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Development of Resistance mechanisms in Pseudomonas aeruginosa. Luis Mart nez Mart nez ... Perron et al, JBC 2004 (Mod.) czcS. czcA. czcB. czcC. czcCBA. czcR. oprD ... – PowerPoint PPT presentation

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Title: Presentacin de PowerPoint


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
Antimicrobial agent
LPS
Outer Memb.
Porina
Phospholípids
Peptidoglycan
Peripl. space
PBP
ß-lactamase
Inner Memb.
Phospholípids
Targets
Cytoplasm
3
Mechanisms of Resistance to ß-lactams
4
Natural Resistance
CXM
AMP
CFZ
AMC
PIP
CAZ
CTX
FOX
OXA
PTZ
5
Mechanisms of natural resistance to ß-lactams
  • AmpC production
  • Low permeability of outer membrane
  • Expression of efflux pumps
  • Altered PBPs?

6
AmpC Basal production
Cell Wall
ampC
ampR
ampD
PG Recycling
AmpC Low Level
Hydrolysis
7
AmpC Induction
ß-lactam
Cell Wall
ampC
ampR
ampD
PG Recycling
AmpC High Level
Partial hydrolysis
8
AmpC Derepression
ß-lactam
Cell Wall
ampC
ampR
ampD
PG Recycling
AmpC Very high level
9
AmpC Resistance phenotypes
10
Inducible AmpC
Deficiente en AmpC
11
AmpC Derepression
CAZ
FEP
IPM
PIP
MPM
12
Secondary ß-lactamases
13
Secondary ß-lactamases
14
ß-lactamasa OXA-31
FEP
ATM
CAZ
15
Metallo-ß-lactamases
IMP VIM SPM GIM SIM
16
VIM METALLO-?-LACTAMASES Kcat/Km (M.s) VALUES
Docquier et al. J-D, JAC 2003
17
VIM METALLO-?-LACTAMASES MIC VALUES
Miriagou V et al, AAC 2003 Jeong SH et al, JAC
2003, Cornaglia G et al, CID 2000
18
MBL Production
CAZ
FEP
PIP
ATM
IPM
MPM
19
MBL Detection Using Etest?
IP imipenem IPI imipenem EDTA
20
OprF P. aeruginosa
21
OmpA E. coli
22
OmpF E. coli
23
(No Transcript)
24
OprD EXPRESSION AND CARBAPENEM RESISTANCE IN P.
aeruginosa
Dib et al. EJCMID 1995
25
AmpC/OprD IN P. aeruginosa
26
OprD Loss
Inducible AmpC
27
OprD loss in clinical isolates of P. aeruginosa
producing VIM-1
OprD
VIM-1()
Cornaglia et al, CID 2000
28
Loss of OprD porin in P. aeruginosa
RNA expression of oprD
IS within oprD
PCR amplification of oprD
Walters et al, FEMS ML 2004
29
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
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
(No Transcript)
32
mexA-mexB-OprM
oprM
mexB
mexA
mexR
nalB
mexE-mexF-OprN
oprN
mexF
mexE
mexT
Nakae, Microbiología SEM, 1997 (Mod.)
33
EFFLUX PROTEINS
Located in Cytoplasmic Membrane Multiprotein
Systems (PUMPMFPOMF) 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
  • BASAL EXPRESSION
  • OVEREXPRESSION
  • Active efflux is present in both (clinically)
    susceptible and resistant organisms

38
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

39
EFFLUX PUMPS
40
OprM/OprD IN P. aeruginosa
41
mexE-mexF-OprN
oprN
mexF
mexE
mexT
oprD
(-)
()
czcA
czcB
czcC
czcS
czcR
oprD
(-)
()
czcCBA
Perron et al, JBC 2004 (Mod.)
42
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.

43
Expression of OprN and OprD
OprM
OprN
OprD
PT75
PAO1
Köhler et al. Mol. Microbiol. 1997
44
MICs of Imipenem against P. aeruginosa
hyperproducing MexCD-OprJ
Wolter et al, AAC 2005
45
Mechanisms of Resistance to other antimicrobials
46
Natural Resistance
DOX
TET
CHL
RIF
SXT
47
Resistance to Aminoglycosides
  • Altered permeability
  • Modifying enzymes
  • Efflux pumps (MexXY-OprM)

48
Resistance to Aminoglycosides
Interpretative reading Difficult to perform
gt10 compounds to be tested) gt80 different
Mechanisms (but...5 of them represent 75 of
cases)
49
Aminoglycosides
50
Resistance to Aminoglycosides
AK
TOB
GM
TOB
GM
AK
ANT(2)-I AAC(6)-II
Combined mechanisms
51
Resistance to Fluoroquinolones Wild-type
phenotype
CIP
NAL
LEV
OFL
NOR
52
Resistance to Fluoroquinolones
  • Efflux pumps
  • Target modification (Gyrase, Topo IV)
  • Altered permeability LPS

53
Resistance to Fluoroquinolones
CIP
OFX
54
EXPRESSION OF EFFLUX PUMPS IN P. aeruginosa
Lee A., J Bacteriol 2000
55
EFFECT OF PAN ON MIC OF LEVOFLOXACIN AGAINST P.
aeruginosa
Lomovskaya O et al, AAC 2001
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