Development of Resistance mechanisms in Pseudomonas aeruginosa

Luis Martínez MartínezService of Microbiology

University Hospital Marqués de ValdecillaSantander, Spain

5th ESCMID School, Santander June 2006

Peptidoglycan

Outer Memb.

PBPß-lactamase

Antimicrobial agent

PorinaPorina

LPS

Phospholípids

Phospholípids

Cytoplasm Targets

Peripl. space

Inner Memb.

Mechanisms of Resistance to ß-lactams

Natural Resistance

PIP

AMP

AMC

OXA

PTZ

FOX

CAZ

CTX

CFZ

CXM

• AmpC production

• Low permeability of outer membrane

• Expression of efflux pumps

• Altered PBPs?

Mechanisms of natural resistance to ß-lactams

ampD ampR ampC

AmpC: Basal production

PG Recycling

Hydrolysis

AmpC: Low Level

Cell Wall

ampD ampR ampC

AmpC: Induction

PG Recycling

AmpC: High Level

ß-lactam

Cell Wall

[Partial hydrolysis]

ampD ampR ampC

AmpC: Derepression

PG Recycling

AmpC: Very high level

[ß-lactam]

Cell Wall

AmpC: Resistance phenotypes

TIC PIP CAZ CPM CBPMINDUCIBLEPRODUCTION

S S S S S/r

PARTIAL DEREP. S I I I S/r

TOTAL DEREP. I R R R S/R

Inducible AmpCDeficiente en AmpC

IPM

MPM

CAZ

FEP

PIPPIPMPM

FEP

CAZIPM

TicarcilinaPiperacilinaPiper/tazobCeftazidimaCefepimeAztreonamImipenemMeropenem

>64>64>6464163212

CMIRRRRIRSS

Interpr.RRRRIRSS

Act.

AmpC Derepression

IPM

MPM

CAZ

FEP

PIP

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,...

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)

Secondary ß-lactamases

ß-lactamasa OXA-31

TicarcillinPiperacilinPiper/tazobCeftazidimeCefepimeAztreonam

641616 116

1

CMISSSSI/RS

Interpr.SSSSIS

Real

CAZ

ATM

FEP

Metallo-ß-lactamases

IMP

VIM

SPM

GIM

SIM

VIM METALLO--LACTAMASES Kcat/Km (M.s) VALUES

Docquier et al. J-D, JAC 2003

VIM-1 VIM-2

IMP 1.3x 105 3.8x 106

MPM 2.6x 105 2.5x 106

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 4

Miriagou V et al, AAC 2003; Jeong SH et al, JAC 2003, Cornaglia G et al, CID 2000

MBL Production

TicarcillinPiperacillinPiper/tazobCeftazidimeCefepimeAztreonamImipenemMeropenem

>64>64>6464324

>16>16

CMI

R RR RR RR RR RS SR RR R

Real Intepr.CAZ

PIP

FEP

ATM

MPM

IPM

MBL Detection ¿Using Etest?

IP: imipenemIPI: imipenem + EDTA

OprF P. aeruginosa

OmpA E. coli

OmpF E. coli

OprD EXPRESSION AND CARBAPENEM RESISTANCE IN P. aeruginosa

MIC IMP OprD ++

OprD +

OprD

- <=1 5 1 0

2 4 5 0

4-8 6 8 5

>=16 0 0 55

Dib et al. EJCMID 1995

AmpC/OprD IN P. aeruginosa

AmpC OprD IMP MPM CRB CAZ BASAL

+

1

0.25

64

1

DEREPRESSED

+

1

0.25

128

32

BASAL

-

0.5

1

32

1

DEREPRESSED

-

16

2

64

32

OprD Loss

TicarcillinPiperacillinPiper/tazobCeftazidimeCefepimeAztreonamImipenemMeropenem

<16<16<16

124

164

CMI

SSSSSSRS

Real

SSSSSSRI

Interpr.

Inducible AmpC

FEP

CAZ

PIP

IPM

MPM

ATM

OprD

VIM-1(+)

OprD loss in clinical isolates of P. aeruginosa producing VIM-1

Cornaglia et al, CID 2000

IS within oprD

RNA expression of oprD

PCR amplification of oprD Walters et al, FEMS ML 2004

Loss of OprD porin in P. aeruginosa

66.2

45.0

31.0

ZINC DECREASES OprD LEVELS IN P. aeruginosa: RESISTANCE TO IMIPENEM

OprD3OprD

P. aeruginosa PAO1 D-P. aeruginosa PAO1

MH Zn2+SLUC MH SLUC Zn2+

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

nalB mexR oprMmexBmexA

mexA-mexB-OprM

mexT oprNmexFmexE

mexE-mexF-OprN

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

PUMP MFP OMP GENES REG SUBSTRATES

MexB MexA OprM mexAmexBoprM mexR Tet, Cl, FQ, -L (MPM...),

Rif, Nov, SXT,...

MexD MexC OprJ mexCmexDoprJ mexS Tet, Cl, FQ, -L

(Cephems), TMP

MexF MexE OprN mexEmexForpN mexT Cl, TMP, FQ

MexY MexX OprM mexXmexY mexZ AmGlu, Tet, Ery

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

1. BASAL EXPRESSION

2. OVEREXPRESSION

Active efflux is present in both (clinically) susceptible and resistant organisms

Antimicrobial Agent mexAB-OprM mexAB-oprM

Nalidixic Acid 512 2Ciprofloxacin 0.25 0.008Tetracycline 64 1Chloramphenicol 512 4Amoxicillin 64 0.13Piperacillin 8 0.06Carbenicillin 128 0.25Ceftazidime 2 0.25Cefepime 2 0.06Meropenem 1 0.016

CONSTITUTIVE EXPRESSION OF EFFLUX PUMP(S) IN P. aeruginosa

EFFLUX PUMPS

SISTEMA MUTACION IMP MPM PEN/CEF/IBL CF4G TET/CLO/TMP ERY FLQ

MexAB/OprM nalB S R R R R R R

MexCD/OprJ nfxB-A S S SS R S R R

nfxB-B SS S SS R R R R

MexEF/OprN* nfxC R R S/SS S/R R S R

*OprD(-)

OprM/OprD IN P. aeruginosa

OprM OprD IMP MPM CRB

+++ + 1 4 256

+++ - 16 16 256

+ + 1 0.5 64

+ - 16 4 64

- + 1 0.125 1

- - 16 0.5 2

oprD mexT oprNmexFmexE

mexE-mexF-OprN

Perron et al, JBC 2004 (Mod.)

(-) (+)

czcS czcAczcBczcC

(-) (+)

czcCBA

czcRoprD

•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

Expression of OprN and OprD

PT75 PAO1

OprD

OprM

Köhler et al. Mol. Microbiol. 1997

OprN

MICs of Imipenem against P. aeruginosa hyperproducing MexCD-OprJ

Strain OprD MexC IMP

164

1.0

1.0

4

164-921C 1.3 736 0.5

164MI 1.0 1.0 4 164-MI-94C 0.8 1332 1

164CD 1.0 1.0 8 164CD-921C 0.8 1304 2

Wolter et al, AAC 2005

Mechanisms of Resistance to other antimicrobials

RIF

TET

SXT

CHL

DOX

Natural Resistance

•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)

Resistance to Aminoglycosides

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

TOB

GM

AK

AK

TOB

GM

ANT(2”)-IAAC(6’)-II

Combined mechanisms

Resistance to Aminoglycosides

Resistance to Fluoroquinolones: Wild-type phenotype

LEV

NOR

OFL

CIPNAL

Resistance to Fluoroquinolones

Efflux pumps

Target modification (Gyrase, Topo IV)

Altered permeability: LPS

Resistance to Fluoroquinolones

CiprofloxacinOfloxacin

14

CMIII

Interpr.SI

Real

Mex AB-OprM

CIP OFX

EXPRESSION OF EFFLUX PUMPS IN P. aeruginosa

OprM OprJ OprN LVFX FEP CARB+ - - 0.125 0.5 64- - - 0.03 0.125 0.5

+++ - - 1 4 256- +++ - 2 4 0.5- - +++ 2 0.25 0.5

+++ +++ - 2 4 256+++ - +++ 4-8 4 256

- +++ +++ 4-8 2 0.5+++ +++ +++ 8 4 256

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 >512 2 0,125 0,03

nfxB >512 2 0,125 0,03

nfxC >512 4 0,25 0,06

Mex(*) 32 0,015 0,015 0,008

Lomovskaya O et al, AAC 2001