Chemotherapy of Bacterial Infections Antibiotics Definitions of Antibiotics

Chemotherapy of Bacterial Infections ~~~~ Antibiotics

Definitions of Antibiotics • OLD: An antibiotic is a chemical substance produced by various species of microorganisms that is capable in small concentrations of inhibiting the growth of other microorganisms • NEW: An antibiotic is a product produced by a microorganism or a similar substance produced wholly or partially by chemical synthesis, which in low concentrations, inhibits the growth of other microorganisms

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History Paul Ehrlich “Magic Bullet” ü Chemicals with selective toxicity ORIGIN: DRUG: NOBEL: 1908 Selective Stains Arsphenamine (1910) “ 606” Salvarsan

History (cont’d) Gerhard Domagk ü Drugs are changed in the body ORIGIN: DRUG: Prontosil (Only active in vivo) Sulfanilamide (1935) NOBEL: 1939

History Alexander Fleming ü Microbes make antibiotics ORIGIN: DRUG: moldy culture plate Penicillin (1928) NOBEL: 1945 (cont’d)

History (cont’d) Selman Waksman ü Soil Streptomyces make antibiotics ü comes up with definition of antibiotic ORIGIN: DRUG: Penicillin development Streptomycin (1943) NOBEL: 1952

The Ideal Drug* 1. Selective toxicity: against target pathogen but not against host ü LD 50 (high) vs. MIC and/or MBC (low) 2. Bactericidal vs. bacteriostatic 3. Favorable pharmacokinetics: reach target site in body with effective concentration 4. Spectrum of activity: broad vs. narrow 5. Lack of “side effects” ü Therapeutic index: effective to toxic dose ratio 6. Little resistance development * There is no perfect drug.

Susceptibility Tests 1. Broth dilution - MIC test 2. Agar dilution - MIC test

Minimal Inhibitory Concentration (MIC) vs. Minimal Bactericidal Concentration (MBC) 32 ug/ml 16 ug/ml 8 ug/ml Sub-culture to agar medium 4 ug/ml 2 ug/ml 1 ug/ml MIC = 8 ug/ml MBC = 16 ug/ml

Susceptibility Tests (cont’d) 3. Agar diffusion Kirby-Bauer Disk Diffusion Test

Susceptibility Tests “Kirby-Bauer Disk-plate test” Diffusion depends upon: 1. 2. 3. 4. 5. Concentration Molecular weight Water solubility p. H and ionization Binding to agar (cont’d)

Susceptibility Tests “Kirby-Bauer Disk-plate test” (cont’d) Zones of Inhibition (~ antimicrobial activity) depend upon: 1. p. H of environment 2. Media components ü Agar depth, nutrients 3. 4. 5. 6. Stability of drug Size of inoculum Length of incubation Metabolic activity of organisms

Antibiotic Mechanisms of Action Alteration of Cell Membrane Polymyxins Bacitracin Neomycin Transcription Translation

Mechanism of Action 1. ANTIMETABOLITE ACTION § Sulfonamides ü an analog of PABA, works by competitive inhibition § Trimethoprim-sulfamethoxazole ü a synergistic combination; useful against UTIs

Mechanism of Action ANTIMETABOLITE ACTION tetrahydrofolic acid (cont’d)

Mechanism of Action (cont’d) 2. ALTERATION OF CELL MEMBRANES § Polymyxins and colistin ü destroys membranes ü active against gram negative bacilli ü serious side effects ü used mostly for skin & eye infections

Mechanism of Action ALTERATION OF CELL MEMBRANES (cont’d)

Mechanism of Action (cont’d) 3. INHIBITION OF PROTEIN SYNTHESIS: Steps in synthesis: 1. 2. 3. 4. Initiation Elongation Translocation Termination 1. Prokaryotes and eukaryotes (80 S) have a different structure to ribosomes so can use antibiotics for selective toxicity against ribosomes of prokaryotes (70 S)

Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS (cont’d) • Aminoglycosides ü bind to bacterial ribosome on 30 S subunit; and blocks formation of initiation complex. Both actions lead to mis-incorporation of amino acids üExamples: Gentamicin Tobramycin Amikacin Streptomycin Kanamycin Spectinomycin Neomycin

Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS • Aminoglycosides (cont’d) ü broad spectrum • • • Gram negative rods P. aeruginosa Drug-resistant gram negative rods Plague, Tularemia, Gonorrhea Pre-op (bowel) External (skin) ü toxic at some level to eighth cranial nerve

Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS (cont’d) • Macrolides: chloramphenicol & erythromycin ü bind to 50 S subunit and blocks the translocation step ü Chloramphenicol: broad spectrum ü Erythromycin: Mycoplasma Legionella S. pyogenes Anaerobes Typhoid Meningitis

Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS (cont’d) • Clindamycin ü binds to 50 S subunit and interferes with binding of the amino acid – acyl-t. RNA complex and so inhibits peptidyl transferase ü works best against • Staphylococcus • Bacteroides & anaerobic gram neg rods ü Penicillin allergic people

Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS (cont’d) • Tetracyclines ü bind to 30 S subunit and interferes with the attachment of the t. RNA carrying amino acids to the ribosome ü effective against: • • Chlamydia Rickettsia Mycoplasma Brucella

Mechanism of Action (cont’d) 4. INHIBITION OF DNA/RNA SYNTHESIS § Rifampin ü binds to RNA polymerase ü active against gram positive cocci ü bactericidal for Mycobacterium ü used for treatment and prevention of meningococcus

Mechanism of Action INHIBITION OF DNA/RNA SYNTHESIS (cont’d) § Metronidazole ü breaks down into intermediate that causes breakage of DNA ü active against: – protozoan infections – anaerobic gram negative infections § Quinolones and fluoroquinolones ü effect DNA gyrase ü broad spectrum

Mechanism of Action INHIBITION OF DNA/RNA SYNTHESIS (cont’d)

Mechanism of Action(cont’d) 5. CELL WALL SYNTHESIS INHIBITORS Steps in synthesis: 1. 2. 3. 4. 5. • • NAM-peptide made in cytoplasm attached to bactoprenol in cell membrane NAG is added whole piece is added to growing cell wall crosslinks added the β-Lactams the non β-Lactams

Mechanism of Action(cont’d) 5. CELL WALL SYNTHESIS INHIBITORS β-Lactam Antibiotics ü Penicillins ü Cephalosporins ü Carbapenems ü Monobactams

Mechanism of Action CELL WALL SYNTHESIS INHIBITORS β-Lactam ring structure (cont’d)

Mechanism of Action CELL WALL SYNTHESIS INHIBITORS (cont’d) Action of β-Lactam antibiotics 1. Bactericidal; growing cells only 2. Drug links covalently to regulatory enzymes called PBPs (penicillinbinding proteins) 3. Blocks cross-linkage of peptidoglycan

Mechanism of Action CELL WALL SYNTHESIS INHIBITORS Action of β-Lactam antibiotics For E. coli > MIC Ø Ø wall damage autolysins spheroplasting cell lysis < MIC Ø no septa Ø filaments (cont’d)

Mechanism of Action CELL WALL SYNTHESIS INHIBITORS (cont’d) Resistance to β-Lactams – Gram pos.

Mechanism of Action CELL WALL SYNTHESIS INHIBITORS (cont’d) Resistance to β-Lactams – Gram neg.

Mechanism of Action CELL WALL SYNTHESIS INHIBITORS Non - β-Lactams § Vancomycin (cont’d) üactive against gram positive cocci, but not gram negative because too large to pass through outer membrane ü interferes with PG elongation § Cycloserine, ethionamide and isoniazid üinhibits enzymes that catalyze cell wall synthesis üfor Mycobacterial infections

Clinical Uses PATHOGENS TYPICAL DRUG Gram positive Pen-ase (-) Pen-ase (+) Penicillin G (oral or IM) Methicillin, Nafcillin Gram negative Enterics, etc. Pseudomonas B. fragilis Ampicillin, gentamicin, etc. Ticarcillin, tobramycin Clindamycin

Clinical Uses (cont’d) PATHOGENS TYPICAL DRUG Mycobacterium Streptomycin Iso-nicotinic hydrazide (INH) Fungi: Cutaneous Deep Parasites: Plasmodium Giardia Nystatin Amphotericin B, ketoconazol Chloroquine Quinacrine

Resistance Physiological Mechanisms 1. Lack of entry – tet, fosfomycin 2. Greater exit ü efflux pumps ü tet (R factors) 3. Enzymatic inactivation ü bla (penase) – hydrolysis ü CAT – chloramphenicol acetyl transferase ü Aminogylcosides & transferases

Resistance Physiological Mechanisms (cont’d) 4. Altered target ü RIF – altered RNA polymerase (mutants) ü NAL – altered DNA gyrase ü STR – altered ribosomal proteins ü ERY – methylation of 23 S r. RNA 5. Synthesis of resistant pathway ü TMPr plasmid has gene for DHF reductase; insensitive to TMP

Origin of Drug Resistance • Non-genetic ü metabolic inactivity • Mycobacteria ü non-genetic loss of target • penicillin – non-growing cells, L-forms ü intrinsic resistance • some species naturally insensitive

Origin of Drug Resistance (cont’d) • Genetic ü spontaneous mutation of old genes • Vertical evolution ü Acquisition of new genes • Horizontal evolution • • Chromosomal Resistance Extrachromosomal Resistance • Plasmids, Transposons, Integrons

Plasmids • independent replicons ü circular DNA • dispensable • several genes ü drug resistance ü metabolic enzymes ü virulence factors • host range ü restricted or broad

Plasmids (cont’d) • size ü small, non-conjugal ü large, conjugal <25 kbp • Transfer between cells: ü CONJUGATION (cell to cell contact) • due to plasmid tra genes (for pili, etc) ü NON-CONJUGAL • transduction • mobilization by conjugation plasmids

Implications of Resistance • Household agents ü they inhibit bacterial growth ü purpose is to prevent transmission of disease-causing microbes to noninfected persons. ü can select for resistant strains • NO evidence that they are useful in a healthy household

Implications of Resistance • Triclosan studies ü effect diluted by water ü one gene mutation for resistance ü contact time exceeds normal handwash time (5 seconds) • Allergies ü link between too much hygiene and increased allergy frequency • http: //www. healthsci. tufts. edu/apua/ROAR/roarhome. htm

Implications of Resistance • www. roar. apua. org

REVIEW

Minimal Inhibitory Concentration (MIC) vs. Minimal Bactericidal Concentration (MBC) 32 ug/ml 16 ug/ml 8 ug/ml Sub-culture to agar medium 4 ug/ml 2 ug/ml 1 ug/ml MIC = 8 ug/ml MBC = 16 ug/ml REVIEW

What are main targets of Antibiotics? REVIEW

Mechanism of Action INHIBITION OF CELL WALL SYNTHESIS • β-Lactams • Non β-Lactams REVIEW

Mechanism of Action CELL WALL SYNTHESIS INHIBITORS (cont’d) β-Lactam ring structure REVIEW

Mechanism of Action INHIBITION OF PROTEIN SYNTHESIS • Aminoglycosides • Macrolides ü Chloramphenicol ü Erythromycin • Tetracyclines • Clindamycin REVIEW

Mechanism of Action INHIBITION OF NUCLEIC ACID SYNTHESIS § Rifampin § Metronidazole § Quinolones and fluoroquinolones REVIEW

Mechanism of Action DISRUPTION OF CELL MEMBRANES § Polymyxins § Colistin REVIEW

Mechanism of Action ANTIMETABOLITE ACTION § Sulfonamides § Trimethoprim-sulfamethoxazole REVIEW

Resistance Physiological Mechanisms 1. Lack of entry – tet, fosfomycin 2. Greater exit ü efflux pumps ü tet (R factors) 3. Enzymatic inactivation ü bla (penase) – hydrolysis ü CAT – chloramphenicol acetyl transferase ü Aminogylcosides & transferases REVIEW

Resistance Physiological Mechanisms (cont’d) 4. Altered target ü RIF – altered RNA polymerase (mutants) ü NAL – altered DNA gyrase ü STR – altered ribosomal proteins ü ERY – methylation of 23 S r. RNA 5. Synthesis of resistant pathway ü TMPr plasmid has gene for DHF reductase; insensitive to TMP REVIEW

Mechanism of Action CELL WALL SYNTHESIS INHIBITORS (cont’d) Resistance to β-Lactams – Gram pos. REVIEW

Mechanism of Action CELL WALL SYNTHESIS INHIBITORS (cont’d) Resistance to β-Lactams – Gram neg. REVIEW