development of resistance mechanisms in pseudomonas aeruginosa luis martínez martínez service of...
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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