Antibiotic Resistance

1. Antibiotic Resistance

2. Alexander Fleming’s Other Experiment • In 1945, a few years after Penicillin was introduced to the world, Fleming created a strain of Staphylococcus aureus that was resistant to penicillin. This was accomplished by exposing s. aureus to sub-optimal doses of Penicillin. Fleming warned the world about antibiotic resistance during an interview with the New York Times.

3. But the world did not listen… • Between 1945 and 1955, Penicillin was available to the public over the counter, without a prescription. • During those ten years the public did in vivo what Fleming did in vitro: People stopped taking Penicillin as soon as they began to feel better, they took Penicillin for viral infections, some reports even say Penicillin was used to treat male pattern baldness…

4. Resistance • By the end of the 1950’s 50% or more of all Staphylococcus aureus strains were resistant to penicillin. • 1975 MRSA (methicillin resistant Staphylococcus aureus). • 1988 VRE (Vancomycin resistant enterococci) . • 2002 VRSA (Vancomycin resistant Staphylococcus aureus). • Today, 70% of all health care facility infections are resistant to one or more antibiotics.

5. Antibiotic Diversity • There is great diversity among antibiotics and among their molecular targets, here are a few examples: 1. Penicillins, Cephalosporins, and Vancomycin- Target the bacterial cell wall. 2. Sulfa drugs: growth factor analogues. 3. Quinolones: effect DNA girase. 4. Tetracycline: effects the bacterial ribosome. 5. Rifampin: targets nucleic acid metabolism. All target processes specific to the bacteria without harming the host

6. Eukaryote vs Prokaryote

8. Schematic Diagrams Of The Two Bacteria Cell Wall Types • A Gram Positive Bacteria Cell and a Gram Negative Bacteria Cell A: Peptidoglycan layer Polymer of sugars and amino acids for structure and support. Note the difference in thickness between the two cells. This difference is what allows gram-positive and gram-negative bacteria to stain in separate colors. • B: Cytoplasmic Membrane Encases the cell’s cytoplasm. • C: Cytoplasm Living cell substance holding all of the cell’s “organs.” • D: Outer membrane Found only in gram-negative bacteria. It holds special chemicals toxic to animals. This membrane is highly resistant to many antibacterial chemicals.

10. Crosslinking of Petidoglycans performed by transeptidases aka penicillin binding proteins (PBP)

11. Amino acids and amino sugars

13. Penicillin binds PBP’s to prevent them from functioning in crosslinking

14. Mechanism of Penicillin • The Beta Lactam (4 membered ring with carbonyl) binds to the active site of the transpeptidase enzyme • Once inside the enzyme’s active site, penicillin doesn’t leave, so it acts as a ‘suicide substrate.’

15. Beta Lactamases (from bacteria) have evolved to break beta lactam ring in penicillins for resistance

16. Vancomycin has no beta- lactam ring

17. Mechanism of Vancomycin • Forms multiple hydrogen bonds to the D-alanyl-D-alanine amino acids of the N-acetyl muramic acid, and N-acetyl glucosamine monomers that are the ‘bricks’ of the cell wall. This prevents the formation of the crosslinking in the walls of the bacteria. They are then unable to produce a wall and they will die.

19. Cell Wall Synthesis • 3 Stages of cell wall synthesis • 1. Synthesis of N-acetylmuramic acid peptide synthesis. • 2. Synthesis of N-acetylmuramic acid-N-acetylglucosamine repeating chains • 3. Cross linking of the MurNAc-GlcNac chains by transpeptidase.

20. Vancomycin Resistance • Vancomycin Resistance is conferred by a change from D-alanyl D-alanine to D-alanyl D-lactate or D-alanyl D-serine. • This decreases the number of hydrogen bonds vancomycin can form with the N-acetylmuramic acid and N-acetyl glucosamine. In effect, increasing the Kd.

21. Types of Antibiotic Resistance • 1. Chemical Warfare, aka the beta lactamase. • 2. Change in target protein structure. • 3. Antibiotic Efflux mechanism • 4. Inaccessibility.

22. Efflux Mechanisms • Pump antibiotics out of the cell, decreasing the intercellular concentration of drug, and increases the changes that the few molecules of the drug that remain inside the cell can be degrade by enzymes (especially beta lactamses).

23. Inaccessibility • If a drug can’t access it’s target, it can’t work. • Example: Gentamicin effects the bacterial ribosome, and is actively transported across the gram negative cell membrane through porin channels. A mutation to one of the proteins involved in this pathway slows entrance of gentamicin into the cell, therefore resulting in resistance.

24. Combating Antibiotic Resistance • CDC’s approach to combat antibiotic resistance: • 1. Vaccination • 2. More appropriate use of and attention to indwelling catheters. • 3. Early involvement of Infectious Disease Specialists • 4. Choosing more appropriate/specialized antibiotics (using ‘broad spectrum drugs as little as possible’) • 5. Appropriate use of prophylactic antibiotics for surgery. • 6. Stricter infection control, especially handwashing!

25. What you can do: • Don’t ask your doctor for antibiotics. • If prescribed antibiotics take them for the full course of treatment, and at equally spaced time intervals as prescribed by your doctor. • WASH YOUR HANDS!!!!

26. Questions? ?????

27. References: • Medical Biochemistry, N.V. Bhagavan. Jones and Bartlett Publishing, 1992. Pages 345-350. • Goodman and Gilman’s The Pharmacological Basis of Therapeutics 11th edition. L. Brunton, J. Lazo, and K. Parker. McGraw-Hill Medical Publishing Division, 2006. Chapters 42, 43, 44. • Cdc.gov • Textbookofbacteriology.net • Also, special thanks to Debra Dunaway-Mariano, PhD professor of chemistry at UNM. (if you’re interested in this topic you might want to take her ‘boilogical chemistry’ course, it was a big help in preparing for this presentation, as well as being a really cool class.)

28. Additional Slides

29. Beta Lactamase • Hydrolyzes the Carbonyl portion of the beta lactam ring. The ring breaks because of strain, and the business end of penicillin is no longer able to bind the transpeptidase enzyme. Kd actually approaches infinity. • Examples: Haemophylis influenzae secretes a beta lactamase, making these strains resistant to all but methicillin. • However, it has been discovered that clavulanic acid acts as a beta-lactamase inhibitor. The antibitoic Augmentin is actually amoxicillin combined with clavulanic acid.

30. Clavulanic acidBeta-lactamase inhibitor.