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Mechanisms of Resistance to Antibiotics

Presentation of different bacterial resistance to antibiotics including extended spectrum beta lactamases, target site modifications and others

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Mechanisms of Resistance to Antibiotics

  1. 1. 1 Antimicrobial Resistance Mechanisms
  2. 2. Outline • Antibiotic selection pressure • Genetic element transfer mechanisms • Antibiotic resistance mechanisms • Resistance in major class of antibiotics 2
  3. 3. Antibiotic Resistance – A Global Problem MRSA MBL VISA VRSA PRP ESBL VRE 1961 1967 1983 1986 1988 1996 2002 All -lactams Penicillin 3rd gen cephalosporin Carbapenem Vancomycin Vancomycin and teicoplanin Vancomycin and teicoplanin Emergence → Spread
  4. 4. Genetic Basis of Resistance • Spontaneous mutations in endogenous genes – Structural genes: expanded spectrum of enzymatic activity, target-site modification, transport defect – Regulatory genes: increased expression • Acquisition of exogenous genes – Usually genes that encode inactivating enzymes or modified targets, regulatory genes – Mechanisms of DNA transfer: conjugation (cell–cell contact); transformation (uptake of DNA in solution); transduction (transfer of DNA in bacteriophages) • Expression of resistance genes – Reversible induction/repression systems can affect resistance phenotypes
  5. 5. evolution.berkeley.edu How do drug resistant bugs arise?
  6. 6. evolution.berkeley.edu How do drug resistant bugs arise?
  7. 7. evolution.berkeley.edu How do drug resistant bugs arise?
  8. 8. evolution.berkeley.edu How do drug resistant bugs arise?
  9. 9. How did that 1st drug resistant bug arise? • A simple error in DNA replication that produced a mutation – Occurs at low frequency – Mutation is on the chromosome • Mutation affects either ribosomal protein S12 or 16S rRNA to produce streptomycin resistance • Does not explain MDR bugs or high rate of spread
  10. 10. A couple of pieces of information • We know that drug resistance spreads at an alarming rate – Far too fast to be the result of single mutations in the chromosome that arise independently • We also know that bacteria become resistant to more than a single drug – If this were the result of point mutations in the chromosome the rate would be even slower
  11. 11. Can you think of other mechanisms of drug resistance? A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms
  12. 12. Horizontal Gene Transfer-Another Mechanism For Resistance http://www.bioteach.ubc.ca/Biodiversity/AttackOf TheSuperbugs
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  14. 14. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. The insertion sequence (IS) transposable element, IS1
  15. 15. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Schematic of the integration of an IS element into chromosomal DNA
  16. 16. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Structure of the composite transposon Tn10
  17. 17. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Structure of the noncomposite transposon Tn3
  18. 18. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. DNA sequence of a target site of Tn3
  19. 19. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Cointegration model for transposition of a transposable element by replicative transposition
  20. 20. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Organizational maps of bacterial plasmids with transposable elements
  21. 21. Mechanisms of Transposition Transposition is catalyzed by an enzyme, transposase, encoded by the transposon The ends of the transposon are critical for transposition
  22. 22. R plasmids
  23. 23. F Plasmid • F+ cells conjugate with F– cells – F+ donates single-stranded copy of F to F– cell (rolling circle) – F+ retains copy of plasmid, F- cell converted to F+ by replication of ssDNA donated to the F- cell – Allows F plasmid to rapidly spread through a bacterial population
  24. 24. R Plasmid
  25. 25. Pumping ssDNA
  26. 26. Mechanisms of Resistance Eliopoulos. Infectious Diseases. 1992.
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  29. 29. 30
  30. 30. Resistance Mechanisms
  31. 31. TEM1 beta-lactamase Structure
  32. 32. TEM1 beta-lactamase Structure
  33. 33. 34
  34. 34. Resistance Mechanisms
  35. 35. Altered Target • Antimicrobics act by binding and inactivating their target, which is typically a crucial enzyme or ribosomal site. • Substitutions of one amino acid in a protein can alter its’ binding. • First generation aminoglycosides and quinolones only bind one site. • Newer agents bind at multiple sites on their target making resistance improbable.
  36. 36. In widely divergent gram-pos and gram- neg species changes in one or more of peptidoglycan transpeptidase penicillin- binding proteins (PBP) have been correlated with decreased susceptibility to multiple -lactams Causes: point mutations, substitutions of amino acid sequence, and synthesis of a new enzyme. An important example : -lactam family
  37. 37. Target Site Alteration • MRSA: the essential function of PBP2 is replaced by PBP2A, the protein product of the resistance gene mecA • Penicillin resistance streptococcus pneumoniae: alteration of: PBP 1A, 2B, 2X • Macrolide resistance: Streptococcus pyogenes, strept pneumo, staphylococcus – MLSB phenotype • Macrolide, Lincomycin and Streptogramin B (quinupristin/dalfopristin) • Methylation of a single adenine on the 23S rRNA of the 50S ribosomal subunit • Encoded by the erm gene (erythromycin ribosomal methylase)
  38. 38. Mechanism Of Resistance http://www.jci.org/cgi/content/full/114/12/1693/F1
  39. 39. Mechanism of Action of Vancomycin Vancomycin binds to the D-alanyl-D-alanine dipeptide on the peptide side chain of newly synthesized peptidoglycan subunits, preventing them from being incorporated into the cell wall by penicillin-binding proteins (PBPs). In many vancomycin-resistant strains of enterococci, the D-alanyl-D-alanine dipeptide is replaced with D-alanyl-D-lactate, which is not recognized by vancomycin. Thus, the peptidoglycan subunit is appropriately incorporated into the cell wall.
  40. 40. N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys d-ala d-ala N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys d-ala d-ala Mechanism of action of Vancomycin Vancomycin blocks cell wall synthesis By binding to the d-alanyl-d-alanine site on the growing peptidoglycan chain
  41. 41. N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys d-ala d-ala Cell membrane Cell wall Transpeptidases (PBP’s) d-lactate
  42. 42. N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys d-ala d-lactate Mechanism of resistance to Vancomycin
  43. 43. Resistance Mechanisms
  44. 44. Multi Drug Efflux Pump 50
  45. 45. 51
  46. 46. 52
  47. 47. Efflux Pump System in Gram Positives and Gram Negatives 53
  48. 48. Efflux Pump – Macrolide resistance in S. aureus, S. pneumoniae and S. pyogenes • Macrolides not lincosamides or streptogramin B • encoded by the mef gene (macrolide efflux pump) • Pumps out the 14 (erythromycin and clarithromycin) and 15 member (azithromycin) rings of macrolides but not 16 member ring macrolides (miocamycin), lincosamides, streptogramins B or ketolides (telithromycin) – ß-lactam resistance in pseudomonas aeruginosa
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  51. 51. Resistance Mechanisms
  52. 52. Major Classes of Antibiotics Antibiotic Mechanism of action Major resistance mechanisms β-Lactams Inactivate PBPs (peptidoglycan synthesis) • β-lactamases • Low affinity PBPs • Efflux pumps Glycopeptides Bind to precursor of peptidoglycan • Modification of precursor Aminoglycosides Inhibit protein synthesis (bind to 30S subunit) • Modifying enzymes (add adenyl or Phosphate) Macrolides Inhibit protein synthesis (bind to 50S subunit) • Methylation of rRNA • Efflux pumps (Fluoro)Quinolones Inhibit topoisomerases (DNA synthesis) • Altered target enzyme • Efflux pumps PBPs penicillin-binding proteins
  53. 53. Major Bacterial Beta-Lactam Resistance Mechanisms MSSA Haemophilus influenzae M. catarrhalis N. gonorrhoeae E. coli Klebsiella pneumoniae Proteus vulgaris Proteus mirabilis Bacteroides fragilis MRSA S. pneumoniae p. aeruginosa
  54. 54. Mechanisms of Resistance: Pseudomonas and Efflux Pumps Adapted with permission from Livermore DM. Clin Infect Dis 2002;34:634-640. Imipenem and meropenem enter here Meropenem is pumped out while imipenem is not Efflux System Exit Portal (OprM) Outer Membrane Periplasm Linker Lipoprotein (Mex A) Cytoplasmic Membrane Efflux System Pump (Mex B) Porin
  55. 55. DNA Staphylococcus aureus MRSA mecA gene Cell Membrane Enzymes: Abnormal Penicillin Binding Protein (PBP2a) Β-Lactam Antibiotics Penicillins, (Methicillin) Cephalosporins, Monobactams, Carbapenems,Staphylococcal Cassette Chromosome (SCC) MSSA
  56. 56. Mechanisms of penicillin resistance in N. Gonorrhoeae (1) 62 Présentation des activités de l’Institut Pasteur Plasmid mediated -lactamases Acquired, Transferable (e.g. TEM) Chromosomally- mediated alterations • Altered penicillin binding proteins • Porins • Efflux pumps
  57. 57. Mechanisms of penicillin resistance in N.gonorrhoeae (2) 63 Présentation des activités de l’Institut Pasteur
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