1. Antimicrobial agents

2. Antibiotics Molecular targets

3. 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.

4. 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.

5. 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

6. Antibiotic Susceptibility Testing-MIC
Chl
Amp
Ery
Str
Tet
Disk Diffusion Test 8 4 2 1
Tetracycline (mg/ml)
MIC = 2 mg/ml
Determination of MIC
Size of zone of inhibition depends on sensitivity, solubility, rate of diffusion. Compare results to MIC tables generated using standards.

7. Zone Diameter Standards for Disk Diffusion Tests

8. 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.

9. 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)

10. Initiation of Protein Synthesis1 3 2
GTP
Initiation Factors
mRNA 3 1 2
GTP
30S Initiation Complex
f-met-tRNA
Spectinomycin 1 2
GDP + Pi
50S
70S Initiation Complex A P
Aminoglycosides

11. Elongation of Protein Synthesis
GTP A P Tu
GDP Ts + Pi
Tetracycline A P
Chloramphenicol G
GTP
GDP + Pi A P
Fusidic Acid
Erythromycin

12. Survey of Antibiotics Discuss one prototype for each category:
Mode of Action
Spectrum of Activity
Resistance
Synergy or Adverse Effects

13. Protein Synthesis Inhibitors
Mostly bacteriostatic
Selectivity due to differences in prokaryotic and eukaryotic ribosomes
Some toxicity - 70S ribosomes eukaryotic in mitochondria

14. Antimicrobials that Bind to the 30S Ribosomal Subunit

15. 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.

16. 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 beta-lactam antibiotics. The beta - lactams inhibit cell wall synthesis and thereby increase the permeability of the membrane to aminoglycosides.

17. 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.

18. 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

19. Antimicrobials that Bind to the 50S Ribosomal Subunit

20. 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.

21. 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

22. Antimicrobials that Interfere with Elongation Factors
Selectivity due to differences in prokaryotic and eukaryotic
elongation factors

23. 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

24. Inhibitors of Nucleic Acid Synthesis

25. Inhibitors of RNA Synthesis
Selectivity due to differences between prokaryotic and eukaryotic
RNA polymerase

26. 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.

27. Inhibitors of DNA Synthesis
Selectivity due to differences between prokaryotic and eukaryotic enzymes

28. 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

29. Antimetabolite Antimicrobials

30. Inhibitors of Folic Acid Synthesis
p-aminobenzoic acid + Pteridine
Dihydropteroic acid
Dihydrofolic acid
Tetrahydrofolic acid
Pteridine synthetase
Dihydrofolate synthetase
Dihydrofolate reductase
Thymidine
Purines
Methionine
Sulfonamides
Tetrahydrofolate required for the methyl group on methionine, and for thymidine and purine synthesis.
Trimethoprim

31. 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.

32. 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.

33. Anti-Mycobacterial Antibiotics

34. 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

35. 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)

36. Isoniazid (INH) (bacteriostatic )
Mode of action - Isoniazid inhibits synthesis of mycolic acids.
Spectrum of activity - Used in treatment of tuberculosis
Resistance - Has developed

37. 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.

38. 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)

39. Antimicrobial Drug Resistance Mechanisms
Inactivation of the antibiotic
b-lactamase
Chloramphenicol Acetyl Transferase

40. Antimicrobial Drug Resistance Mechanisms
Mutation in the target site.
Penicillin binding proteins (penicillins)
RNA polymerase (rifampin)
30S ribosome (streptomycin)

41. Antimicrobial Drug Resistance Mechanisms
Replacement of a sensitive enzyme with a resistant enzyme
Plasmid mediated acquisition of a resistant enzyme (sulfonamides, trimethoprim)