Lecture # 37 Dr. Buckhaults

1. Lecture # 37Dr. Buckhaults

2. Antibiotics Disrupt Cell Wall Synthesis, Protein Synthesis, Nucleic Acid Synthesis and Metabolism

3. Principles and Definitions • Selectivity • Selectivityvstoxicity • 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. Disk Diffusion Test Determination of MIC Str Tet Ery 4 2 1 0 8 Tetracycline (g/ml) Chl Amp MIC = 2 g/ml Antibiotic Susceptibility Testing-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. 3 1 GTP 2 30S GTP 1 2 3 Initiation Factors f-met-tRNA mRNA Spectinomycin 3 50S GDP + Pi GTP 2 2 P A 1 1 Aminoglycosides 70S Initiation Complex 30S Initiation Complex Review of Initiation of Protein Synthesis

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

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

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 b-lactam antibiotics. The b-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 negative-supercoiling of DNA, causing positive supercoils to accumulate in advance of the replication fork. This inhibits DNA synthesis. • Spectrum of activity - Gram-positive cocci and urinary tract infections • Resistance - Common for nalidixic acid; developing for ciprofloxacin

29. Antimetabolite Antimicrobials

30. Sulfonamides p-aminobenzoic acid + Pteridine Pteridine synthetase Dihydropteroic acid Dihydrofolate synthetase Dihydrofolic acid Dihydrofolate reductase Trimethoprim Tetrahydrofolic acid Methionine Thymidine Purines 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.

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. 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.

37. 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)

38. Antimicrobial Drug ResistanceMechanisms • Inactivation of the antibiotic b-lactamase Chloramphenicol Acetyl Transferase

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

40. Antimicrobial Drug ResistanceMechanisms • Replacement of a sensitive enzyme with a resistant enzyme • Plasmid mediated acquisition of a resistant enzyme (sulfonamides, trimethoprim)

41. Lecture # 38Dr. Buckhaults

42. Exchange of Genetic Information

43. Mutations in Bacteria • Mutations arise in bacterial populations • Induced • Spontaneous • Rare mutations are expressed • Bacteria are haploid • Rapid growth rate • Selective advantage enriches for mutants • Gene transfer occurs in bacteria

44. General Features of Gene Transfer in Bacteria • Unidirectional • Donor to recipient • Donor does not give an entire chromosome • Merozygotes • Gene transfer can occur between species

45. Transformation • Definition: Gene transfer resulting from the uptake of DNA from a donor. • Factors affecting transformation • DNA size and state • Sensitive to nucleases • Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus) • Competence factors • Induced competence

46. Transformation • Steps • Uptake of DNA • Gram + • Gram - • Recombination • Legitimate, homologous or general • recA, recB and recC genes • Significance • Phase variation in Neiseseria • Recombinant DNA technology

47. Transduction • Definition: Gene transfer from a donor to a recipient by way of a bacteriophage • Bacteriophage (phage): A virus that infects bcteria

48. Head/Capsid Contractile Sheath Tail Tail Fibers Base Plate Phage Composition and Structure • Composition • Nucleic acid • Genome size • Modified bases • Protein • Protection • Infection • Structure (T4) • Size (80 X 100 nm) • Head or capsid • Tail

49. Infection of Host Cells by Phages • Adsorption • Tail fibers • Receptor is LPS for T4 • Irreversible attachment • Base plate • Sheath Contraction • Nucleic acid injection • DNA uptake

50. Microbe Library, American Society for Microbiology www.microbelibrary.org