Presentacin de PowerPoint

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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
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Antimicrobial agent
LPS
Outer Memb.
Porina
Phospholípids
Peptidoglycan
Peripl. space
PBP
ß-lactamase
Inner Memb.
Phospholípids
Targets
Cytoplasm
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Mechanisms of Resistance to ß-lactams
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Natural Resistance
CXM
AMP
CFZ
AMC
PIP
CAZ
CTX
FOX
OXA
PTZ
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Mechanisms of natural resistance to ß-lactams

• AmpC production
• Low permeability of outer membrane
• Expression of efflux pumps
• Altered PBPs?

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AmpC Basal production
Cell Wall
ampC
ampR
ampD
PG Recycling
AmpC Low Level
Hydrolysis
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AmpC Induction
ß-lactam
Cell Wall
ampC
ampR
ampD
PG Recycling
AmpC High Level
Partial hydrolysis
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AmpC Derepression
ß-lactam
Cell Wall
ampC
ampR
ampD
PG Recycling
AmpC Very high level
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AmpC Resistance phenotypes
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Inducible AmpC
Deficiente en AmpC
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AmpC Derepression
CAZ
FEP
IPM
PIP
MPM
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Secondary ß-lactamases
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Secondary ß-lactamases
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ß-lactamasa OXA-31
FEP
ATM
CAZ
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Metallo-ß-lactamases
IMP VIM SPM GIM SIM
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VIM METALLO-?-LACTAMASES Kcat/Km (M.s) VALUES
Docquier et al. J-D, JAC 2003
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VIM METALLO-?-LACTAMASES MIC VALUES
Miriagou V et al, AAC 2003 Jeong SH et al, JAC
2003, Cornaglia G et al, CID 2000
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MBL Production
CAZ
FEP
PIP
ATM
IPM
MPM
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MBL Detection Using Etest?
IP imipenem IPI imipenem EDTA
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OprF P. aeruginosa
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OmpA E. coli
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OmpF E. coli
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(No Transcript)
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OprD EXPRESSION AND CARBAPENEM RESISTANCE IN P.
aeruginosa
Dib et al. EJCMID 1995
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AmpC/OprD IN P. aeruginosa
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OprD Loss
Inducible AmpC
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OprD loss in clinical isolates of P. aeruginosa
producing VIM-1
OprD
VIM-1()
Cornaglia et al, CID 2000
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Loss of OprD porin in P. aeruginosa
RNA expression of oprD
IS within oprD
PCR amplification of oprD
Walters et al, FEMS ML 2004
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ZINC DECREASES OprD LEVELS IN P. aeruginosa
RESISTANCE TO IMIPENEM
OprD3
OprD
MH
Zn2
SLUC
MH
SLUC
Zn2
P. aeruginosa PAO1 D-
P. aeruginosa PAO1
Conejo MC et al, AAC 2003
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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
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(No Transcript)
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mexA-mexB-OprM
oprM
mexB
mexA
mexR
nalB
mexE-mexF-OprN
oprN
mexF
mexE
mexT
Nakae, Microbiología SEM, 1997 (Mod.)
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EFFLUX PROTEINS
Located in Cytoplasmic Membrane Multiprotein
Systems (PUMPMFPOMF) Multiple variants in the
same host Broad/Narrow substrate profile
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OprM P. aeruginosa
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MULTIDRUG EEFLUX PUMPS IN P. aeruginosa
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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
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EXPRESSION OF ACTIVE EFFLUX

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

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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 0.0
6 Carbenicillin 128 0.25 Ceftazidime 2 0.2
5 Cefepime 2 0.06 Meropenem 1 0.016

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EFFLUX PUMPS
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OprM/OprD IN P. aeruginosa
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mexE-mexF-OprN
oprN
mexF
mexE
mexT
oprD
(-)
()
czcA
czcB
czcC
czcS
czcR
oprD
(-)
()
czcCBA
Perron et al, JBC 2004 (Mod.)
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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.

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Expression of OprN and OprD
OprM
OprN
OprD
PT75
PAO1
Köhler et al. Mol. Microbiol. 1997
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MICs of Imipenem against P. aeruginosa
hyperproducing MexCD-OprJ
Wolter et al, AAC 2005
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Mechanisms of Resistance to other antimicrobials
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Natural Resistance
DOX
TET
CHL
RIF
SXT
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Resistance to Aminoglycosides

• Altered permeability
• Modifying enzymes
• Efflux pumps (MexXY-OprM)

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Resistance to Aminoglycosides
Interpretative reading Difficult to perform
gt10 compounds to be tested) gt80 different
Mechanisms (but...5 of them represent 75 of
cases)
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Aminoglycosides
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Resistance to Aminoglycosides
AK
TOB
GM
TOB
GM
AK
ANT(2)-I AAC(6)-II
Combined mechanisms
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Resistance to Fluoroquinolones Wild-type
phenotype
CIP
NAL
LEV
OFL
NOR
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Resistance to Fluoroquinolones

• Efflux pumps
• Target modification (Gyrase, Topo IV)
• Altered permeability LPS

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Resistance to Fluoroquinolones
CIP
OFX
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EXPRESSION OF EFFLUX PUMPS IN P. aeruginosa
Lee A., J Bacteriol 2000
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EFFECT OF PAN ON MIC OF LEVOFLOXACIN AGAINST P.
aeruginosa
Lomovskaya O et al, AAC 2001