Lecture # 37

Dr. Buckhaults
 Antibiotics Disrupt Cell Wall Synthesis, Protein
Synthesis, Nucleic Acid Synthesis and Metabolism
 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 Ery

8 4 2 1 0 Chl Amp
Tetracycline (g/ml)
MIC = 2 g/ml

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) Approx. MIC

Antimicrobial agent (ug/ml) for:
(amt. per disk)
and organism R I MS S R S
Ampicillin (10ug/disk)

Enerobacteriacae <11 12-13 >14 >32 <8

Haemophilus spp. <19 >20 >4 <2

Enterococci <16 >17 >16

Tetracycline (30 :γ ) <14 15 −18 >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)
Review of Initiation of Protein Synthesis
1 3
30S 2 GTP

1 2 3 GTP
Initiation Factors
f-met-tRNA
mRNA
Spectinomycin

GDP + Pi
2
50S
P A
1 1
2 GTP

70S Aminoglycosides
30S
Initiation Initiation
Complex Complex
Review of Elongation of Protein Synthesis

P A Tetracycline P A

Tu GTP Tu GDP + Pi

GTP Ts
Ts Tu
Ts GDP
Chloramphenicol

GDP
Fusidic Acid +
GTP
G

G GDP + Pi
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
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are needed to see this picture. 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 -lactam
antibiotics. The -lactams inhibit cell wall synthesis and
thereby increase the permeability of the membrane to
aminoglycosides.
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Tetracyclines (bacteriostatic)
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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.
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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
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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.
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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
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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
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(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)
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nalidixic acid, ciprofloxacin, ofloxacin,
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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
Basis of Selectivity-Bacteria synthesize folic
•
acid, humans do not. We get it from our diet. p-aminobenzoic acid + Pteridine
Review of Folic Acid Metabolism
• Pteridine
•
Tetrahydrofolate required for the methyl Sulfonamides
group on methionine, and for thymidine and synthetase
purine synthesis.

Dihydropteroic acid

Dihydrofolate
synthetase

Dihydrofolic acid
Dihydrofolate
Trimethoprim reductase

Tetrahydrofolic acid

Thymidine Methionine
Purines
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Sulfonamides,
Sulfones
(bacteriostatic)
• Mode of action - These antimicrobials are analogues of para-
aminobenzoic 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.
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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 gram-positive 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
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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

 QuickTimeª and a
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are needed to see this picture. 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)
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Isoniazid (INH)
(bacteriostatic )

• Mode of action - Isoniazid inhibits synthesis of mycolic acids.

• Spectrum of activity - Used in treatment of tuberculosis

• Resistance - Has developed

 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
β -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)