Antibiotics: Protein Synthesis, Nucleic Acid Synthesis, and Metabolism Dr. Jeffrey Patton Associate Professor Pathology, Microbiology, and Immunology USC-School of Medicine

1
Antibiotics Protein Synthesis, Nucleic Acid
Synthesis, and Metabolism Dr. Jeffrey
PattonAssociate ProfessorPathology,
Microbiology, and ImmunologyUSC-School of
Medicine
2
(No Transcript)
3
Antibiotics disrupt cell wall synthesis, protein
synthesis, nucleic acid synthesis, and metabolism
Basic sites of antibiotic activity
4
Principles and Definitions

• Selectivity
• Selectivity versus 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.

5
Principles and Definitions

• Selectivity
• Therapeutic index

• Categories of antibiotics
• Use of bacteriostatic versus 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.

6
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

7
Antibiotic Susceptibility Testing-MIC
Size of zone of inhibition depends on
sensitivity, solubility, rate of diffusion.
Compare results to MIC tables generated using
standards.
8
Zone Diameter Standards for Disk Diffusion Tests
R resistant, I intermediate susceptibility,
MS moderately susceptible, S susceptible
9
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.

10
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)

11
Review of Initiation of Protein Synthesis
12
Review of Elongation of Protein Synthesis
13
Survey of Antibiotics

• Discuss one prototype for each category
• Mode of Action
• Spectrum of Activity
• Resistance
• Synergy or Adverse Effects

14
Protein Synthesis Inhibitors

• Mostly bacteriostatic
• Selectivity due to differences in prokaryotic and
  eukaryotic ribosomes
• Some toxicity - 70S ribosomes
• eukaryotic mitochondria

15
Antimicrobials that Bind to the 30S Ribosomal
Subunit
16
Aminoglycosides (only bactericidal protein
synthesis inhibitor)streptomycin, kanamycin,
gentamicin, tobramycin, amikacin, netilmicin,
neomycin (topical)

• Modes of action -
• Irreversibly bind to proteins of the 30S subunit
  and freeze the 30S initiation complex
  (30S-mRNA-tRNA) and prevent initiation of
  translation.
• Increase the affinity of the A site for t-RNA
  regardless of the anticodon specificity. Induce
  misreading of the mRNA for proteins already being
  synthesized.
• Destabilizes microbial membranes
• Multiple modes of action is the reason this
  protein synthesis inhibitor is bactericidal.

17
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
  bacteria or in anaerobic environments (oxygen
  required for uptake of antibiotic)
• or intracellular bacteria.
• Resistance - enzymatic modification of antibiotic
  is most common
• Synergy - The aminoglycosides synergize with
  ß-lactam antibiotics. The ß-lactams inhibit cell
  wall synthesis and thereby increase the
  permeability of the membrane to aminoglycosides.

18
Tetracyclines (bacteriostatic)tetracycline,
minocycline, and doxycycline

• Mode of action - The tetracyclines reversibly
  bind to the 30S ribosome and inhibit binding of
  aminoacyl-t-RNA.
• Spectrum of activity - Broad spectrum Useful
  against intracellular bacteria such as Chlamydia,
  Mycoplasma, and Rickettsia
• Resistance - most common mode is active efflux of
  antibiotic out the cell
• 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
  unless necessary.

19
Spectinomycin (bacteriostatic)

• Mode of action - Spectinomycin reversibly
  interferes with mRNA 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

20
Antimicrobials that Bind to the 50S Ribosomal
Subunit
21
Chloramphenicol, Lincomycin, Clindamycin
(bacteriostatic)

• Mode of action - These antimicrobials bind to the
  50S ribosome and inhibit peptidyl transferase
  activity. No new peptide bonds formed, no
  elongation.
• Spectrum of activity - Chloramphenicol - Broad
  range Lincomycin and clindamycin - Restricted
  range
• Resistance - plamid-encoded acetyltransferase
  that renders the antibiotic incapable of binding
  to 50S subunit
• Adverse effects - Chloramphenicol is toxic (can
  suppress protein synthesis in bone marrow cells
  possibly producing aplastic anemia) but is used
  in life threatening situations such as the
  treatment of bacterial meningitis.

22
Macrolides (bacteriostatic)erythromycin,
clarithromycin, azithromycin, spiramycin

• Mode of action - The macrolides inhibit
  translocation of the ribosome, binding to 23S
  rRNA.
• Spectrum of activity - useful for treating Gram
  positive bacteria (in patients with penicillin
  allergies), Mycoplasma, Chlamydia, Legionella.
  Most Gram negative bacteria are resistant.
• Resistance - most common mechanism of resistance
  is methylation of the 23S rRNA inhibiting the
  binding of the drug.

23
Antimicrobials that Interfere with Elongation
Factors
Selectivity due to differences in prokaryotic and
eukaryotic elongation factors
24
Fusidic acid (bacteriostatic)

• Mode of action - Fusidic acid binds to elongation
  factor G (EF-G) and inhibits release of EF-G from
  the EF-G/GDP complex. Cant reload EF-G with GTP.
• Spectrum of activity - Gram-positive cocci like
  Staphylococcus

25
Inhibitors of Nucleic Acid Synthesis
26
Inhibitors of RNA Synthesis
Selectivity due to differences between
prokaryotic and eukaryotic RNA polymerase
27
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
  and aerobic gram-positive cocci.
• Resistance - Develops rapidly through mutations
  of RNA polymerase.
• Combination therapy - Since resistance is
  common, rifampin is usually used in combination
  therapy to treat tuberculosis.

28
Inhibitors of DNA Synthesis
Selectivity due to differences between
prokaryotic and eukaryotic enzymes
29
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 negative-supercoiling of DNA, causing
  positive supercoils to accumulate in advance of
  the replication fork. This inhibits DNA
  synthesis. Also inhibit DNA recombination and
  repair.
• Spectrum of activity - Gram-positive and
  -negative bacteria
• Resistance - mutations in topo genes as well as
  blocking uptake

30
Antimetabolite Antimicrobials
31
Inhibitors of Folic Acid Synthesis

• Basis of Selectivity-Bacteria synthesize folic
  acid, humans do not. We get it from our diet.
• Review of Folic Acid Metabolism
• Tetrahydrofolate required for the methyl group on
  methionine, and for thymidine and purine
  synthesis.

32
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 - due to permeability barriers
• 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.

33
Trimethoprim, Methotrexate, Pyrimethamine
(bacteriostatic)

• Mode of action - These antimicrobials bind 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 - due to decreased affinity for
  substrates
• 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.

34
Anti-Mycobacterial Antibiotics
35
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

36
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)

37
Isoniazid (INH) (bacteriostatic )

• Mode of action - Isoniazid inhibits synthesis of
  mycolic acids.
• Spectrum of activity - Used in treatment of
  tuberculosis

38
Antimicrobial Drug ResistancePrinciples 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.

39
Antimicrobial Drug ResistanceMechanisms

• 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)

40
Antimicrobial Drug ResistanceMechanisms

• Inactivation of the antibiotic
• ß-lactamase
• Chloramphenicol Acetyl Transferase

41
Antimicrobial Drug ResistanceMechanisms

• Mutation in the target site.
• Penicillin binding proteins (penicillins)
• RNA polymerase (rifampin)
• 30S ribosome (streptomycin)

42
Antimicrobial Drug ResistanceMechanisms

• Replacement of a sensitive enzyme with a
  resistant enzyme
• Plasmid mediated acquisition of a resistant
  enzyme (sulfonamides, trimethoprim)

43
Summary

• Antibiotics disrupt cell wall synthesis, protein
  synthesis, nucleic acid synthesis, and
  metabolism.
• These drugs take advantage of the differences
  between prokaryotic (them) and eukaryotic (us)
  cell metabolism.
• A bacteriostatic drug inhibits growth and a
  bactericidal drug kills the bacteria.
• The combination of two drugs is often used to
  treat an infection, for example a combination of
  bacteriostatic and bactericidal drugs or two
  inhibitors of folic acid synthesis.
• Bacteria use a number of ways to render
  antibiotics ineffective, from enzymatically
  destroying the drug to altering transport of the
  drug into or out of the cell.