1. Antibiotics
Hugh B. Fackrell
Filename: antibiot.ppt

2. Outline History Ideal properties Sources “Sulfas”
Antimetabolites
antibiotic synergism
Major Groups of antibiotics
Mechanisms of action

3. History
Salvarsan 606
Prontosil
Penicillin

4. Salvarsan 606 Paul Ehrlich early 1900’s syphilis
arsenic + organic compound
Aniline dyes -
wasn't able to find the "magic bullet”

5. Prontosil 1930's, Gerhard Domagk 1935, Jacques and Therese Trefoncel
discovered that the active compound in Prontosil was Sulfanilamide
sulfanilamide “ Sulfas”

6. Penicillin 1928, Alexander Fleming 1938, Howard Florey and Ernst Chain
antibacterial activity in Penicillium mold (called it Penicillin)
1938, Howard Florey and Ernst Chain
developed Penicillin as an effective antibiotic

7. Antimicrobial Therapy
Antimicrobics
substances produced by microbes that inhibit other microbes
Semi-synthetic antibiotics
naturally produced but altered
Synthetic antibiotics:
derived from chemicals

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

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

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

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...

12. Mechanisms of Drug Action
inhibit cell wall synthesis
inhibit nucleic acid synthesis
inhibit protein synthesis
interfere with cell membrane function

13. Sulfa Drugs

14. Sulfa vs PABA
Sulfanilamide
NH2
NH2SO2
PABA
NH2
HOOC

15. Structure of Sulfa Drugs
Sulfanilamide
Sulfisoxazole
Prontosil

16. Folic Acid Metabolism PABA + pteridine Dihydrofolic Acid
Pteridine synthetase
Dihydropteroic acid
[GTP]
Sulfonamide
Dihydrofolic Acid
Dihydrofolate Synthetase
L- Glutamine
Tetrahydrofolic Acid
Dihydrofolate synthetase
2 NADPH
2 NADP+
Trimethoprim
Thymidine
DNA
Purines
DNA, RNA
Methionine
tRNa, Proteins

17. Folic Acid Inhibition PABA + pteridine Dihydrofolic Acid
Sulfonamide
Dihydropteroic acid
Dihydrofolic Acid
Trimethoprim
Tetrahydrofolic Acid
Thymidine
DNA
Purines
DNA, RNA
Methionine
tRNa, Proteins

18. Antibiotic Synergism
Sulfisoxazole
Trimethoprim

19. Antibiotic Synergism Sulfonamide + trimethoprim
Effective dosage 10% of two separately
Broader spectrum of action
Reduce emergence of resistant strains

20. Major Groups of Antibiotics

21. Major Groups of Antibiotics
Aminoglycosides
streptomycin, kanamycin, neomycin, gentamicin, spectinomycin, tobramycin, amikacin
Beta lactams
Penicillins, cephalosporins
Lincomycins
lincomycin clindamycin

22. Major Groups of Antibiotics
Macrolides
erythromycin, carbomycin
Polypeptides
polymyxin, colimycin, bacitracin, tyrothricin
Polyenes
amphotericin B, nystatin
Rifamycins
Rifampin

23. Major Groups of Antibiotics
Synthetic
pyridine
isoniazid, ethambutol
sulfonamides
sulfanilamide, sulphisoxazole
misc
nitrofurans, metronidazole, nalidixic acid
Tetracyclines
oxytetracline, chlortetracycline
Unclassified
Chloramphenicol, vancomycin

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

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
hepato toxic

26. Adverse Effects of Antibiotics
Tetraclycine
Depress bone marrow
“Yellow teeth”
Pregnant women
children <7 years
Broad spectrum
Super infections
Candida albicans
Clostridium difficle
Staphylococcus
Gram -ve

27. Mode of Action of Antibiotics

28. Mode of Action of Antibiotics
Inhibit Synthesis of Cell Wall
Damage Cell Membrane
Inhibit Protein Synthesis
Inhibit Nucleic acid Synthesis

29. Bacterial Cell Wall Peptidoglycan
many layers in gram positives
thin in gram negative
protects the cell against rupture from hypotonic environments

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)

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 NAM-pentapeptide to form the peptidoglycan repeat unit

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.

33. Synthesis of peptidoglycan (4/4)
The bactoprenol carrier returns to the inside of the membrane to collect another NAM-pentapeptide. Bactoprenol pyrophosphate must give up phosphate to connect Bacitracin
Finally, transpeptidization - interbridges formed

34. Inhibit Synthesis of Cell Wall
penicillin, bacitracin, vancomycin, cephalosporin, carbapenems

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.

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

37. Inhibition of Cell Wall Synthesis

38. Natural Penicillins

39. Semi Synthetic Penicillins

40. Semi Sythetic Penicillins -2

41. Structure of Penicillin

42. Hydrolysis of Beta Lactam Ring

43. Comparison of Structures

44. Pen G in Blood

45. Damage Cell Membrane
polymyxin, colimycin, nystatin, amphoteracin, tyrothricin

46. Injury of Plasma Membrane

47. Polymyxin action
Polymyxin B binds to the cell membrane to disrupts its structural and permeability properties
Polymyxin
Membrane
Cytoplasm

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

49. Inhibition of Translation

50. Translation

51. Inhibition of Peptide Bond

52. Inhibition of Ribosome Movement

53. Inhibition of tRNA Attachment

54. Misreading mRNA

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

56. Inhibition of DNA Replication

57. Inhibit Nucleic Acid Synthesis
Rifamycin
Inhibits DNA dependent RNA polymerase
Blocks transcription DNA ->RNA

58. Acylovir vs Deoxyguanosine

59. Inhibition of Transcription

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

61. Inhibited metabolites,Synthesis

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

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

64. Plasmids
resistance factors or R factors transfered by conjugation, transformation or transduction

65. Transposons sequences that can move from plasmid >> chromosome
plasmid>> plasmid

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

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
0.1 ml removed and plated on suitably rich medium - usually the agar version of the liquid growth medium
Reference: lab manual p. 270

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 entry - oral or parenteral (injected subcutaneously, intramuscularly, etc.)
pretrials determine the carrier substance
Reference: lab manual p. 270

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

70. Disk Diffusion Tests each antibiotic is unique
diffusion of antibiotic from disk controlled by agar concentration
Zone of Inhibition
controlled by diffusion rate
level of sensitivity
each antibiotic is unique