Role of the Efflux Pumps in
Antimicrobial Resistance in E. coli
Patrick Plésiat
Bacteriology Department
Teaching Hospital
Besançon, France
ANTIBIOTIC
TARGET
Bacterial targets for antibiotics
Chromosome
Cell wall
Ribosomes
Cytoplasmic membrane
Main resistance mechanisms to drugs
Inactivation
Modification
ANTIBIOTIC
Efflux
Impermeability
Protection
TARGET
Substitution
Amplification
Reduced affinity
- mutations
- recombinaisons
- enzymatic modification
Gram-negative species with known efflux systems
Escherichia coli
Salmonella Typhimurium
Shigella dysenteriae
Klebsiella pneumoniae
Enterobacter aerogenes
Serratia marcescens
Proteus vulgaris
Citrobacter freundii...


Bacteroides fragilis...
Pseudomonas aeruginosa
Pseudomonas putida
Burkholderia cepacia
Burkholderia pseudomallei
Stenotrophomonas maltophilia
Alcaligenes eutrophus...

Haemophilus influenzae
Campylobacter jejuni
Helicobacter pylori
Vibrio parahaemolyticus
Vibrio cholerae
Neisseria gonorrhoeae...

Efflux mechanisms: practical implications

Do efflux systems produce clinically relevant levels of
resistance ?

Does the expression of drug transporters somewhat impair the
virulence of bacterial pathogens ?

What is the prevalence of efflux systems relative to other
resistance mechanisms among the clinical isolates ?

How to recognize efflux mutants in laboratory practice ?

What recommendations can be made to the physician for the
treatment of patients infected with mdr strains ?
Intracellular accumulation
Drug accumulation experiments
S
ATP
glucose
R
CCCP
Time
Structure of bacterial efflux systems

One component systems
– Mostly in Gram positive species (except Tet...)
– A single transporter protein in the cytoplasmic membrane
– Determines the substrate specificity and resistance

Three component (tripartite) systems
– Exclusively in Gram negative species (GNB)

A transporter protein

A periplasmic adaptor lipoprotein

A outer membrane channel protein
Energy sources

Antiporters
– PMF transporters (proton motive force)
– Na+-antibiotic antiporters

ABC transporters
– ATP binding cassette pumps
– Hydrolysis of ATP into ADP + Pi
– Mostly in Gram positive species
PMF transporters

Major Facilitator Superfamily (MFS)
– Drug efflux


12 TMS transporters
14 TMS transporters
– Active uptake/export



Small Multidrug Resistance Family (SMR)


4 TMS transporters
Resistance/Nodulation Cell Division Family (RND)


sugars...
amino acids, secondary metabolites...
12 TMS transporters
Multi Antimicrobial Extrusion Family (MATE)

12 TMS transporters
Structure of drug efflux systems
antibiotic
antibiotic
H+
Na+
H+
ATP
MFS, SMR
MATE
ADP
ABC
RND, MFS, ABC
Fernandez-Recio J. et al. FEBS 2004, 578: 5-9
Murakami S. et al. Nature 2002, 419: 587
Murakami S. et al. Curr Opinion Struct. Biol. 2003, 13: 443
Murakami S. et al. Curr Opinion Struct. Biol. 2003, 13: 443
Efflux systems in E. coli

Chromosomally encoded pumps
– 37 putative drug transporters: 19 MFS, 3 SMR, 7 RND, 7 ABC,
1 MATE
– 20 pumps are able to transport toxic/antibiotic molecules
– 15-17 pumps may provide with some resistance to antibiotics when
overproduced from cloned genes (Nishino K et al. J. Bacteriol. 2001)
– Upregulation of a single pump may result in increased drug efflux

Acquisition of exogenous pump encoding genes
– Genes carried by mobile elements (plasmids, transposons,
integrons)
Efflux pumps coded by mobile genetic elements
Species
System
E. coli
E. coli
E. coli
E. coli
TetA/B/E
CmlA
Flo
OqxAB-TolC
Family
MFS
MFS
MFS
RND
Substrates
Tc, Min
Cmp
Cmp, Flo
Olaquindox, Cmp
Tc: tetracycline; Min: minocycline; Cmp: chloramphenicol; Flo: florfenicol
Efflux pumps of MFS, MATE, SMR, or ABC family
Species
System
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
EmrAB-TolC
Bcr
MdfA
MdtG
MdtH
MdtL
MdtM
NorE
EmrE
MdtJK
MacAB-TolC
Family
MFS
MFS
MFS
MFS
MFS
MFS
MFS
MATE
SMR
SMR
ABC
Substrates
Genes
Nal
Tc, Km, Fos
Tc, Rif, Cmp, Ery, Neo, Fq...
Fos
Fq
Cmp
Cmp, Fq
Cmp, Fq, Fos, Tmp
Tc
Nal, Fos
Ery
C
C
C
C
C
C
C
C
C
C
C
Nal: nalidixic acid; Tc: tetracycline + glycylcyclines; Km: kanamycin; Fos: fosfomycin; Rif: rifampicin; Cmp: chloramphenicol;
Ery: erythromycin; Neo: neomycin; Fq: fluoroquinolones; Tmp: trimethoprim
Efflux pumps of the RND family
Bacteria
System
Substrates
E. coli
AcrAB-TolC1
Fq, ß-lactams3, Tc, Cmp, Nov, Ery, Fus, Rif…
E. coli
AcrEF-TolC2
Fq, ß-lactams3, Tc, Cmp, Nov, Ery, Fus, Rif…
E. coli
AcrD2-AcrA-TolC
AGs, Ery, PolyB
E. coli
CusAB-?2
Fos
E. coli
MdtABC-TolC2
Fq
E. coli
MdtEF-TolC2
Ery
P. aeruginosa
MexAB-OprM1
Fq, ß-lactams1, Tc, Cmp, Nov, Ery, Fus, Tm...
P. aeruginosa
MexCD-OprJ2
Fq, 3rd GC, Tc, Cmp, Ery, Tmp
P. aeruginosa
MexEF-OprN2
Fq, Cmp, Tmp
P. aeruginosa
MexXY2-OprM
Fq, AGs, 3rdGC, Ery, Tc
N. gonorrhoeae
MtrCDE1
Tc, Cmp, ß-lactams1, Ery, Fus, Rif...
Fq: (fluoro)quinolones; Tc: tetracycline; Cmp: chloramphenicol; Nov: novobiocin; Ery: erythromycin; Fus: fusidic acid; Rif:
rifampicin; AGs: aminoglycosides; PolyB: polymyxin B; Tmp: trimethoprim; Sulf: sulfamethoxazole; 3rdGC: cefepime, cefpirome.
expressed constitutively in wild type cells, 2 inducible expression, 3 except imipenem.
1
Induction of acrAB-tolC expression
tetracycline
chloramphenicol
salicylate-acetylsalicylate
benzoate
stress...
MarROAB
SoxSR
Rob
 Porin OmpF
 TolC
 AcrAB
EmrAB
Other proteins
Mar regulon
oxidative stress
bile salts
tetracycliner
chloramphenicolr
quinolonesr
erythromycinr
solvants, pine oil...
Overexpression of acrAB and mtrCDE operons
_
MarA
(MppA)
MarR
_
+
-
SoxS
acrA
SoxR
acrB
acrR
MtrA
+
-
mtrC
mtrD
mtrE
mtrR
mutations mdr
*
*
*
*
*
*
*
*
*
*
*
Webber M. et al. Antimicrob. Agents Chemother. 2001, 45: 1550
Systems MtrCDE and FarAB in N.
gonorrhoeae
wild type
CDE++
CDE-
FarAB-
Penicillin G
0.008
0.032
0.008
nd
Erythromycin
0.25
1-2
0.06
0.25
Tetracycline
0.25
0.5
nd
nd
Rifampicin
0.06
0.25
0.015
nd
Linoleic acid
1600
nd
25 - 50
50
Palmitic acid
100
nd
12.5
12.5
Antibiotics
System AcrAB-TolC in E. coli
Antibiotics
wild type
AcrAB++
AcrAB-
Nalidixic acid
4-6
8.5 - 32
0.6
Norfloxacin
0.025 - 0.1
0.3 - 1.25
nd
Ofloxacin
0.06 - 0.07
0.25 - 0.3
nd
Ciprofloxacin
0.02
0.15
nd
Ampicillin
2-4
5-6
0.6 - 2
128 - 256
> 512
<2-8
Tetracycline
1.25 - 3
5 - 16
0.25 - 0.3
Chloramphenicol
4 - 7.5
10 - 28
0.6
Erythromycin
System MexAB-OprM in P. aeruginosa
Antibiotics
wild type
MexAB++
MexAB-
Norfloxacin
0.25 - 1
2-4
0.05 - 0.25
Ofloxacin
0.4 - 1
1.6 - 8
0.025 - 0.05
Ciprofloxacin
0.03 - 0.25
0.4 - 1.6
0.012 - 0.03
Carbenicillin
12.5 - 64
50 - 256
0.4 - 1
Aztreonam
1.6 - 4
12.5 - 32
0.1 - 0.2
Ceftazidime
0.4 - 2
1.6 - 8
0.2 - 0.4
Cefepime
0.8 - 2
3-4
0.1 - 0.5
Meropenem
0.2 - 0.5
0.8 - 2
0.1 - 0.2
Tetracycline
6.25 - 16
25 - 64
0.2 - 1.2
Chloramphenicol
12.5 - 32
100 - 512
0.8 - 2
Interplays between resistance mechanisms in GNB
Outer membrane
permeability
Other mechanisms
Active efflux
Efflux/target double mutants of E. coli
Genotype/Phenotype
Oflo
Cipro
wild type AG100
0.03
≤0.015
AcrAB++
0.125
0.06
gyrA (Asp87->Gly)
0.25
0.25
gyrA (Asp87->Gly; Ser83->Leu)
4
2
gyrA (Asp87->Gly), AcrAB++
8
4
0.06
0.03
gyrA (Asp87->Gly), AcrAB-
Oethinger et al. Antimicrob. Agents Chemother. 2000, 44: 10-13
Therapeutic implications of efflux systems

Resistance levels conferred by intrinsic pumps
– Low to moderate drug resistance (MIC x 2 - 16)
– Clinical significance




Lack of clinical data !
Poor response to treatment when the concentrations of
antibiotics are low at the infection site (insufficient dosage,
inappropriate drug, abcess...)
Increased emergence of target mutants ?
Emergence of efflux mutants under treatment
– Cross resistance to structurally unrelated molecules
– Role of fluoroquinolones
PK/PD Monte Carlo
MI C (mg/L)
Treatment
Drug
Ciproflox.
total daily dosage
(mg)
unitary dose interval
(hours)
1200
8
1600
2400
Levoflox.
500
1000
6
8
24
12
Target Attainm ent Rate (%)
Cmax/MIC > 10
AUC/MIC > 125
0.12
66
87
0.25
6
7
0.12
66
90
0.25
5
12
0.12
98
100
0.25
60
85
0.5
4.2
3.7
0.5
70
40
1
4
3
0.5
72
72
1
4
5
Dupont P. et al. J. Antimicrob. Chemother. 2005
Efflux mutants, are they virulent ?

Clinical experience
– Many examples of mdr isolates recovered from clinical specimens
(blood, urine, sputums…)

Other considerations
– marA disruption mutants of S. Typhimurium remain fully virulent
in a murine BALB/c infection model (Sulavik, J. Bacteriol. 1997,
179: 1857)
– First step fluoroquinolone resistant mutants with mutations in
gyrA, gyrB or marOR do not display significant loss of fitness (in
vitro competition experiments, experimental urinary tract infection
in mouse) (Komp Lindgren P., AAC 2005, 49: 2343)
– Role of secondary mutations ?
How to characterize efflux mechanisms

Plasmid or transposon encoded efflux systems
– Multiresistance phenotype
– Detection of efflux gene(s): PCR, nucleic probes

Upregulation of intrinsic efflux systems
– Protein levels

Western blotting of membrane extracts with specific antibodies
– mRNA levels


Northern blot, MacroArray, MicroArray
Real Time RT-PCR (Light Cycler, Taq Man, I Cycler…)
– Intracellular accumulation of antibiotics

[3H] ou [14C] radiolabeled or fluorescent compounds (BET,
acriflavine…)
– Sequencing of regulatory genes
Efflux inhibitors
Phenyl-Arginyl ß N-naphtylamide