzyme (sulfonamides, trimethoprim)