Resistance to antibiotics: the rise of efflux…
Paul M. Tulkens, MD, PhD
Cellular and Molecular Pharmacology &
Center for Clinical Pharmacy
Louvain Drug Research Institute,
Université catholique de Louvain
Bruxelles
22 May 2015
22/5/2015
Resistance to antibiotics: the rise of efflux…
1
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered ?
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (examples: AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Inhibitors of efflux ?
Resistance to antibiotics: the rise of efflux…
2
A very short (pictorial) (selective) survey of
antibacterial chemotherapy
Cell wall
Protein synthesis
(-lactams, glycopeptides)
tetracyclines, macrolides, aminoglycosides, …)
Replication
& transcription
(fluoroquinolones, ansamycines)
22/5/2015
Membrane
Enzymes
(polymyxines, lipopeptides, …)
(sulfamides, diaminopyridines)
Resistance to antibiotics: the rise of efflux…
3
You said "antibiotic eflux"
1750
No. of publications / 2 years
1500
1250
1000
750
No. of publications in PubMed with
keywords:
"antibiotic AND (efflux OR transporter)"
500
250
0
1960
1970
1980
1990
2000
2010
Year
22/5/2015
Resistance to antibiotics: the rise of efflux…
4
Historical landmarks …
daptomycin *
linezolid
1750
aminoglycosides
1500
No. of publications / 2 years
* in eucaryotic cells
only (so far)
rifampin
-lactams
1250
macrolides
Successive
description of effluxmediated resistance
for major classes of
antibiotics
1000
fluoroquinolones
750
tetracyclines
500
250
0
1960
1970
1980
1990
2000
2010
Year
22/5/2015
Resistance to antibiotics: the rise of efflux…
5
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Inhibitors of efflux ?
Resistance to antibiotics: the rise of efflux…
6
An original observation with cancer cells…
22/5/2015
Resistance to antibiotics: the rise of efflux…
7
Most chemotherapeutic agents must reach an
intracellular target…
Conclusion #1: in order to survive to anticancer agents, cells "invented" efflux…
Inaba and Johnson, Cancer Res, 1977; 37:4629-34.
22/5/2015
Resistance to antibiotics: the rise of efflux…
8
But antibiotics were first ...
Who remembers that car ?
22/5/2015
Resistance to antibiotics: the rise of efflux…
9
Historical observations on tetracyclines …
Who remembers that graph ?
22/5/2015
Resistance to antibiotics: the rise of efflux…
10
Historical observations on tetracyclines …
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Resistance to antibiotics: the rise of efflux…
11
Historical observations on tetracyclines …
Whole bacteria
Everted membranes
McMurry et al., PNAS 1980; 77:3974-3977
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Resistance to antibiotics: the rise of efflux…
12
Historical observations
…
Parkinson et al. Antimicrob Agents Chemother. 1995 Aug;39(8):1696-9
antibiotics
1965
antifungal drugs
1980
1980
1995
1977
anticancer drugs
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Resistance to antibiotics: the rise of efflux…
13
Historical trends …
3000
bacteria
cancer
fungi
No. of publications in 10 years
2500
2000
1500
No. of publications
in PubMed with
keywords: efflux AND
• bacteria
• cancerl
• fungi
1000
500
0
1969
1979
1989
1999
2009
Year (10 years interval: 0-9)
22/5/2015
Resistance to antibiotics: the rise of efflux…
14
Most chemotherapeutic agents must reach
an intracellular target…
enzymes
nucleic acids
ribosomes
How can these drugs
reach their target inside the cells ?
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Resistance to antibiotics: the rise of efflux…
15
Reaching an intracellular target …
polar drug
lipophilic drug
physico-chemical properties are inadequate
for reaching an intracellular target !
Van Bambeke et al., Biochem. Pharmacol (2000) 60:457-70
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Resistance to antibiotics: the rise of efflux…
16
Reaching an intracellular target …
amphipathic drug
most drugs are amphipathic by design,
to be able to cross membrane barriers !
Van Bambeke et al., Biochem. Pharmacol (2000) 60:457-70
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Resistance to antibiotics: the rise of efflux…
17
Intracellular chemotherapeutic agents
But a diffusible compound
may have
potentially harmful effects !
Van Bambeke et al., Biochem. Pharmacol (2000) 60:457-70
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Resistance to antibiotics: the rise of efflux…
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Why efflux transporters ?
Extrusion by efflux pumps
Van Bambeke et al., Biochem. Pharmacol (2000) 60:457-70
22/5/2015
Resistance to antibiotics: the rise of efflux…
19
Why efflux transporters ?
Extrusion by efflux pumps
general mean of protection
against cell invasion by diffusible molecules
Van Bambeke et al., Biochem. Pharmacol (2000) 60:457-70
22/5/2015
Resistance to antibiotics: the rise of efflux…
20
Typical ‘toxic’ diffusible substances as
substrates for efflux pumps
antifungals
antibiotics
anticancer agents
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Resistance to antibiotics: the rise of efflux…
21
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Inhibitors of efflux
Resistance to antibiotics: the rise of efflux…
22
Most antibiotics are amphiphilic !
Neutral
apolar
polar
chloramphenicol
Van Bambeke et al. Biochem. Pharmacol. (2000) 60: 457-470
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Resistance to antibiotics: the rise of efflux…
23
Most antibiotics are amphiphilic !
anionic
fluoroquinolones
beta-lactams
fusidic acid
Van Bambeke et al. Biochem. Pharmacol. (2000) 60: 457-470
22/5/2015
Resistance to antibiotics: the rise of efflux…
24
Most antibiotics are amphiphilic !
cationic
lincosamides
tetracyclines
macrolides
rifampicin
fluoroquinolones
22/5/2015
sulfamides
Resistance to antibiotics: the rise of efflux…
25
Antibiotic classes recognized by efflux pumps
in different types of organisms
Antibiotic
class
bacteria
Gram (+)
Gram(-)
fungi
superior
eucaryotes
-lactams
fusidic acid
macrolides
streptogramins
tetracyclines
aminoglycosides
chloramphenicol
rifamycins
sulfamides
trimethoprim
fluoroquinolones
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Resistance to antibiotics: the rise of efflux…
26
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Inhibitors of efflux ?
Resistance to antibiotics: the rise of efflux…
27
Structure of pumps in procaryotic cells
Van Bambeke et al. JAC (2003) 51: 1055-1065
AB
AB
pore
porin
H+
AB
Na+
AB
AB
H+
lipoprotein
H+
RND
MFS
SMR
22/5/2015
Na+
MATE
ATP
ADP
pump
H+
RND, MFS, ABC
ABC
Gram-positive
periplasm
Gram-negative
Resistance to antibiotics: the rise of efflux…
28
Some abbreviations
•
•
•
•
•
22/5/2015
ABC:
MATE:
MFS:
RND:
SMR:
ATP Binding Cassette
Multi AnTimicrobial Extrusion
Major Facilitator Superfamily
Resistance Nodulation Division
Small Multidrug Resistance
Resistance to antibiotics: the rise of efflux…
29
Antibiotic efflux transporters are ubiquitous
distribution
spectrum
narrow
prokaryotes
Gram (+)
eukaryotes
Gram (-)
MATE
SMR
MFS
ABC
RND
broad
22/5/2015
Mesaros et al., Louvain médical (2005) 124:308-20
Resistance to antibiotics: the rise of efflux…
30
Efflux and resistance in pathogenic bacteria
1 bacteria
several pumps
multiresistance
1 pump
several classes
of antibiotics
crossresistance
1 class
of antibiotics
several pumps
efficacy of
inhibitors ?
22/5/2015
Resistance to antibiotics: the rise of efflux…
31
A brief survey of the many transporters (2003)
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Resistance to antibiotics: the rise of efflux…
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A brief survey of the many transporters (2003)
1. Gram +
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Resistance to antibiotics: the rise of efflux…
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A brief survey of the many transporters (2003)
2. Gram - (part #1)
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Resistance to antibiotics: the rise of efflux…
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A brief survey of the many transporters (2003)
2. Gram - (part #2)
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Resistance to antibiotics: the rise of efflux…
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A brief survey of the many transporters (2009)
809
references
22/5/2015
Resistance to antibiotics: the rise of efflux…
36
A brief survey of the many transporters: S. aureus
14 distinct
transporters for S.
aureus (only) in 2009
vs. 4 in 2003
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Resistance to antibiotics: the rise of efflux…
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What do you wish to know ?
•
Specific information about antibiotic transporters in procaryotes
http://ardb.cbcb.umd.edu/browse/multidrug.shtml
22/5/2015
Resistance to antibiotics: the rise of efflux…
38
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Inhibitors of efflux ?
Resistance to antibiotics: the rise of efflux…
39
Mechanisms of transport
Murakami S., Current Opinion in Structural Biology 2008, 18:459–465
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Resistance to antibiotics: the rise of efflux…
40
General structure of an RND (AcrAB-TolC)
Murakami S., Current Opinion in Structural Biology 2008, 18:459–465
Nikaido & Takatsuka, Biochimica et Biophysica Acta 1794 (2009) 769–781
22/5/2015
Resistance to antibiotics: the rise of efflux…
41
AcrB in more
details
Nikaido & Takatsuka, Biochimica et Biophysica Acta 1794 (2009) 769–781
22/5/2015
Resistance to antibiotics: the rise of efflux…
42
Pos K. Biochimica et Biophysica Acta 1794 (2009) 782–793
Seeger et al. Curr. Drug Targets 9 (2008) 729–749.
Proposed AcrB drug / H+ exchange
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Resistance to antibiotics: the rise of efflux…
43
AcrB-TolC
is a
multidrug
transporter
Pos K. Biochimica et Biophysica Acta 1794 (2009) 782–793 / Seeger et al. Curr. Drug Targets 9 (2008) 729–749.
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Resistance to antibiotics: the rise of efflux…
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How can AcrB be a multi-drug ?
Nature. 2011 Nov 27;480(7378):565-9.
• Our structures indicate that there are two discrete multisite binding pockets
along the intramolecular channel.
• High-molecular-mass drugs (rifampicin1, erythromycin2) first bind to the
proximal pocket in the access state and are then forced into the distal pocket
in the binding state by a peristaltic mechanism involving subdomain
movements that include a shift of the Phe-617 loop.
• By contrast, low-molecular-mass drugs, such as minocycline3 and
doxorubicin4, travel through the proximal pocket without specific binding and
immediately bind to the distal pocket.
• The presence of two discrete, high-volume multisite binding pockets
contributes to the remarkably broad substrate recognition of AcrB.
1
22/5/2015
822; 2 733; 3 457; 4 543
Resistance to antibiotics: the rise of efflux…
45
Interplay of RND and porins
22/5/2015
Resistance to antibiotics: the rise of efflux…
46
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Inhibitors of efflux ?
Resistance to antibiotics: the rise of efflux…
47
Efflux as a significant mechanism of resistance
in Gram-positive bacteria
spectrum
narrow
specific for one (or a few) families of drugs
ABC
PatA/PatB of S. pneumoniae
 FQ, chl
MsrA of S. epidermidis
 erythromycin
broad
22/5/2015
MFS
NorA of S. aureus
 FQ,Tet, chl
MefE of S. pneumoniae
 ML
PmrA of S. pneumoniae
 FQ
MefA of S. pyogenes
 ML
Resistance to antibiotics: the rise of efflux…
48
FQ efflux pumps in S. pneumoniae – S. aureus
Primary transporters
« ATP-Binding Cassette »
PatA/PatB (Sp)
Marrer et al, AAC 2006; 50:685-93
Secondary transporters
(Proton motive force)
PmrA (Sp)
Gill et al, AAC 1999; 43:187-9
NorA (Sa)
Gill et al, AAC 1999; 43:187-9
Terry et al., Nature Reviews Microbiology 2005; 3: 566-572
22/5/2015
Resistance to antibiotics: the rise of efflux…
49
Efflux as a a significant mechanism of resistance
in Gram-negative bacteria
spectrum
narrow
specific for one (or a few) families of drugs
MFS
TetA of E. coli
Tet
broad spectrum, conferring cross-resistance
RND
MexAB-OprM of P. aeruginosa
 -lac, FQ,Tet, ML, chl, rif, sulf
AcrAB-TolC of E. coli
 -lac, FQ,Tet, ML, chl, rif, sulf
broad
22/5/2015
Resistance to antibiotics: the rise of efflux…
50
Efflux and resistance in P. aeruginosa
Constitutive
basal expression
overexpressed
upon induction
No basal
expression;
expression
upon induction
MexB
MexY
MexD
MexF
MexA
MexX
MexC
MexE
OprM
OprM
OprJ
OprN
Mesaros et al. (2005) Louvain médical. 124:308-20
22/5/2015
Resistance to antibiotics: the rise of efflux…
51
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Inhibitors of efflux
Resistance to antibiotics: the rise of efflux…
52
Early data with -lactams
logD pH
7.4 = 2.25
(REAXYS)
logD pH
7.4 = 0.25
(REAXYS)
Mazzariol et al. Antimicrob Agents
Chemother. 2000 May;44(5):1387-90.
22/5/2015
Resistance to antibiotics: the rise of efflux…
53
Substrate specificity of efflux pumps
14 fluoroquinolones; Gram + versus Gram +
S. aureus vs S. pneumoniae
S. aureus vs S. pneumoniae
5
MIC change [PatB+/-] (fold dilutions)
MIC change [PatA+/-] (fold dilutions)
5
R² = 0.8290
CIP
NOR
4
SAR
3
LVX
2
1
PEF
GAR
SPX
ENX
LOM
GMF
FIN
MAR
0
DIF MXF
0
1
2
3
4
5
6
MIC change [NorA+/-] (fold dilutions)
7
R² = 0.7943
CIP
NOR
4
SAR GMF
3
LVX
FIN
2
1
ENX
LOM
PEF
MAR
DIF GAR
SPX
0
MXF
0
1
2
3
4
5
6
7
MIC change [NorA+/-] (fold dilutions)
Similar recognition for non phylogenitically-related transporters
Vallet et al. ECCMID 2011
22/5/2015
Resistance to antibiotics: the rise of efflux…
54
Substrate specificity of efflux pumps
14 fluoroquinolones; Gram + versus Gram P. aeruginosa vs S. aureus
P. aeruginosa vs S. pneumoniae
5
R² = 0.6807
MIC change [PatA+/-] (fold dilutions)
MIC change [NorA+/-] (fold dilutions)
7
NOR
CIP
6
5
4
GMF
3
SAR
ENX
LOM
LVX
MAR
2
FIN
PEF
MXF
GAR
1
0
SPX
DIF
0
1
2
3
4
5
6
7
MIC change [Mex+/-] (fold dilutions)
8
R² = 0.6702
NOR
CIP
4
3
SAR
2
GMF
1
ENX
LOM
LVX
FIN
MAR
PEF
GAR
SPX
DIF
MXF
0
0
1
2
3
4
5
6
7
8
MIC change [Mex+/-] (fold dilutions)
All fluoroquinolones are substrates for broad spectrum transporters from Gram Vallet et al. (2011) ECCMID
22/5/2015
Resistance to antibiotics: the rise of efflux…
55
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Inhibitors of efflux ?
Resistance to antibiotics: the rise of efflux…
56
Pseudomonas and penem efflux
MICs
Mex pumps
AB CD XY
-
MERO
IMI
BIA
PANI FARO RITI
-
-
0.032
0.25
0.25
0.25
+* -
-
0.25
1
0.5
++ -
-
1
0.25
0.25
-
++
-
0.25
0.125 0.063 0.25
-
-
++
0.063
0.25
0.25
1
2
4
512
128
1
4096
256
16
4
4
8
2
* clinical isolate, basal level of expression
Okamoto et al. J. Infect. Chemother (2002) 8: 371-373
Okamoto et al. AAC (2002) 46:2696-2699
22/5/2015
Resistance to antibiotics: the rise of efflux…
57
Pseudomonas and penem efflux
MICs
Mex pumps
AB CD XY
-
MERO
IMI
BIA
PANI FARO RITI
-
-
0.032
0.25
0.25
0.25
+* -
-
0.25
1
0.5
++ -
-
1
0.25
0.25
-
++
-
0.25
0.125 0.063 0.25
-
-
++
0.063
0.25
0.25
1
2
4
512
128
1
4096
256
16
4
4
8
2
* clinical isolate, basal level of expression
Okamoto et al. J. Infect. Chemother (2002) 8: 371-373
Okamoto et al. AAC (2002) 46:2696-2699
22/5/2015
Resistance to antibiotics: the rise of efflux…
58
S. pneumoniae and fluoroquinolones
MIC distribution for 184 isolates from community-acquired pneumonia
• Efflux (+) strains
considered
as susceptible
• FQ with high intrinsic
activity can be
substrates for efflux !
22/5/2015
Resistance to antibiotics: the rise of efflux…
Lismond et al., JAC (2011) 66:948-951
60
P. aeruginosa and temocillin
Pseudomonas aeruginosa and temocillin
MexAB-OprM mutants are highly susceptible !
 Efflux responsible for intrinsic resistance
Buyck et al, J. Antimicrob. Chemother. (2012) 67(3):771-5
22/5/2015
Resistance to antibiotics: the rise of efflux…
61
Intrinsic resistance of Pseudomonas to
temocillin
But temocillin is used successfully in Cystic Fibrosis patients …
Natural mutations in MexAB-OprM restore temocillin activity
Buyck et al, J. Antimicrob. Chemother. (2012) 67(3):771-5
22/5/2015
Resistance to antibiotics: the rise of efflux…
62
Intrinsic resistance of Pseudomonas to temocillin
Is this clinically relevant ?
1/4 of CF strains susceptible!
22/5/2015
Resistance to antibiotics: the rise of efflux…
Chalhoub, unpublished
63
Conditions modulating efflux and susceptibility
Azithromycin is widely and successfully used in Cystic Fibrosis patients
BUT Pseudomonas is supposedly intrinsically resistant ….
22/5/2015
Resistance to antibiotics: the rise of efflux…
64
Intrinsic resistance of Pseudomonas to macrolides
Is Pseudomonas « intrinsically » resistant to macrolides ?
MHB-OprM(-)
MHB-OprM(+)
cumulative percentage
100
75
Major role
of constitutively-expressed
transporters!
50
25
32
64
12
8
25
6
51
10 2
24
8
16
4
2
1
0.
25
0.
5
0
MIC (mg/L)
Buyck et al. Clin Infect Dis. 2012; 55:534-42
22/5/2015
Resistance to antibiotics: the rise of efflux…
65
An intriguing observation …
MIC (mg/L)
CA-MHB
Antibiotic
RPMI-1640
pH 7.4
pH 5.5
Gentamicin
2
8
4
Amikacin
4
64
4
Tobramycin
1
8
1
Piperacillin/Tazobactam
16
16
16
Cefepime
4
8
4
Ceftazidime
2
4
2
Aztreonam
8
16
8
Meropenem
1
1
2
0.125
0.25
0.125
1
2
2
Aminoglycosides
-lactams
Fluoroquinolones
Ciprofloxacin
Macrolides regain
activity against
P. aeruginosa in
« eukaryotic » media
Polymyxins
Colistin
Azithromycin
128
>512
16
Buyck et al. Clin Infect Dis. 2012; 55:534-42
22/5/2015
Resistance to antibiotics: the rise of efflux…
66
Why do macrolides express their activity
against P. aeruginosa in « eukaryotic » media ?
ML
ML
OprM
X
1
5
ML
X
prot
prot
ML
2
3
ML
Buyck et al.
Clin Infect Dis. 2012; 55:534-42
22/5/2015
X
MexAB-OprM
MexXY-OprM
oprM
MHB
RPMI
high MIC
low MIC
Resistance to antibiotics: the rise of efflux…
4
67
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Cooperation between procaryotic and eucaryotic transporters
Resistance to antibiotics: the rise of efflux…
68
Efflux of fluroquinolones in S. pneumoniae:
is transporter more expressed in patients chronically treated
Suspected efflux based on phenotypic analysis (CIP MIC +/- reserpine)
reserpine effect on MIC (x dilutions)
1
2
2
100
% strains
80
acute pathology

« one shot »
antibiotic exposure
60
40
chronic pathology

repetitive
antibiotic exposures
20
0
CAP
BPCO
origin of isolates
183 strains
22/5/2015
107 strains
Resistance to antibiotics: the rise of efflux…
Lismond & Degives, unpublished
69
Impact of efflux on clinical susceptibility
of P. aeruginosa
MICs vs EUCAST breakpoints for 109 P. aeruginosa without or with efflux
mechanisms, isolated from ICU patients (VAP)
amikacin
256
128
64
32
MIC
16
8
4
2
1
0.5
0.25
mexX < 5
mexX  5
mexX expression level
pip-tazo
meropenem
256
128
64
32
16
8
4
2
1
0.5
0.25
0.125
0.0625
0.03125
mexA < 2
mexA  2
256
128
64
32
16
8
4
2
1
0.5
0.25
0.125
0.0625
0.03125
mexA expression level
mexA < 2
mexA  2
mexA expression level
Riou et al, ECCMID 2010
22/5/2015
Resistance to antibiotics: the rise of efflux…
70
Increase of P. aeruginosa during treatment:
is efflux involved ?
Prevalence of MexA and MexX overexpressers in 62 phylogentically-related
pairs of P. aeruginosa isolated from ICU patients (VAP)
DAY x (%)
DAY 0 (%)
17.74%
9.68%
MexA-/MexX-
11.29%
38.71%
MexA+/MexXMexX+/MexA12.90%
20.97%
MexA+/MexX+
66.13%
22.58%
Riou et al, ECCMID 2010
Riou et al. submitted for publication
22/5/2015
Resistance to antibiotics: the rise of efflux…
71
Early diagnosis could be implemented in the clinics
22/5/2015
Resistance to antibiotics: the rise of efflux…
72
Early diagnosis could be implemented in the clinics
22/5/2015
Resistance to antibiotics: the rise of efflux…
73
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Cooperation between procaryotic and eucaryotic transporters
Resistance to antibiotics: the rise of efflux…
74
Efflux cooperates
with other mechanisms of bacterial resistance
R
physical
clearance
C
O
COOH
R
R
-lactamase
C
N
C HN
O OH
O
COOH
22/5/2015
chemical
clearance
N
Resistance to antibiotics: the rise of efflux…
COOH
75
Efflux cooperates
with other mechanisms of resistance
Contributions of the AmpC -lactamase and the AcrAB Multidrug
Efflux System in Intrinsic Resistance of E. coli to  -lactams
Efflux
-lactamase
-
-
CMI carbenicillin
0.2
CMI ofloxacin
0.05
+
-
12.5
0.2
+++
+
+++
+
+
+
50
100
200
400
1.56
0.05
0.39
1.56
WT:
intrinsic
resistance !
Mazzariol et al, AAC (2000) 44:1387-1390
22/5/2015
Resistance to antibiotics: the rise of efflux…
76
Efflux and selection of resistance to FQ
Exposure to subMIC
concentrations favors
the selection of
target mutations !
Gyrase/ Topoisomerase
22/5/2015
Resistance to antibiotics: the rise of efflux…
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Efflux and selection of resistance
Frequency of Levofloxacin-resistant mutants in
Pseudomonas aeruginosa with deletions of the efflux pump operons
Pump status
WT
 mexAB-oprM
 mexCD-oprJ
 mexEF-oprN
 mexAB-oprM; mexEF-oprN
 mexCD-oprJ;  mexEF-oprN
 mexAB-oprM;  mexCD-oprJ
 mexAB-oprM;  mexCD-oprJ;
 mexEF-oprN
Lomovskaya et al,
AAC (1999) 43:1340-1346
22/5/2015
LVX MIC
Frequency of LVXresistant mutants
0.25
0.015
0.25
0.25
0.015
0.25
0.015
0.015
2 × 107 - 4 × 107
2 × 107 - 4 × 107
2 × 107 - 4 × 107
2 × 107 - 4 × 107
2 × 107 - 107
2 × 106
1 × 109
<1 × 1011
Selection of mutants in FQ target
undetectable if ALL pumps are disrupted
Resistance to antibiotics: the rise of efflux…
78
What is in the menu ?
•
•
•
•
•
•
•
•
•
•
•
22/5/2015
Brief overview of antibiotics and resistance
Efflux: why does it exist and how has it been discovered
Why antibiotics ?
Main antibiotic efflux transporters
Structure and mechanisms (an example with AcrAB-TolC)
Antibiotic transporters important for the clinical microbiologist
Substrate specificities
Efflux and intrinsic susceptibility
Efflux and clinical susceptibility and impact of treatment
Cooperation with other mechanisms of resistance
Inhibitors of efflux ?
Resistance to antibiotics: the rise of efflux…
79
And now, can we make inhibitors of efflux ?
• There are a LARGE number of inhibitors
• Many are endowed with other pharmacological activities
that appear already at lower concentrations that what is
needed to impair efflux (e.g., reserpine)
• Others are very effective but also very toxic (e.g.
Phenylalanine-arginine--naphthylamide [PAN; MCMC-207110]).
• The search for microbiologically-active and safe-to-host
inhibitors is ongoing but with little “drug” success so far…
22/5/2015
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