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Development of Resistance mechanisms in Pseudomonas aeruginosa Luis Martínez Martínez

Development of Resistance mechanisms in Pseudomonas aeruginosa Luis Martínez Martínez Service of Microbiology University Hospital Marqués de Valdecilla Santander, Spain. 5th ESCMID School, Santander June 2006. Antimicrobial agent. LPS. Outer Memb. Porina. Phospholípids. Peripl. space.

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Development of Resistance mechanisms in Pseudomonas aeruginosa Luis Martínez Martínez

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  1. Development of Resistance mechanisms in Pseudomonas aeruginosa Luis Martínez Martínez Service of Microbiology University Hospital Marqués de Valdecilla Santander, Spain 5th ESCMID School, Santander June 2006

  2. Antimicrobial agent LPS Outer Memb. Porina Phospholípids Peripl. space Peptidoglycan PBP ß-lactamase Inner Memb. Phospholípids Targets Cytoplasm

  3. Mechanisms of Resistance to ß-lactams

  4. Natural Resistance CXM AMP CFZ AMC PIP CAZ CTX FOX OXA PTZ

  5. Mechanisms of natural resistance to ß-lactams • AmpC production • Low permeability of outer membrane • Expression of efflux pumps • Altered PBPs?

  6. AmpC: Basal production Cell Wall ampC ampR ampD PG Recycling AmpC: Low Level Hydrolysis

  7. AmpC: Induction ß-lactam Cell Wall ampC ampR ampD PG Recycling AmpC: High Level [Partial hydrolysis]

  8. AmpC: Derepression [ß-lactam] Cell Wall ampC ampR ampD PG Recycling AmpC: Very high level

  9. TIC PIP CAZ CPM CBPM INDUCIBLE PRODUCTION S S S S S/r PARTIAL DEREP. S I I I S/r TOTAL DEREP. I R R R S/R AmpC: Resistance phenotypes

  10. CAZ CAZ IPM IPM FEP FEP MPM MPM PIP PIP Inducible AmpC Deficiente en AmpC

  11. AmpC Derepression Interpr. CMI Act. R R R R I R S S Ticarcilina Piperacilina Piper/tazob Ceftazidima Cefepime Aztreonam Imipenem Meropenem R R R R I R S S >64 >64 >64 64 16 32 1 2 CAZ FEP IPM PIP MPM

  12. B-J-M Group PROPERTIES EXAMPLES 2a PenSe/CLV (S) NPS-1 2b BroadEsp/CLV (S) TEM 1/2 2be ExtEsp/CLV (S) PER-1; TEM-42 2c CARBse/CLV (S) PSE-n, CARB-n 2d OXAse OXA-n, LCR-1 IMP,VIM, SPM,... 3 MetEnz/EDTA (S) Secondaryß-lactamases

  13. ANTIM. WILD TYPE PSE1 TEM1 OXA3 OXA11 PER1 IMP1 TIC 16 1024 2048 512 256 256 1024 TIC-CLV 32 64 32 512 128 16 1024 PIP 4 64 128 64 64 8 32 CAZ 1 1 1 1 1024 128 512 PIM 2 2 2 2 64 32 ND AZT 4 4 4 4 32 128 [4] IMP 2 2 2 2 2 2 16(var) Secondaryß-lactamases

  14. CMI Real Interpr. Ticarcillin Piperacilin Piper/tazob Ceftazidime Cefepime Aztreonam S S S S I/R S S S S S I S 64 16 16  1 16  1 ß-lactamasa OXA-31 FEP ATM CAZ

  15. Metallo-ß-lactamases IMP VIM SPM GIM SIM

  16. VIM METALLO--LACTAMASESKcat/Km (M.s) VALUES Docquier et al. J-D, JAC 2003

  17. VIM METALLO--LACTAMASESMIC VALUES Miriagou V et al, AAC 2003; Jeong SH et al, JAC 2003, Cornaglia G et al, CID 2000

  18. CMI Real Intepr. Ticarcillin Piperacillin Piper/tazob Ceftazidime Cefepime Aztreonam Imipenem Meropenem >64 >64 >64 64 32 4 >16 >16 R R R R R R R R R R S S R R R R MBL Production CAZ FEP PIP ATM IPM MPM

  19. MBL Detection ¿Using Etest? IP: imipenem IPI: imipenem + EDTA

  20. OprF P. aeruginosa

  21. OmpA E. coli

  22. OmpF E. coli

  23. OprD EXPRESSION AND CARBAPENEM RESISTANCE IN P. aeruginosa Dib et al. EJCMID 1995

  24. AmpC/OprD IN P. aeruginosa

  25. PIP CMI Interpr. Real IPM Ticarcillin Piperacillin Piper/tazob Ceftazidime Cefepime Aztreonam Imipenem Meropenem <16 <16 <16 1 2 4 16 4 CAZ S S S S S S R S S S S S S S R I MPM FEP ATM OprD Loss Inducible AmpC

  26. OprD loss in clinical isolates of P. aeruginosa producing VIM-1 OprD VIM-1(+) Cornaglia et al, CID 2000

  27. Loss of OprD porin in P. aeruginosa RNA expression of oprD IS within oprD PCR amplification of oprD Walters et al, FEMS ML 2004

  28. 66.2 45.0 31.0 ZINC DECREASES OprD LEVELS IN P. aeruginosa: RESISTANCE TO IMIPENEM OprD OprD3 MH SLUC Zn2+ MH SLUC Zn2+ P. aeruginosa PAO1 P. aeruginosa PAO1 D- Conejo MC et al, AAC 2003

  29. EFFLUX SYSTEMS MAJOR FAMILIES 1. MFS: Major Facilitator Superfamily 2. RND: Resistance Nodulation-Division 3. SMR: Small Multidrug Resistance 4. ABC: ATP-Binding Cassette 5. MATE: Multidrug And Toxic Extrusion

  30. mexA-mexB-OprM mexA mexB oprM mexR nalB mexE-mexF-OprN mexE mexF oprN mexT Nakae, Microbiología SEM, 1997 (Mod.)

  31. EFFLUX PROTEINS Located in Cytoplasmic Membrane Multiprotein Systems (PUMP+MFP+OMF) Multiple variants in the same host Broad/Narrow substrate profile

  32. OprM P. aeruginosa

  33. MULTIDRUG EEFLUX PUMPS IN P. aeruginosa

  34. BASAL EXPRESSION OF EFFLUX SYSTEMS Constitutively Expressed: MexAB-OprM, AcrAB-TolC,… Inducible systems: MexXY-OprM,… Silent in wild type strains: MexCD-OprJ, MexEF-OprN,… INTRINSIC RESISTANCE CONTRIBUTED BY CONSTITUTIVE AND INDUCIBLE SYSTEMS

  35. EXPRESSION OF ACTIVE EFFLUX • BASAL EXPRESSION • OVEREXPRESSION • Active efflux is present in both (clinically) susceptible and resistant organisms

  36. CONSTITUTIVE EXPRESSION OF EFFLUX PUMP(S) IN P. aeruginosa Antimicrobial Agent mexAB-OprMDmexAB-oprM Nalidixic Acid 512 2 Ciprofloxacin 0.25 0.008 Tetracycline 64 1 Chloramphenicol 512 4 Amoxicillin 64 0.13 Piperacillin 8 0.06 Carbenicillin 128 0.25 Ceftazidime 2 0.25 Cefepime 2 0.06 Meropenem 1 0.016

  37. SISTEMA MUTACION IMP MPM PEN/CEF/IBL CF4G TET/CLO/TMP ERY FLQ MexAB/OprM S R R R R R R nalB MexCD/OprJ S S SS R S R R nfxB-A SS S SS R R R R nfxB-B MexEF/OprN* R R S/SS S/R R S R nfxC *OprD(-) EFFLUX PUMPS

  38. OprM/OprD IN P. aeruginosa

  39. mexE-mexF-OprN mexE mexF oprN mexT oprD (-) (+) czcC czcB czcA oprD czcS czcR (-) (+) czcCBA Perron et al, JBC 2004 (Mod.)

  40. ZINC ANDRESISTANCE TO IMIPENEM. ROLE OF THE CzcCBA SYSTEM • Sublethal [Zn] induce resistance to Zn and to IMP • Imipenem may also select for Zn-R • Lethal [Zn] select stable mutants resistant to Zn and to IMP, because of mutation (V194L) in CzcS • Expression of mutated CzcS causes increased expression of czcC and decreased expression of oprD.

  41. Expression of OprN and OprD OprM OprN OprD PT75 PAO1 Köhler et al. Mol. Microbiol. 1997

  42. MICs of Imipenem against P. aeruginosahyperproducing MexCD-OprJ Wolter et al, AAC 2005

  43. Mechanisms of Resistance to other antimicrobials

  44. Natural Resistance DOX TET CHL RIF SXT

  45. Resistance to Aminoglycosides • Altered permeability • Modifying enzymes • Efflux pumps (MexXY-OprM)

  46. Resistance to Aminoglycosides • Interpretative reading: Difficult to perform • >10 compounds to be tested) • >80 different Mechanisms (but...5 of them represent 75% of cases)

  47. GM TOB Nt AK Enzyme R S S S AAC(3)-I R R S S ANT(2”)-I R R R S AAC(6’)-II, AAC(3)-II,IV,V S R R R AAC(6’)-I S S S R APH(3’)-VI Aminoglycosides

  48. Resistance to Aminoglycosides AK TOB GM TOB GM AK ANT(2”)-I AAC(6’)-II Combined mechanisms

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