1. Mechanisms of antimicrobial action directed against the bacterial cell wall and corresponding resistance mechanisms M-4 Advanced Therapeutics Course

2. Mechanisms of antimicrobial resistance Drug-modifying enzymes (e.g.,  - lactamases, aminoglycoside- modifying enzymes) Altered drug targets (e.g., PBPs ribosomes, DNA gyrase) Altered uptake or accumulation of drug (e.g., altered porins, membrane efflux pumps)

3. Subunits for cell wall construction D-ala-D-ala pentapeptide N-acetylmuramic acidN-acetylglucosamine

4. Cell Wall Assembly Layer of cell wall with cross links of 5 glycines (gray) Second layer of cell wall cross-linked to the lower layer Transpeptidase (PBP) forms a 5-glycine bridge between peptides A subunit is added to the growing chain

5. Transpeptidase, or PBP (orange sunburst) is bound by beta-lactam antibiotic (light blue) and its activity is inhibited (turns gray)

6. 5-glycine crosslinking bridges cannot form in the presence of a beta-lactam, and the cell wall is deformed and weakened

7. Mechanisms of beta-lactam resistance Drug-modifying enzymes (beta-lactamases) – Gram-positives(e.g., S. aureus) excrete the enzyme – Gram-negative (e.g., E. coli) retain the enzyme in the periplasm Overexpression of cell wall synthetic enzymes – e.g., vancomycin-intermediate S. aureus (VISA) Alteration of the PBPs so antibiotic cannot bind – e.g., S. pneumoniae, gonococcus Exclusion from the site of cell wall synthesis – Porin mutations in the outer membrane of Gram- negative bacteria only (e.g., Ps. aeruginosa)

8. Beta-lactamases (dark orange) bind to the antibiotics (light blue) and cleave the beta-lactam ring. The antibiotic is no longer able to inhibit the function of PBP (orange sunburst) Beta-lactamases

9. Beta-lactamase activity

10. Altered drug targets

11. Vancomycin-intermediate S. aureus vancomycin MIC = 2 µg/ml vancomycin MIC =8 µg/ml MRSAVISA Production of excessive cell wall; the antibiotic cannot keep up

12. MRSAVISA

13. Mechanism of vancomycin action D-ala-D-ala V

14. Mechanism of vancomycin resistance V D-ala-D-lactate Vancomycin is unable to bind to the D-ala-D- lactate structure

15. · June 2002: isolated from the catheter exit site in a chronic dialysis patient · The patient had received multiple courses of abx since April 2001; toe amputation in April 2002 --> MRSA bacteremia · VRSA also found at amputation stump wound (with VRE and Klebsiella); not in the patient’s nose · Vancomycin MIC >128mcg/ml!! (contains vanA) · Sensitive to trim/sulfa, chloro, tetracyclines, Synercid, linezolid

16. MRSA and penicillin-resistant S. pneumoniae These bacteria are both resistant because they have altered bacterial targets -- penicillin-binding proteins (PBPs or transpeptidases) In MRSA, the altered PBP2 (mecA) gene is acquired by gene transfer from another bacterium. In pneumococci, the alteration in PBP is generated by uptake of DNA released by dead oral streptococci and recombination at the pneumococcal pbp gene to create a new, chimeric protein that does not bind penicillin. – depicted on the next slide...

17. S. pneumoniae chromosomal pbp; penicillin-sensitive alpha-strep pbp Chimeric pbp (resistant to penicillin) Alpha-strep transformation S. pneumoniae DNA

18. Outer membrane permeability in Gram-negative bacteria Inner membrane Outer membrane Cell wall (peptidoglycan) Cytoplasm Beta-lactam (blue) enters through an outer membrane porin channel Altered porin channel prevents access of the antibiotic to the cell wall Bacterium