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1 Antibiotics • Hugh B. Fackrell • Filename: antibiot.ppt 5/23/2017 2 Outline • • • • History Ideal properties Sources “Sulfas” – Antimetabolites – antibiotic synergism • Major Groups of antibiotics • Mechanisms of action 5/23/2017 3 History • Salvarsan 606 • Prontosil • Penicillin 5/23/2017 4 Salvarsan 606 • Paul Ehrlich – early 1900’s – syphilis – arsenic + organic compound • Aniline dyes - – wasn't able to find the "magic bullet” 5/23/2017 5 Prontosil • 1930's, Gerhard Domagk – Prontosil • 1935, Jacques and Therese Trefoncel – discovered that the active compound in Prontosil was Sulfanilamide • sulfanilamide “ Sulfas” 5/23/2017 6 Penicillin • 1928, Alexander Fleming – antibacterial activity in Penicillium mold (called it Penicillin) • 1938, Howard Florey and Ernst Chain – developed Penicillin as an effective antibiotic 5/23/2017 7 Antimicrobial Therapy • Antimicrobics – substances produced by microbes that inhibit other microbes • Semi-synthetic antibiotics – naturally produced but altered • Synthetic antibiotics: – derived from chemicals 5/23/2017 8 Ideal Properties of an Antibiotic • Low toxicity for patient – kills the invading microorganism without damaging the host – no adverse side reactions – non allergenic • High toxicity for microbe – bactericidal not bacteriostatic – broad spectrum • Low risk of other infections 5/23/2017 9 More Characteristics • drug can be administered orally or parenterally (by injection) – Soluble in tissue fluids – absorbed by and dissolved in tissues or body fluids • levels of active drug sustained long enough to kill the invading agent • Long “Shelf” life 5/23/2017 10 Still More Characteristics • • • • • Low probability of resistance Microbial drug resistance develops slowly microbicidal rather than microbistatic Not inactivated by organic material Assists the host in eliminating the infecting microbe • Not a powerful allergen 5/23/2017 11 Sources of Antibiotics Most spore-forming microorganisms • Fungi – Penicillium penicillin, – Cephalosporium griseofulvin • Bacteria – Bacillus bacitracin, polymyxin, tyrothricin, colimycin, gramicidin – Streptomycetes Aminoglycosides, nystatin, chloramphenicol, erythromycin, tetracylcine... 5/23/2017 12 Mechanisms of Drug Action • • • • inhibit cell wall synthesis inhibit nucleic acid synthesis inhibit protein synthesis interfere with cell membrane function 5/23/2017 Sulfa Drugs 14 Sulfa vs PABA NH2SO2 NH2 Sulfanilamide NH2 HOOC PABA 5/23/2017 15 Structure of Sulfa Drugs Sulfisoxazole Sulfanilamide Prontosil 5/23/2017 16 Folic Acid Metabolism PABA + pteridine Sulfonamide [GTP] Pteridine synthetase Dihydropteroic acid Dihydrofolate L- Glutamine Synthetase Dihydrofolic Acid 2 NADPH Trimethoprim 2 NADP+ Dihydrofolate synthetase Tetrahydrofolic Acid Thymidine DNA Purines DNA, RNA Methionine tRNa, 5/23/2017 Proteins 17 Folic Acid Inhibition PABA + pteridine Sulfonamide Dihydropteroic acid Dihydrofolic Acid Trimethoprim Tetrahydrofolic Acid Thymidine DNA Purines DNA, RNA Methionine tRNa, Proteins 5/23/2017 18 Antibiotic Synergism Sulfisoxazole Trimethoprim 5/23/2017 19 Antibiotic Synergism • • • • Sulfonamide + trimethoprim Effective dosage 10% of two separately Broader spectrum of action Reduce emergence of resistant strains 5/23/2017 Major Groups of Antibiotics 21 Major Groups of Antibiotics • Aminoglycosides – streptomycin, kanamycin, neomycin, gentamicin, spectinomycin, tobramycin, amikacin • Beta lactams – Penicillins, cephalosporins • Lincomycins – lincomycin clindamycin 5/23/2017 22 Major Groups of Antibiotics • Macrolides – erythromycin, carbomycin • Polypeptides – polymyxin, colimycin, bacitracin, tyrothricin • Polyenes – amphotericin B, nystatin • Rifamycins – Rifampin 5/23/2017 23 Major Groups of Antibiotics • Synthetic – pyridine • isoniazid, ethambutol – sulfonamides • sulfanilamide, sulphisoxazole – misc • nitrofurans, metronidazole, nalidixic acid • Tetracyclines – oxytetracline, chlortetracycline • Unclassified – Chloramphenicol, vancomycin 5/23/2017 24 PENEMS • Carbapenems • “Ideal” antibiotics – – – – non toxic broad spectrum good “Shelf” life effective at very low conc • Attach to Penicillin Binding Proteins – found in cell membrane – Gm+ve lysis through loss of cell wall integrity – Gm -ve filamentous bacteria loss of septum formation 5/23/2017 25 Adverse Effects of Antibiotics • Aminoglycosides – Ototoxic- destroys cochlear hair cells – renal toxic • Chloramphenicol – depresses bone marrow – aplastic anemia – fatal “Grey baby” syndrome • Penicillins – allergy anaphylaxis • Vancomycin – thrombophlebitis – ototoxic – renal toxic • Polymyxin, bacitracin colimycin – renal toxic • Sulfas – – – – skin allergy anemia renal toxic hepato toxic 5/23/2017 26 Adverse Effects of Antibiotics • Broad spectrum – – – – – Super infections Candida albicans Clostridium difficle Staphylococcus Gram -ve Tetraclycine – Depress bone marrow – “Yellow teeth” Pregnant women children <7 years 5/23/2017 27 Mode of Action of Antibiotics 5/23/2017 28 Mode of Action of Antibiotics • • • • Inhibit Synthesis of Cell Wall Damage Cell Membrane Inhibit Protein Synthesis Inhibit Nucleic acid Synthesis 5/23/2017 29 Bacterial Cell Wall • Peptidoglycan – many layers in gram positives – thin in gram negative • protects the cell against rupture from hypotonic environments 5/23/2017 30 Synthesis of peptidoglycan (1/4) • Uridine diphosphate (UDP) derivatives of NAM and NAG are synthesized in the cytoplasm • Amino acids are sequentially added to UDP-NAM to form the pentapeptide chain using ATP as an energy source. The two terminal D-alanines are added as a dipeptide (Cycloserine) 5/23/2017 31 Synthesis of peptidoglycan (2/4) • The NAM- pentapeptide is transferred from UDP to a bactoprenol PO4 at the membrane surface. Bactoprenol is a 55-Carbon alcohol that attaches to NAM by a pyrophosphate group and moves peptidoglycan components through the hydrophobic membrane • UDP-NAG adds NAG to the NAMpentapeptide to form the peptidoglycan repeat unit 5/23/2017 32 Synthesis of peptidoglycan (3/4) • The completed NAM-NAG peptidoglycan repeat unit is transported across the membrane to its outer surface by the bactoprenol pyrophosphate carrier • The peptidoglycan unit is attached to the growing end. 5/23/2017 33 Synthesis of peptidoglycan (4/4) • The bactoprenol carrier returns to the inside of the membrane to collect another NAMpentapeptide. Bactoprenol pyrophosphate must give up phosphate to connect Bacitracin • Finally, transpeptidization - interbridges formed 5/23/2017 34 Inhibit Synthesis of Cell Wall • penicillin, bacitracin, vancomycin, cephalosporin, carbapenems 5/23/2017 35 Inhibition of Cell Wall Synthesis • Cycloserine - inhibits peptidoglycan sub-unit formation • Vancomycin - inhibits peptidoglycan elongation • Beta-lactam antibiotics - Penicillins – lactam antibiotics block peptidases required to connect inter bridges • Cephalosporins bind to the peptidases that are essential to cross link the glycan molecules. 5/23/2017 36 Inhibition of cell wall synthesis • Cycloserine - inhibits the addition of the two terminal D-alanines • Bacitracin - inhibits the transport of the subunits to their position in the cell wall • Vancomycin - inhibits the elongation of the peptidoglycan to form connecting units Murray 2.4 & 5.4, p. 10 5/23/2017 37 Inhibition of Cell Wall Synthesis 5/23/2017 38 Natural Penicillins 5/23/2017 39 Semi Synthetic Penicillins 5/23/2017 40 Semi Sythetic Penicillins -2 5/23/2017 41 Structure of Penicillin 5/23/2017 42 Hydrolysis of Beta Lactam Ring 5/23/2017 43 Comparison of Structures 5/23/2017 44 Pen G in Blood 5/23/2017 45 Damage Cell Membrane polymyxin, colimycin, nystatin, amphoteracin, tyrothricin 5/23/2017 46 Injury of Plasma Membrane 5/23/2017 47 Polymyxin action • Polymyxin B binds to the cell membrane to disrupts its structural and permeability properties Polymyxin Membrane Cytoplasm 5/23/2017 48 Inhibit Protein Synthesis • Binds to 50S ribosomal subunit – prevents peptide chain elongation • clindamycin, chlorampenicol, erythromycin – block rRNA(23S) • lincomycin, macrolides • Binds to 30S ribosomal subunit – misreading of mRNA • aminoglycosides- genetamcin – Blocks binding of tRNA-AA to 30S • tetraclyclines 5/23/2017 49 Inhibition of Translation 5/23/2017 50 Translation 5/23/2017 51 Inhibition of Peptide Bond 5/23/2017 52 Inhibition of Ribosome Movement 5/23/2017 53 Inhibition of tRNA Attachment 5/23/2017 54 Misreading mRNA 5/23/2017 55 Inhibit Nucleic acid Synthesis Quinolones – Ciprofloxacin and other quinolones • Inhibits DNA gyrase • Blocks DNA replication • Inhibits mitochondrial DNA – conc in tissues too low for toxicity • Urinary and intestinal infections 5/23/2017 56 Inhibition of DNA Replication 5/23/2017 57 Inhibit Nucleic Acid Synthesis • Rifamycin – Inhibits DNA dependent RNA polymerase – Blocks transcription DNA ->RNA 5/23/2017 58 Acylovir vs Deoxyguanosine 5/23/2017 59 Inhibition of Transcription 5/23/2017 60 Antimetabolites • Sulfonamides • Donald D. Woods • Sulfanilamide blocks folic acid – folic acid is essential to the synthesis of DNA and RNA – Para amino benzoic acid (PABA) not incorporated into folic acid • Reversible inhibition – High [PABA] competitively inhibit sulfanilamide 5/23/2017 61 Inhibited metabolites,Synthesis 5/23/2017 62 Drug Resistance • synthesis of enzymes that inactivate the drug • decrease in cell permeability and uptake of the drug • change in the number or affinity of drug receptor sites • modification of an essential metabolic pathway 5/23/2017 63 Development of Drug Resistance • intrinsic – chromosomal mutations - low probability • acquired – transfer of extra chromosomal DNA from a resistant species to a sensitive one – Plasmids – Transposons 5/23/2017 64 Plasmids – resistance factors or R factors transfered by conjugation, transformation or transduction 5/23/2017 65 Transposons – sequences that can move from – plasmid >> chromosome – plasmid>> plasmid 5/23/2017 66 Clinical Trials • patient - has a diagnosed infection - two possibilities: – a) the new drug is the drug of choice by testing – b) the patient has not responded to other drugs and the new drug is testing well in the lab • samples of blood etc. taken to determine all the possible parameters: – level of antimicrobial and presence of agent – cultures of infecting agent taken 2 times per day • disappearance of the bacteria and patient recovery conclude a successful trial 5/23/2017 67 Minimum Inhibitory Concentration • 1. test for antimicrobial activity • 2. dilute antibiotic (pictures of tubes) • 3. range selected obtained from therapeutic index • 4. add to medium • 5. add pure culture of isolated bacteria • 6. incubate - tubes that are clear after 16 hours incubation at 35° C are subcultured Reference: lab manual p. 270 – 0.1 ml removed and plated on suitably rich medium usually the agar version of the liquid growth medium 5/23/2017 68 Kirby-Bauer Plate Sensitivity • disks impregnated with various concentrations of appropriate antibiotics are placed aseptically on innoculated plates • measurement of drug concentrations in the blood preclinical trials • subjects receive varying dose levels and intervals of dosage • pretrials usually determine the route of Reference: lab manual p. 270 entry - oral or parenteral (injected subcutaneously, intramuscularly, etc.) • pretrials determine the carrier substance 5/23/2017 69 Subjects Tested for Antimicrobial Levels • blood, lymph, urine, feces tested for effective levels depending on disease • also of concern is rapid metabolism (catabolism) of the drug and also rapid excretion 5/23/2017 70 Disk Diffusion Tests • diffusion of antibiotic from disk controlled by agar concentration • Zone of Inhibition – controlled by diffusion rate – level of sensitivity • each antibiotic is unique 5/23/2017