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

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

Mechanisms of Resistance to ß-lactams

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

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

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

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

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

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

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

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

Secondary ß-lactamases 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 3 MetEnz/EDTA (S) IMP,VIM, SPM,...

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

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

Metallo-ß-lactamases IMP VIM SPM GIM SIM

VIM METALLO--LACTAMASES Kcat/Km (M.s) VALUES IMP 1.3x 105 3.8x 106 MPM 2.6x 105 2.5x 106 Docquier et al. J-D, JAC 2003

VIM METALLO--LACTAMASES MIC VALUES IMP MPM P. aeruginosa (VIM-1) >128 128->128 E. coli (VIM-1) 8 2 E. cloacae (VIM-2) 4 Miriagou V et al, AAC 2003; Jeong SH et al, JAC 2003, Cornaglia G et al, CID 2000

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

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

OprF P. aeruginosa

OmpA E. coli

OmpF E. coli

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

AmpC/OprD IN P. aeruginosa

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

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

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

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

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

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

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

OprM P. aeruginosa

MULTIDRUG EEFLUX PUMPS IN P. aeruginosa

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

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

CONSTITUTIVE EXPRESSION OF EFFLUX PUMP(S) IN P. aeruginosa Antimicrobial Agent mexAB-OprM DmexAB-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 19

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

OprM/OprD IN P. aeruginosa

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

ZINC AND RESISTANCE 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.

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

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

Mechanisms of Resistance to other antimicrobials

Natural Resistance DOX TET CHL RIF SXT

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

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

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

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

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

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

Resistance to Fluoroquinolones CIP OFX Ciprofloxacin Ofloxacin 1 4 CMI I Interpr. S Real Mex AB-OprM

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

EFFECT OF PAN ON MIC OF LEVOFLOXACIN AGAINST P. aeruginosa Strain PAN MIC of Levofloxacin+[PAN] 0 mg/L 10 mg/L 20 mg/L WT >512 0,125 0,015 0,008 nalB 2 0,03 nfxB nfxC 4 0,25 0,06 Mex(*) 32 Lomovskaya O et al, AAC 2001