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 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.

Zone Diameter Standards for Disk Diffusion Tests

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 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

Elongation of Protein Synthesis GTP A P Tu GDP Ts + Pi Tetracycline A P Chloramphenicol G GTP GDP + Pi A P Fusidic Acid 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 beta-lactam 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 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

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.

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

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)

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 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)