Doctrine about antibiotics. Antimicrobial Chemotherapy . Clinical use of antibiotics

1. Chair of Microbiology, Virology, and Immunology Doctrine about antibiotics. Antimicrobial Chemotherapy. Clinical use of antibiotics

2. Lecture schedule 1. History of antibiotics discovery. 2. Classification of antibiotics. 3. Examination of bacterial susceptibility to antibiotics. Complication of antibioticotherapy.

3. - Diarrheal diseases - 4 billionscases, • Malaria - 500 mln, • acute infection of respiratory tract - 395 mln, • sexual transmitted diseases - 330 mln, • measles - 42 mln, • whooping cough - 40 mln • tuberculosis – 1,9 bln of infected persons, • 9 mln of new cases of diseases • AIDS – 50 mln cases, 6 mln people died • SARS, hemorrhagic fever

4. Tremendous quantities of antibiotics are produced and released into the environment. 90 – 180 million kg/year of antibiotics are used (enough for 25 BILLION full treatment courses ~ 4 per human/yr!)About 10 X more antibiotics are used in agriculture than to treat people. (Levy 1997 estimated 30 X more in animals than in people).

6. Paul Ehrlich “Magic bullet" concept

7. Sulfonamides Analogues of para-aminobenzoic acid Broad spectrum Competitive inhibitors of dihydropteroate synthase – needed for folic acid synthesis Cidal in urine Mechanisms of resistance Altered affinity of enzyme for drug Decreased permeability or active efflux New pathway of folic acid synthesis Gerhard Domagk gets a Nobel for Medicine, 1939. It had little success until the 1930s, when Gerhard Domagk discovered the protective effects of prontosil, the forerunner of sulfonamide.

8. Sir A. Fleming and Penicillin Ironically, penicillin G was discovered fortuitously in 1929 by Fleming, who did not initially appreciate the magnitude of his discovery.

9. Sir A. Fleming

10. In 1939 Florey and colleagues at Oxford University again isolated penicillin G. Florey E. Chainy

11. In 1944 S. Waksman isolated streptomycin and subsequently found agents such as chloramphenicol, tetracyclines, and erythromycin in soil samples. -Born of Streptomycin -

12. What are Antibiotics? • Antibiotics means: anti – against, bios – life = “against life” The term “antibiotics” was proposed in 1942 by S. Waksman • Antibiotics are molecules that stop microbes, both bacteria, viruses, protozoa, and fungi, from growing or kill them outright. • Antibiotics can be either natural products or man-made synthetic chemicals.

13. Microbial antagonism is the basis of modern use of antibiotics L. Pasteur

14. Peculiarities of antibiotics • - high level of biological activity • high election specificity • Activity of antibiotics is evaluated in International Unit or µg/ml

16. Spectrum – range of activity of a drug • Narrow-spectrum– effective on a small range of microbes • target a specific cell component that is found only in certain microbes • Broad-spectrum – greatest range of activity • target cell components common to most pathogens

17. Antimicrobial agents may be either: Bactericidal agents are more effective, but bacteriostatic agents can be extremely beneficial since they permit the normal defenses of the host to destroy the microorganisms

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

20. Drugs that affect the bacterial cell wall • Most bacterial cell walls contain a rigid girdle of peptidoglycan. • Penicillin and cephalosporin block synthesis of peptidoglycan, causing the cell wall to lyse. • Penicillins do not penetrate the outer membrane and are less effective against gram-negative bacteria. • Broad spectrum penicillins and cephalosporins can cross the cell walls of gram-negative bacteria.

21. Other Inhibitors of Cell Wall Synthesis • Antibiotics effective against Mycobacteria: interfere with mycolic acid synthesis or incorporation • Isoniazid (INH) • Ethambutol

22. Drugs that disrupt cell membrane function • A cell with a damaged membrane dies from disruption in metabolism or lysis. • These drugs have specificity for a particular microbial group, based on differences in types of lipids in their cell membranes. • Polymyxins interact with phospholipids and cause leakage, particularly in gram-negative bacteria • Amphotericin B and nystatin form complexes with sterols on fungal membranes which causes leakage.

23. Inhibitors of Nucleic Acid Synthesis • Rifamycin • Inhibits RNA synthesis • Antituberculosis • Quinolones and fluoroquinolones • Ciprofloxacin • Inhibits DNA gyrase • Urinary tract infections

24. Drugs that inhibit nucleic acid synthesis • may block synthesis of nucleotides, inhibit replication, or stop transcription • Sulfonamides and trimethoprim block enzymes required for tetrahydrofolate synthesis needed for DNA & RNA synthesis. • competitive inhibition – drug competes with normal substrate for enzyme’s active site • synergistic effect – an additive effect, achieved by multiple drugs working together, requiring a lower dose of each

25. Drugs that block protein synthesis Ribosomes of eucaryotes differ in size and structure from procaryotes, so antimicrobics usually have a selective action against procaryotes. But they can also damage the eucaryotic mitochondria. • Aminoglycosides(streptomycin, gentamicin) insert on sites on the 30S subunit and cause misreading of mRNA. • Tetracyclines block attachment of tRNA on the A acceptor site and stop further synthesis. • Macrolides: Erythromycin (gram +, used in children) • Chloramphenicol

26. Drugs that block protein synthesis

27. Figure 20.4b

28. Penicillins • Large diverse group of compounds • The R group is responsible for the activity of the drug, and cleavage of the beta-lactam ring will render the drug inactive. • Penicillins G and V most important natural forms • Penicillin is the drug of choice for gram-positive cocci (streptococci) and some gram-negative bacteria (meningococci and syphilis spirochete) • Semisynthetic penicillins – ampicillin, carbenicillin & amoxicillin have broader spectra – gram negative enterics rods • Penicillinase-resistant – methicillin, nafcillin, cloxacillin • Primary problems – allergies and resistant strains of bacteria

29. Beta()-lactams

30. Cephalosporins Isolated from Cephalosporium acremonium mold Beta-lactam ring that can be altered Relatively broad-spectrum, resistant to most penicillinases, & cause fewer allergic reactions

31. Cephalosporins 5 generations exist • 1st generation – cefazolin, cephalothin – most effective against gram-positive cocci • 2nd generation – cefuroxime, cefaclor, cefonacid – more effective against gram-negative bacteria • 3rd generation – ceftriaxone, cephalexin, cefotaxime – broad-spectrum activity against enteric bacteria with beta-lactamases Ceftriaxone – semisynthetic broad-spectrum drug for treating wide variety of infections • 4th generation - cefepime • 5th generation - ceftobiprole • Differ in spectrum, resistance to beta lactamases, penetration into CNS

32. Vancomycin (Vancocin) • Mechanism of action • Inhibits bacterial cell wall synthesis • Bactericidal? • Spectrum • Gram positive bacteria • Methicillin resistant Staphylococcus aureus • Clostridium difficile (oral)

33. Carbapenems • Specific agents • Imipenem • With cilastin (Primaxin) • Meropenem (Merrem) • Ertapenem (Invanz) • Doripenem (Doribax) • Mechanism of action • Inhibit bacterial cell wall synthesis • Bactericidal • Broad spectrum • Gram positives • Gram negatives • Pseudomonas (except ertapenem

34. Aminoglycosides Cmposed of 2 or more amino sugars and an aminocyclitol (6C) ring • products of various species of soil actinomycetes in genera Streptomyces & Micromonospora • Broad-spectrum, inhibit protein synthesis, especially useful against aerobic gram-negative rods & certain gram-positive bacteria • Streptomycin – bubonic plague, tularemia, TB • Gentamicin – less toxic, used against gram-negative rods • Tobramycin & Amikacin gram-negative bacteria

35. Macrolides Mechanism of action • Inhibit bacterial protein synthesis • Bacteriostatic • Mechanism of resistance • Decreased permeability of drug into the microbe • Modification of target sites • Hydrolysis of macrolide by bacterial enzymes • Specific agents: • Erythromycin (attaches to ribosomal 50s subunit) • Azithromycin (Zithromax) • Clarithromycin (Biaxin)

36. Clindamycin • Antimicrobial spectrum • Anaerobes • Some gram positives • Inhibits bacterial protein synthesis • Bacteriostatic • Adverse effects • Nausea, diarrhea • Clostridium difficile

37. Tetracycline antibiotics • Broad-spectrum, block protein synthesis • Specific agents; Aureomycin, Terramycin, Tetracycline, Doxycycline and Minocycline • Doxycycline & minocycline – oral drugs taken for STDs, Rocky Mountain spotted fever, Lyme disease, typhus, acne & protozoa

38. Chloramphenicol • Isolated from Streptomyces venezuelae • Potent broad-spectrum drug with unique nitrobenzene structure • Blocks peptide bond formation • Very toxic, restricted uses, can cause irreversible damage to bone marrow • Typhoid fever, brain abscesses, rickettsial & chlamydial infections

39. Quinolones Spectrum of action Broad spectrum (varies by agent) • Inhibit DNA synthesis - Interact with bacterial gyrase to prevent supercoiling during DNA synthesis; • Targets DNA gyrase (G-) and topoisomerase IV (G+) • Bactericidal • Mechanisms of resistance • Change in target enzyme • Change in permeability of organism • Specific agents • Norfloxacin • Ciprofloxacin • Levofloxacin (Levaquin) • Gatifloxacin (Tequin) • Moxifloxacin (Avelox) • Nalidixic acid

40. Spectrum of Antimicrobial Activity

41. Considerations in Selecting an Antimicrobial Drug Testing for Drug Susceptibility: The MIC and Therapeutic Index • In vitro activity of a drug is not always correlated with in vivo effect. • If therapy fails, a different drug, combination of drugs, or different administration must be considered. • Best to choose a drug with highest level of selectivity but lowest level toxicity – measured by therapeutic index – the ratio of the dose of the drug that is toxic to humans as compared to its minimum effective dose • High index is desirable.

42. Antimicrobial Susceptibility Tests Provide information for selection ofan appropriate agent for antimicrobial therapy Serial dilutions: - in a liquid medium - in a solid medium Disc diffusion method Rapid methods 42

43. Demands to nutrient media • to be standard and provide optimal conditions for microbial growth (Mueller Hinton agar); • do not have inhibitors of bacterial growth and a lot of stimulators; • do not have substances, which inhibit antibiotic activity

44. Selection of a Colony to Test MacFarland 0.5 and Adjusted Test Organism 44

45. Use of Disk Dispensers Advantages practical, rapid increase reproducibility Risks: contamination reduces personal judgment skills Use Mueller Hinton agar

46. The Kirby-Bauer Test

47. Measuring Conditions Ruler Calipers • read with good light, and from the back of the plate • zone size reading is drug specific • magnification may help • millimeters matter 47

49. AST Methods

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