Faculty: Dr. Alvin Fox

1. Antibiotics that affect the Cell Envelope Faculty: Dr. Alvin Fox Suggested reading: Murray, Third edition Chapters 10 & 20

2. Key Words Sterilization/disinfection/antisepsis Bacitracin Antibiotic Vancomycin Selective toxicity beta lactam Bactericidal Penicillins Bacteriostatic Cephalosporins Minimal inhibitory concentration (MIC) Monobactam Susceptibility Clavulinic acid Penicillin binding proteins Penicillinase/beta lactamase Autolysins Polymyxin B Cycloserine Resistance

3. STERILIZATION: Killing of ALL bacteria in a non-selective fashion.

4. There are 4 major methods for sterilization: 1. Autoclaving which involves heating liquids (e.g. media) or solids to 121oC under steam pressure. However, the materials must be heat resistant. 2. Ethylene oxide is sometimes used in hospitals for equipment that cannot be heated. 3. Membrane filters have pores that trap bacteria, but allow drugs and small chemicals to pass through; thus pre-sterilized filters can be used to sterilize delicate solutions. 4a. UV light is used for decreasing bacterial levels on surfaces such as in operating rooms; however it is not totally effective. 4b. Ionizing radiation is more efficient and can be used for sterilizing instruments and food.

5. Disinfectants: Can be used in killing many bacteria on certain instruments, but cannot be used for internal consumption or on skin (e.g. phenol-based products)

6. Antiseptics: These products are used topically (e.g. on skin surfaces) to reduce bacterial load. (e.g. iodine or 70% alcohol)

7. ANTIBIOTICS Antibiotics are agents that are "selectively" toxic for bacteria (either killing them [bactericidal] or inhibiting their growth [bacteriostatic]) without harm to the patient.

8. 1. Antibiotics must act on structures found in bacteria, but not in the host. 2. Antibiotics work most efficiently in conjunction with an active immune system to kill infecting bacteria in the host.

9. The minimal inhibitory • concentration (MIC) refers to the • lowest concentration of an antibiotic • that stops visible growth. • The zone of inhibition around a disk • impregnated with antibiotic • (Kirby-Bauer) is another measure of • antibiotic activity.

10. INHIBITORS OF CELL WALL SYNTHESIS 1. One major class of antibiotics inhibit the synthesis of peptidoglycan. 2. Once cell wall synthesis (involving penicillin binding proteins) is inhibited, enzymatic autolysis of the cell wall can occur.

11. Without the restraining influence of the cell • wall the high osmotic pressure inside the cell • bursts the inner and/or outer membranes of • bacteria. • Antibiotics which are inhibitors of cell wall • biosynthesis are generally bactericidal.

12. Mechanisms involved in the inhibition of peptidoglycan synthesis include: • The terminal two amino acids of a peptide side chain of • peptidoglycan are unusual amino acids (D-alanine as • opposed to its isomer L-alanine). • The antibiotic cycloserine is an analog of D-alanine and • interferes with enzymatic conversion of L-alanine to • D-alanine in the cytoplasm. • Thus subsequent synthesis of peptidoglycan cannot occur.

13. 2. The peptidoglycan subunit (containing one side-chain and an attached peptide to be used in cross-bridge formation) is passed across the cytoplasmic membrane attached to undecaprenol diphosphate. After the nascent peptidoglycan monomer leaves the carrier on reaching the cell wall, the undecaprenol diphosphate is dephosphorylated to its monophosphate form. Bacitracininhibits the dephosphorylation reaction and in the absence of monophosphorylated carrier peptidoglycan subunit synthesis stops.

14. 3. The final step in peptidoglycan synthesis involves linking the sugar portion of the peptidoglycan subunit to the glycan backbone of the existing cell wall polymer. Cross-linking of the peptide portion of the subunit to a peptide in the cell wall then occurs. During this process D-alanine is enzymatically excised from the end of a pre-existing peptide side chain allowing it to be cross-linked (by a new peptide bond) to the recently synthesized peptidoglycan subunit. Vancomycin binds to D-alanine-D-alanine thus sterically inhibits transpeptidation (cross-linking).

15. Cross-linking of peptidoglycan

16. The beta lactam antibiotics include penicillins • (e.g. ampicillin), cephalosporins and monobactams. • They bind to and inhibit enzymes (penicillin binding • proteins) involved in the transpeptidation (cross-linking) of • peptidoglycan. These antibiotics have in common the four • membered lactam ring. • Attached to the lactam, penicillins have an additional five • membered ring and cephalosporins a six membered ring. • Monobactams consist of the lactam ring alone and display • antibiotic activity.

17. Various chemical side chains have been synthetically linked to the ring structures producing a host of antibiotics with different properties in the host. Many penicillins display little activity against Gram negative bacteria, since they do not penetrate the outer membrane. Cephalosporins and other newer penicillins are active against Gram negative bacteria, since they can penetrate the outer membrane.

18. O S CH3 C NH CH C CH CH3 O C N CH COOH STRUCTURE OF PENICILLIN Site of penicillinase action. Breakage of the  lactam ring.

19. Penicillins can be destroyed by beta lactamase (penicillinase) produced by resistant bacterial strains. Clavulinic acid, also has a beta lactam component which binds strongly to beta lactamases inhibiting their activity. Another form of resistance involves a change in the structure of penicillin binding proteins such that the antibiotic does not bind efficiently. In the case of Gram negative bacteria, penicillins pass across the outer membrane using porins. Resistance may develop from mutation leading to modified porins.

22. POLYMYXIN B Polymyxin B binds to the lipid A portion of lipopolysaccharide and also to phospholipids. This disrupts the outer membrane of Gram negative bacteria. Since the cell membrane is not exposed in Gram positive bacteria polymyxin has little activity against them. This drug is toxic to human cells, since it can also lyse eukaryotic membranes; this explains its limited clinical use.

23. STRUCTURE OF POLYMYXIN