Post on 11-Jan-2020
1INRA-Toulouse21/05/2010
AntibioticAntibiotic efflux efflux in in procaryoticprocaryotic and and eucaryoticeucaryotic cellscells::
fromfrom molecularmolecular mechanismsmechanisms toto pharmacologicalpharmacological consequencesconsequences
Françoise Van Bambeke, PharmD, PhD
Pharmacologie cellulaire et moléculaireLouvain Drug Research
Institute
Université
catholique de LouvainBruxelles, Belgium
www.facm.ucl.ac.be
2INRA-Toulouse21/05/2010
Where do I come from ?
"corpora non aguntnisi fixata"
"The goal is
… to findchemical
substances that
have special
affinities
for pathogenic
organisms
and that, like
magic
bullets, go straight to their
targets"
Université
catholique de LouvainLouvain Drug Research
InstituteBrussels
3INRA-Toulouse21/05/2010
WhyWhy active efflux ?active efflux ?
Manneken Pis, who saved Brussels from fire
4INRA-Toulouse21/05/2010
Chemotherapeutic agents exert toxic effects on specific target cells
How can these drugsreach their target inside the cells ?
nucleic acids
ribosomes
enzymes
5INRA-Toulouse21/05/2010
Reaching an intracellular target …
polar drug
lipophilic drug
physico-chemical properties are inadequate for reaching an intracellular target !
Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70
6INRA-Toulouse21/05/2010
Reaching an intracellular target …
amphipathic drug
most drugs are amphipathic by design,to be able to cross membrane barriers !
Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70
7INRA-Toulouse21/05/2010
Intracellular chemotherapeutic agents
But a diffusible compound may have
potentially harmful effects !Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70
8INRA-Toulouse21/05/2010
Why efflux transporters ?
Extrusion by efflux pumps
Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70
9INRA-Toulouse21/05/2010
Why efflux transporters ?
Extrusion by efflux pumps
Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70
general mean of protectionagainst cell invasion by diffusible molecules
10INRA-Toulouse21/05/2010
Typical ‘toxic’ diffusible substances as substrates for efflux pumps
antifungals
anticancer
agents
antibiotics
11INRA-Toulouse21/05/2010
Most antibiotics do act on intracellular targets
(lipo- and glycopeptides) (-lactams)
Efflux as a mechanism
of resistanceby reducing
antibiotic
concentration inside
the bacteria
quinolones
macrolides aminoglycosides
tetracyclines
sulfamides
14INRA-Toulouse21/05/2010
Active efflux in Active efflux in bacteriabacteria:: rolerole in in antibioticantibiotic resistanceresistance
Niagara Falls, Canada
15INRA-Toulouse21/05/2010
FQ efflux pumps in S. pneumoniae
Terry et al., Nature Reviews Microbiology 2005; 3: 566-572
Primary
transporters«
ATP-Binding
Cassette
»
Secondary
transporters(Proton motive force)
PmrAGill et al, AAC 1999; 43:187-9
PatA/PatBMarrer et al, AAC 2006; 50:685-93
16INRA-Toulouse21/05/2010
Fluoroquinolone antibiotics
ciprofloxacin
N
C
O
FOH
O
OCH3
HN N
moxifloxacin
N
O
OH
O
HNN
F
17INRA-Toulouse21/05/2010
Selection of resistance by subMIC concentrations of FQ
ciprofloxacin moxifloxacin
Loss of susceptibility; efflux selected by ciprofloxacin Avrain et al, JAC (2007) 60:965-972
no efflux
basal efflux
18INRA-Toulouse21/05/2010
Selection of resistance by subMIC concentrations of FQ
ciprofloxacin moxifloxacin
Loss of susceptibility; efflux selected by ciprofloxacin only Avrain et al, JAC (2007) 60:965-972
no efflux
basal efflux
19INRA-Toulouse21/05/2010
Selection of resistance by subMIC concentrations of FQ
Expression of genes coding
for efflux pumps
in the absence of fluoroquinolones
Increased efflux due toPatA/PatB
Avrain et al, JAC (2007) 60:965-972
24INRA-Toulouse21/05/2010
PatA/B are inducible by fluoroquinolones
0 1 2 3 4 5 60
25
50
75
100
125
150
175
MXFCIP
expr
essi
on ra
tio (%
val
ue a
t 4 h
)
0 1 2 3 4 5 60
25
50
75
100
125
150
175CIPMXF
patA patB
time (h) time (h)
Bacteria in the presence of ½ MIC of FQ ATCC49619
El garch et al, ECCMID (2009) O495
25INRA-Toulouse21/05/2010
Kinetics of induction & reversibility
0 1 2 3 4 5 60
25
50
75
100
125
150
175
MXFCIP
expr
essi
on ra
tio (%
val
ue a
t 4 h
)
0 1 2 3 4 50
25
50
75
100
125
150
175
0 1 2 3 4 5 60
25
50
75
100
125
150
175CIPMXF
0 1 2 3 4 50
25
50
75
100
125
150
175patA patB
time (h) time (h)
4 h with ½ MIC; up to 5 h without FQ ATCC49619
Overexpression
may contribute to resistance during treatment !El garch et al, ECCMID (2009) O495
26INRA-Toulouse21/05/2010
Efflux in S. pneumoniae: is it important in the clinics ?
MIC of fluoroquinolones in 107 strains collected from patients with AECB
Reserpine reduces MICs !
27INRA-Toulouse21/05/2010
Efflux in S. pneumoniae: is it important in the clinics ?
Suspected efflux based on phenotypic analysis
28INRA-Toulouse21/05/2010
Efflux and resistance in P. aeruginosa
Mesaros et al. (2005) Louvain médical. 124:308-20
29INRA-Toulouse21/05/2010
Efflux and low level of resistance in Pseudomonas aeruginosa
MICs
vs EUCAST breakpoints
for 109 P. aeruginosa without
or with
efflux mechanisms,
isolated
from
ICU patients (VAP)
Riou et al, ECCMID 2010
meropenem
mexA < 2 mexA 20.03125
0.06250.125
0.250.5
1248
163264
128256
mexA expression level
amikacin
mexX < 5 mexX 50.25
0.51248
163264
128256
mexX expression level
MIC
pip-tazo
mexA < 2 mexA 20.03125
0.06250.125
0.250.5
1248
163264
128256
mexA expression level
MexX substr.
MexA substr.
MexA substr.
30INRA-Toulouse21/05/2010
Efflux selection during treatment
Prevalence of MexA
and MexX
overexpressers
in 62 phylogentically-related pairs of P. aeruginosa isolated from ICU patients (VAP)
DAY x (%)
38.71%
22.58%
20.97%
17.74%
DAY 0 (%)
66.13%
12.90%
11.29%
9.68%
MexA-/MexX-
MexA+/MexX-
MexX+/MexA-
MexA+/MexX+
Riou et al, ECCMID 2010
31INRA-Toulouse21/05/2010
Efflux selection during treatment
Riou et al, ECCMID 2010
Antibiotic no. patients
Piperacillin-tazobactam
(TZP) 26Amikacin
(AMK) 22Meropenem
(MEM) 20Cefepime
(CEF) 19Ciprofloxacin (CIP) 6
Antipseudomonal antibiotics received by the patients during
treatment
DAY 0 DAY X05
1015
202530
3540
455055
60
0
234
1
nb effluxsystems
num
ber o
f str
ains
global influence of treatment
number of efflux systems detected at day 0 and day X
69% com
binations
32INRA-Toulouse21/05/2010
Active efflux in Active efflux in eucaryoticeucaryotic cellscells:: rolerole in in antibioticantibiotic PK/PDPK/PD
Old Faithful Geyser
33INRA-Toulouse21/05/2010
Target accessibility is critical for intracellular activity
34INRA-Toulouse21/05/2010
Active efflux reduces antibiotic cellular concentration
35INRA-Toulouse21/05/2010
Consequences of antibiotic efflux from eucaryotic cells
• alteration of pharmacokinetics
• alteration of pharmacodynamics
• single cell: accumulation, localization
• whole
organism: absorption, distribution, elimination
• cellular level: activity
against
intracellular
bacteria
• body level: drug
concentration in the infected
compartment
Van Bambeke et al, JAC (2003) 51:1067-1077
36INRA-Toulouse21/05/2010
Characterization of antibiotic Characterization of antibiotic efflux pumps in macrophages efflux pumps in macrophages
Belgian beer
37INRA-Toulouse21/05/2010 37
ATP-Binding Cassette transporters
ABCs transporters
ABCA ABCB ABCC ABCD ABCE ABCF ABCG
P-gp MRPs BCRP
multidrug transporters
38INRA-Toulouse21/05/2010
Fluoroquinolone antibiotics
ciprofloxacin
moxifloxacin
N
O
OH
O
HNN
F
N
C
O
FOH
O
OCH3
HN N
39INRA-Toulouse21/05/2010
Kinetics of accumulation and efflux for ciprofloxacin
Michot et al. (2004) AAC 48:2673-82
both accumulation and efflux markedly affectedBy probenecid (inhibitors of Mrps)
extracell. conc. 17 mg/L; probenecid 5 mM
40INRA-Toulouse21/05/2010
Ciprofloxacin, classical model
Kolaczkowski & Goffeau (1997) Pharmacol. Ther. 76:219-42
41INRA-Toulouse21/05/2010
Kinetics of accumulation and efflux for moxifloxacin
Michot et al. AAC (2005) 49:2429-37
neither accumulation nor efflux affectedBy probenecid
extracell. conc. 17 mg/L; probenecid
5 mM
42INRA-Toulouse21/05/2010
Quinolones as inhibitors of ciprofloxacin efflux
• ciprofloxacin efflux inhibited by ciprofloxacin
Michot et al. AAC (2005) 49:2429-37
43INRA-Toulouse21/05/2010
Quinolones as inhibitors of ciprofloxacin efflux
• ciprofloxacin efflux inhibited by ciprofloxacin• moxifloxacin not affected
Michot et al. AAC (2005) 49:2429-37
44INRA-Toulouse21/05/2010
Quinolones as inhibitors of ciprofloxacin efflux
• ciprofloxacin efflux inhibited by ciprofloxacinmoxifloxacin
CIP MXF
CIP
Michot et al. AAC (2005) 49:2429-37
45INRA-Toulouse21/05/2010
Quinolones as inhibitors of ciprofloxacin efflux
• ciprofloxacin efflux inhibited by ciprofloxacinmoxifloxacin
CIP MXF
moxifloxacinalso able
to interactwith the transporter !
CIP
Michot et al. AAC (2005) 49:2429-37
46INRA-Toulouse21/05/2010
Moxifloxacin, ‘futile-cycle’ model
Eytan et al. (1996) JBC 271:12897-902
47INRA-Toulouse21/05/2010
Influence of efflux pumps Influence of efflux pumps on antibiotic activityon antibiotic activity against intracellular against intracellular
infections infections
Belgian chocolate
48INRA-Toulouse21/05/2010
Active efflux reduces antibiotic intracellular activity
49INRA-Toulouse21/05/2010
Models of intracellular infection
L. monocytogenes S. aureus
cytosol phagolysosomes
50INRA-Toulouse21/05/2010
Influence of pump inhibitors on intracellular activity
azithromycin and L. monocytogenes
verapamil 20 µM; 24 h
L. monocytogenes
AZMAZM
Seral et al. (2003) JAC 51:1167-73
51INRA-Toulouse21/05/2010
azithromycin and S. aureus
verapamil 20 µM; 24 h
AZMAZM
S. aureus
Influence of pump inhibitors on intracellular activity
Seral et al. (2003) JAC 51:1167-73
52INRA-Toulouse21/05/2010
ciprofloxacin and L. monocytogenes
gemfibrozil 250 µM; 24 h
L. monocytogenes
CIP
Influence of pump inhibitors on intracellular activity
Seral et al. (2003) JAC 51:1167-73
53INRA-Toulouse21/05/2010
ciprofloxacin and S. aureus
gemfibrozil 250 µM; 24 h
CIP
S. aureus
Influence of pump inhibitors on intracellular activity
Seral et al. (2003) JAC 51:1167-73
54INRA-Toulouse21/05/2010
Influence of pump inhibitors on antibiotic distribution
L. monocytogenes
AZMAZM
S. aureus
verapamil enhances azithromycin concentrationIn cytosol and vacuoles
Seral et al. (2003) JAC 51:1167-73
55INRA-Toulouse21/05/2010
L. monocytogenes
CIP
S. aureus
gemfibrozil enhances ciprofloxacin cytosolic content
Influence of pump inhibitors on antibiotic distribution
Seral et al. (2003) JAC 51:1167-73
56INRA-Toulouse21/05/2010
Can we make Can we make eucaryoticeucaryotic cells cells «« resistantresistant »» to antibiotics ?to antibiotics ? A way to further characterize A way to further characterize
efflux transporters efflux transporters
J.M. Folon, La Hulpe, Belgium
57INRA-Toulouse21/05/2010
Over-expression of efflux pumps as mechanism of resistance
How to get resistant cells ?
Gottesman et al, Methods Enzymol. (1998) 292: 248-58
•
step-wise
increase in AB concentration
•
several
passages at
each
step
time and drugconsumming!
multifactorial
multidrug
resistance
58INRA-Toulouse21/05/2010
Identification of the ciprofloxacin transporter: « resistant » cells as a tool
Chronical exposure to increasing concentrations
Michot et al., Antimicrob. Ag. Chemother. (2006) 50:1689-1695
0.1 mM
0.15 mM
0.2 mM
59INRA-Toulouse21/05/2010
Ciprofloxacin « resistant » cells: phenotypic analysis
ATP-dependent
reduction
in cell
accumulation of CIP; MXF non affected
control ATP-depleted
CIP MXF0
250
500
750
1000 WT
cellu
lar c
once
ntra
tion
(ng/
mg
prot
)
CIP MXF0
250
500
750
1000 WT
Michot et al., Antimicrob. Ag. Chemother. (2006) 50:1689-1695
60INRA-Toulouse21/05/2010
Ciprofloxacin « resistant » cells: phenotypic analysis
CIP MXF0
250
500
750
1000 WT RS
cellu
lar c
once
ntra
tion
(ng/
mg
prot
)
CIP MXF0
250
500
750
1000 WT RS
ATP-dependent
reduction
in cell
accumulation of CIP; MXF non affected
control ATP-depleted
Michot et al., Antimicrob. Ag. Chemother. (2006) 50:1689-1695
61INRA-Toulouse21/05/2010
Ciprofloxacin « resistant » cells: phenotypic analysis
efflux rate
IC50
gemfibrozil
Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6
62INRA-Toulouse21/05/2010
ARNm levels (Real-Time PCR)
expression Mrp2 and Mrp4, but Mrp4 predominates
Ciprofloxacin « resistant » cells: genotypic analysis
Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6
63INRA-Toulouse21/05/2010
Ciprofloxacin « resistant » cells: proteomic analysis
Mrp2
µg 5 15 30 5 15 30
µg 5 15 30 5 15 30
Mrp4
WT RS
WT RS
detection of the proteins by Western-Blot of membrane fraction Confocal
microscopy
Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6
64INRA-Toulouse21/05/2010
Ciprofloxacin « resistant » cells : which transporter ?
Mrp2 Mrp4- CT 10 25 10 25 - CT 5 10 25 2510
Specific extinction of gene expression by siRNA
Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6
65INRA-Toulouse21/05/2010
Acquisition of resistance is a stepwise process
0 50 100 150 2000
20
40
60
80
100
0
5
10
15
ciprofloxacin concentration (µM)to which cells are resistant
cipr
oflo
xaci
n re
sidu
al a
ccum
ulat
ion
(% w
ild-ty
pe c
ells
) Abcc4 m
RN
A ratio
Accumulation and Mrp4 expression during selection of resistance
204060801000
5
10
15
20R2 = 0.994
ciprofloxacin residual accumulation(% wild-type cells)
Abc
c4 m
RN
A ra
tio
66INRA-Toulouse21/05/2010
Can we select for moxifloxacin « resistant » cells ?
ciprofloxacin moxifloxacin
FQ accumulation and gemfibrozil effect
WT
Vallet et al., FEBS-ABC meeting. (2010)
67INRA-Toulouse21/05/2010
Can we select for moxifloxacin « resistant » cells ?
ciprofloxacin moxifloxacin
WT
MXF-RS
FQ accumulation and gemfibrozil effect
Vallet et al., FEBS-ABC meeting. (2010)
68INRA-Toulouse21/05/2010
Can we select for moxifloxacin « resistant » cells ?
Ciprofloxacin efflux
T1/2
WT >> T1/2
WT+GEM = T1/2
MXF-RSVallet et al., FEBS-ABC meeting. (2010)
69INRA-Toulouse21/05/2010
Moxifloxacin-exposed cells are « anti » resistant!
Mrp4 expression
WT MXF-RS CIP RS
Mrp4
WT MXF- -
20µg 40µg 60µg 20µg 40µg 60µg 10µg
Actin
Western-Blot ARNm
(Real Time PCR)
Vallet et al., FEBS-ABC meeting. (2010)
70INRA-Toulouse21/05/2010
Fluoroquinolone transport: model
ciprofloxacin
inout
41
inout
0.21
inout
151
WT CIP-RS MXF-RS
71INRA-Toulouse21/05/2010
Fluoroquinolone transport: model
moxifloxacin
inout
151
inout
151
inout
151
WT CIP-RS MXF-RS
72INRA-Toulouse21/05/2010
Efflux pumps … and what next ?
How to get resistant cells ?
Gottesman et al, Methods Enzymol. (1998) 292: 248-58
•
step-wise
increase in AB concentration
•
several
passages at
each
step
time and drugconsumming!
multifactorial
multidrug
resistance
73INRA-Toulouse21/05/2010
13C6
-Lys13C6
-Arg12C6
-Lys12C6
-Arg
WT CIP-macrophages
Stable Isotope Labeling Aminoacid in Culture
sample
mixing
1:1
protein
digestion
identification in mass spectrometryand determination
of the relative abundance
74INRA-Toulouse21/05/2010
13C6
-Lys13C6
-Arg12C6
-Lys12C6
-Arg
WT CIP-macrophages
DiscontinuousSucrose gradient
Enriched plasmamembrane fraction
Stable Isotope Labeling Aminoacid in Culture
post-nuclearhomogenate
1.10
1.13
1.15
1.17
1.19
ultracentrifugation
1.10
1.13
1.15
1.17
1.19
F1
F2
F3
F4
F5
MRP1
Prohibitin
190 -
kDa
40 -
F1 F2 F3 F4 F5
75INRA-Toulouse21/05/2010
13C6
-Lys13C6
-Arg12C6
-Lys12C6
-Arg
WT CIP-macrophages
DiscontinuousSucrose gradient
Enriched plasmamembrane fraction
Stable Isotope Labeling Aminoacid in Culture
Combine 1:1
SDS-PAGE/in-gel digestion
Protein ID & Quantification
-
200
-
150
-
120-
100
-
85
-
70-
60
-
40
-
50
-
30
A
B
C
F
DE
G
H
I
J
F2
AB
C
F
DE
F1
G
HIJ
k
76INRA-Toulouse21/05/2010
SILAC: global data
Identification of 900 proteins with
3 uniques peptides
Among
proteins
detected
in both
fractions
15
expression in CIP-macrophages
as compared
to WT
13
expression in CIP-macrophages
as compared
to WT
Among
proteins
detected
in one of the two
fractions
37/36
expression in CIP-macrophages
as compared
to WT
29/34
expression in CIP-macrophages
as compared
to WT
Marquez et al, FEBS 2009
77INRA-Toulouse21/05/2010
Proteins with modified expression in CIP-resistant cells
78INRA-Toulouse21/05/2010
Proteins with modified expression in CIP-resistant cells
Dnajc3
WT CIP-R25 µg 50 µg 25 µg 50 µg
►
WT CIP-R 25 µg 50 µg 25 µg 50 µg
kDa
►Tlr7- 130 -- 100 -
On the same chromosome as mrp4 !
Transports nucleosides/tides
(substrates for Mrp4 !)
79INRA-Toulouse21/05/2010
Gene amplification in CIP-resistant cellschromosome 14 mrp4 FISH analysis
WT cells
chromosome 14
mrp4
Cells resistant to 0.15 mM
Cells resistant to 0.1 mM
80INRA-Toulouse21/05/2010
Gene amplification in CIP-resistant cells
chromosome 5
chromosome 13 chromosome 16
Heterogeneity of cell population !
mFISH analysis of CIP-R cells
81INRA-Toulouse21/05/2010
Gene amplification in CIP-resistant cells
Mrp4 and Dnaj3 co-amplified in CIP-resistant cells
FISH analysis of CIP-R cells
Dnajc3
WT CIP-R25 µg 50 µg 25 µg 50 µg
► Mrp4
Dnajc3
82INRA-Toulouse21/05/2010
and in moxifloxacin-exposed cells ?
TaqMan Low Density Array of ABC transporters
Cell line gene ciprofloxacin-
resistant moxifloxacin-
resistant Abca1 -1.53a -3.51 Abca2 -1.13 -1.12 Abca3 1.10 -1.56 Abca4 nd nd Abca5 -1.85 2.31 Abca6 nd nd Abca7 -1.36 -1.20 Abca8a nd nd Abca8b (1.39) 76.20 Abca9 (-17.45) (-10.48) Abca13 1.66 2.02 Abca14 nd nd Abca15 nd nd Abcb1a* (-1.38) 76.35 (-1.89) 84.42 Abcb1b 1.03 1.72 Abcb2 1.10 4.30 Abcb3 -1.35 3.17 Abcb4 1.01 2.48 Abcb6 -1.12 -1.22 Abcb8 1.00 -1.25 Abcb9 6.08 9.83 Abcb10 1.40 1.43 Abcb11 (1.15) (1.19)
Cell line gene ciprofloxacin-
resistant moxifloxacin-
resistant Abcc1 1.08 -1.26 Abcc2* (1.44) (-1.26) (9.25) (5.09) Abcc3 -1.15 1.09 Abcc4 14.59 -1.82 Abcc5 -1.26 1.29 Abcc6 nd nd Abcc7 nd nd Abcc8 (1.22) (7.74) Abcc9 nd nd Abcc10 1.01 -1.40 Abcc12 nd nd Abcd1 1.35 2.22 Abcd2 1.10 1.79 Abcd3 -1.04 -1.51 Abcd4 -1.43 -2.03 Abce1 -1.04 -1.00 Abcf2 -1.01 1.21 Abcf3 -1.08 1.11 Abcg1 -1.93 -4.92 Abcg2* (1.12) 108.41 (1.17) 99.47 Abcg3 nd nd Abcg4 (1.62) (-1.07) Abcg5 nd nd Abcg8 nd nd
Vallet et al., FEBS-ABC meeting. (2010)
83INRA-Toulouse21/05/2010
and in moxifloxacin-exposed cells ?
CT + VER0
50
100
WT cells
MXF-R cells
a
A
b
B*
*
Rhodamine 123
200
300
400
Subs
trat
e ac
cum
ulat
ion
(% o
f con
trol
in w
ild-ty
pe c
ells
CT + FTC
a
a
bB
*
Bodipy prasozin
A
0
50
100
150
P-gp and Bcrp are functional
Vallet et al., FEBS-ABC meeting. (2010)
84INRA-Toulouse21/05/2010
and in moxifloxacin-exposed cells ?
P-gp and Bcrp are functionalbut do not play a major role in FQ accumulation
CT+ G
EM+ V
ER+ F
TC+ G
EM + VER
+ GEM +
FTC
0
250
500
750
1000
1250
1500 WT cellsMXF-R cells
a
b* **
* *
cd
e
f
AB B
B
C C
ciprofloxacin
cellu
lar c
once
ntra
tion
(ng/
mg
prot
)
CT+ G
EM+ V
ER+ F
TC
a
b
*
bb
A
AA
A
0
250
500
750
1000
1250
1500
moxifloxacin
Vallet et al., FEBS-ABC meeting. (2010)
85INRA-Toulouse21/05/2010
IncreasedIncreased efflux efflux …….. ConsequencesConsequences for for activityactivity
againstagainst intracellularintracellular bacteriabacteria andand
CooperationCooperation betweenbetween procaryoticprocaryotic and and eucaryoticeucaryotic transporterstransporters
Les aventures de Tintin, Hergé, Belgium
86INRA-Toulouse21/05/2010
Increased efflux in ciprofloxacin-resistant cells : consequence for antibiotic activity
Lismond et al., Antimicrob. Ag. Chemother. (2008) 52:3040-46
Ciprofloxacin
and Listeria
Wild-type cells
and bacteria
87INRA-Toulouse21/05/2010
Increased efflux in bacteria
MIC of Listeria strains and effect of reserpine
MIC (mg/L)
quinolone
EGD CLIP
Res. (-) Res. (+) Res. (-) Res. (+)
CIP 1.2 1.0 5.0 1.0
MXF 0.6 0.6 0.5 0.25
Lismond et al., Antimicrob. Ag. Chemother. (2008) 52:3040-46
88INRA-Toulouse21/05/2010
Increased efflux in ciprofloxacin-resistant cells : consequence for antibiotic activity
Ciprofloxacin
and Listeria
cells
and overproducing efflux pumps
for ciprofloxacin
Lismond et al., Antimicrob. Ag. Chemother. (2008) 52:3040-46
89INRA-Toulouse21/05/2010
Increased efflux in ciprofloxacin-resistant cells : consequence for antibiotic activity
Ciprofloxacin
and Listeria
bacteria
overproducing efflux pumps
for ciprofloxacin
bacteria
and cells
overproducing efflux pumps
for ciprofloxacin
Lismond et al., Antimicrob. Ag. Chemother. (2008) 52:3040-46
90INRA-Toulouse21/05/2010
Increased efflux in ciprofloxacin-resistant cells : consequence for antibiotic activity
Moxifloxacin
and Listeria
Wild-type cells
and bacteriacells
and bacteria
overproducing
efflux pumps
for ciprofloxacin
Lismond et al., Antimicrob. Ag. Chemother. (2008) 52:3040-46
91INRA-Toulouse21/05/2010
ConclusionsConclusions
Toulouse: fontaines
92INRA-Toulouse21/05/2010
Food for thought …
• in procaryotic
cells, active efflux truly
contributes
to resistance
need
of appropriate
diagnostic tools selection
of antibiotics
that
are poor
substrates
• in eucaryotic
cells,
active efflux can
modify• pharmacokinetics
(cellular accumulation; barrier
effects)
• pharmacodynamics
(intracellular
activity)
selection
of antibiotics
that
are poor
substrates combinations
with
efflux pumps
inhibitors
?
• expression of efflux pumps
can
be
modified
by exposure
to drugs
reduced
susceptibility
during
treatment cell
metabolism
modifications
same ?
93INRA-Toulouse21/05/2010
CoworkersCoworkers ……
•
B. Devreese and M. Aerts Proteomics Laboratory for Protein Biochemistry and Biomolecular
Engineering, Ghent University, Belgium
•
E. Jacquet and N. Nhiri TaqMan Low Density Array Institut de Chimie des Substances Naturelles, CNRS, Gif
sur Yvette, France
•
P. Courvalin Resistant strains Unité
des Agents antibactériens,
Institut Pasteur, Paris, France
•
L. Piddock and M. Garvey PatA/PatB Antimicrobial Agents Research Group, University
of Birmingham, UK
94INRA-Toulouse21/05/2010
Efflux in Efflux in ourour team team ……
www.facm.ucl.ac.be
HOOC
NH2
95INRA-Toulouse21/05/2010
Let’s dream to the next pump we will discover …
Toulouse: première pompe à gaz naturel pour véhicules