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Development of Resistance mechanisms in Pseudomonas aeruginosa Luis Martínez Martínez Service of Microbiology University Hospital Marqués de Valdecilla.

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Presentation on theme: "Development of Resistance mechanisms in Pseudomonas aeruginosa Luis Martínez Martínez Service of Microbiology University Hospital Marqués de Valdecilla."— Presentation transcript:

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

3 Mechanisms of Resistance to ß-lactams

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

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

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

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

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

9 AmpC: Resistance phenotypes TICPIPCAZCPMCBPM INDUCIBLE PRODUCTION SSSSS/r PARTIAL DEREP.SIIIS/r TOTAL DEREP.IRRRS/R

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

11 Ticarcilina Piperacilina Piper/tazob Ceftazidima Cefepime Aztreonam Imipenem Meropenem > CMI RRRRIRSSRRRRIRSS Interpr. RRRRIRSSRRRRIRSS Act. AmpC Derepression IPM MPM CAZ FEP PIP

12 Secondary ß-lactamases B-J-M GroupPROPERTIESEXAMPLES 2aPenSe/CLV (S)NPS-1 2bBroadEsp/CLV (S)TEM 1/2 2beExtEsp/CLV (S)PER-1; TEM-42 2cCARBse/CLV (S)PSE-n, CARB-n 2dOXAseOXA-n, LCR-1 3MetEnz/EDTA (S) IMP,VIM, SPM,...

13 ANTIM.WILD TYPEPSE1TEM1OXA3OXA11PER1IMP1 TIC TIC-CLV PIP CAZ PIM ND AZT [4] IMP (var) Secondary ß-lactamases

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

15 Metallo-ß-lactamases IMP VIM SPM GIM SIM

16 VIM METALLO- -LACTAMASES K cat /K m (M.s) VALUES Docquier et al. J-D, JAC 2003 VIM-1VIM-2 IMP1.3x x 10 6 MPM2.6x x 10 6

17 VIM METALLO- -LACTAMASES MIC VALUES IMPMPM P. aeruginosa (VIM-1)> >128 E. coli (VIM-1)82 E. cloacae (VIM-2)44 Miriagou V et al, AAC 2003; Jeong SH et al, JAC 2003, Cornaglia G et al, CID 2000

18 MBL Production Ticarcillin Piperacillin Piper/tazob Ceftazidime Cefepime Aztreonam Imipenem Meropenem > >16 CMIRRRRRSRR Real Intepr. CAZ PIP FEP ATM MPM IPM

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

20 OprF P. aeruginosa

21 OmpA E. coli

22 OmpF E. coli

23

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

25 AmpC/OprD IN P. aeruginosa

26 OprD Loss Ticarcillin Piperacillin Piper/tazob Ceftazidime Cefepime Aztreonam Imipenem Meropenem < CMI SSSSSSRSSSSSSSRS Real SSSSSSRISSSSSSRI Interpr. Inducible AmpC FEP CAZ PIP IPM MPM ATM

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

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

29 ZINC DECREASES OprD LEVELS IN P. aeruginosa: RESISTANCE TO IMIPENEM OprD3OprD P. aeruginosa PAO1 D-P. aeruginosa PAO1 MHZn 2+ SLUCMHSLUCZn 2+ Conejo MC et al, AAC 2003

30 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

31

32 nalB mexR oprMmexBmexA mexA-mexB-OprM mexT oprNmexFmexE mexE-mexF-OprN Nakae, Microbiología SEM, 1997 (Mod.)

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

34 OprM P. aeruginosa

35 MULTIDRUG EEFLUX PUMPS IN P. aeruginosa

36 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

37 EXPRESSION OF ACTIVE EFFLUX 1.BASAL EXPRESSION 2.OVEREXPRESSION Active efflux is present in both (clinically) susceptible and resistant organisms

38 Antimicrobial AgentmexAB-OprM mexAB-oprM Nalidixic Acid5122 Ciprofloxacin Tetracycline641 Chloramphenicol5124 Amoxicillin Piperacillin80.06 Carbenicillin Ceftazidime20.25 Cefepime20.06 Meropenem CONSTITUTIVE EXPRESSION OF EFFLUX PUMP(S) IN P. aeruginosa

39 EFFLUX PUMPS SISTEMAMUTACIONIMPMPMPEN/CEF/IBLCF4GTET/CLO/TMPERYFLQ MexAB/OprM nalB SRRRRRR MexCD/OprJ nfxB-A SSSSRSRR nfxB-B SSS RRRR MexEF/OprN* nfxC RRS/SSS/RRSR *OprD(-)

40 OprM/OprD IN P. aeruginosa

41 oprD mexT oprNmexFmexE mexE-mexF-OprN Perron et al, JBC 2004 (Mod.) (-)(+) czcS czcAczcBczcC (-)(+) czcCBA czcRoprD

42 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. ZINC AND RESISTANCE TO IMIPENEM. ROLE OF THE CzcCBA SYSTEM

43 Expression of OprN and OprD PT75PAO1 OprD OprM Köhler et al. Mol. Microbiol OprN

44 MICs of Imipenem against P. aeruginosa hyperproducing MexCD-OprJ Wolter et al, AAC 2005

45 Mechanisms of Resistance to other antimicrobials

46 RIF TET SXT CHL DOX Natural Resistance

47 Altered permeability Modifying enzymes Efflux pumps (MexXY-OprM) Resistance to Aminoglycosides

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

49 Aminoglycosides S R R S S Nt AAC(3)-ISSR APH(3)-VIRSS AAC(6)-IRRS AAC(6)-II, AAC(3)-II,IV,VSRR ANT(2)-ISRR EnzymeAKTOBGM

50 TOB GM AK TOB GM ANT(2)-I AAC(6)-II Combined mechanisms Resistance to Aminoglycosides

51 Resistance to Fluoroquinolones: Wild-type phenotype LEV NOR OFL CIP NAL

52 Resistance to Fluoroquinolones Efflux pumps Target modification (Gyrase, Topo IV) Altered permeability: LPS

53 Resistance to Fluoroquinolones Ciprofloxacin Ofloxacin 1414 CMI IIII Interpr. SISI Real Mex AB-OprM CIPOFX

54 EXPRESSION OF EFFLUX PUMPS IN P. aeruginosa Lee A., J Bacteriol 2000

55 EFFECT OF PAN ON MIC OF LEVOFLOXACIN AGAINST P. aeruginosa StrainPANMIC of Levofloxacin+[PAN] 0 mg/L10 mg/L20 mg/L WT >5120,1250,0150,008 nalB >51220,1250,03 nfxB >51220,1250,03 nfxC >51240,250,06 Mex(*) 320,015 0,008 Lomovskaya O et al, AAC 2001


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