2 - BACTERIOWEB

Role of the Efflux Pumps in
Antimicrobial Resistance
Patrick Plésiat
Bacteriology Department
Teaching Hospital
Besançon, France
1

ANTIBIOTIC
[C int ]
TARGET
[n]
affinity
2

Cell wall
Bacterial targets for antibiotics
Chromosome
Cytoplasmic membrane
Ribosomes
3

Main resistance mechanisms to drugs
Inactivation
Modification
Cleaning
Protection
ANTIBIOTIC
TARGET
Substitution
Amplification
Efflux
Impermeability
Reduced affinity
- mutations
- recombinaisons
- enzymatic modification
4

Drug inactivation
Target alteration
Decreased mb permeability
Active efflux
Drug resistance mechanisms
+++
+++
+
+
ß-lactams
+++
+
+
+
Aminoglycosides
Quinolones
+/-
+++
+
+
+/-
+++
+
+
Macrolides
+/-
++
+
+++
5
Tetracyclines
Tetracyclines

First description
Antibiotic efflux
60’: E. coli strains resistant to nalidixic acid (K. Arima)
80’: Tet determinants (S. Levy)
Since early 90’
Several hundreds of characterized or putative efflux systems
reported in the literature…
Definition of efflux systems
Transmembrane proteins able to actively transport diverse
substrate molecules from the cell interior to the external medium
Pumps : functional export systems
6

Intracellular accumulation
Drug accumulation experiments
S
R
CCCP
Time
ATP
glucose
7

8
Gram-negative species with known efflux systems
−Escherichia coli
−Salmonella Typhimurium
−Shigella dysenteriae
−Klebsiella pneumoniae
−Enterobacter aerogenes
−Serratia marcescens
−Proteus sp.
−Citrobacter freundii...
−Bacteroides fragilis...
−Pseudomonas aeruginosa
−Pseudomonas putida
−Burkholderia cepacia
−Burkholderia pseudomallei
−Stenotrophomonas maltophilia
−Alcaligenes eutrophus
−Acinetobacter baumannii...
−Neisseria gonorrhoeae
−Haemophilus influenzae
−Campylobacter coli, jejuni
−Helicobacter pylori
−Vibrio parahaemolyticus
−Vibrio cholerae
−Yersinia pestis...

9
Other bacterial species with known efflux systems
−Staphylococcus aureus
−Staphylococcus sp.
−Streptococcus pneumoniae
−Streptococcus pyogenes
−Streptococcus agalactiae
−Enterococcus sp…
−Mycoplasma hominis...
−Bacillus subtilis
−Listeria monocytogenes
−Corynebacterium sp
−Lactococcus lactis
−Lactobacillus brevis...
-Mycobacterium smegmatis
-Mycobacterium tuberculosis...

Efflux mechanisms: practical implications
Do efflux systems produce clinically relevant levels of resistance ?
Does the expression of drug transporters 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 ?
10

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
11

ABC transporters
Energy sources
– ATP binding cassette pumps
– Hydrolysis of ATP into ADP + Pi
– Mostly in Gram positive species
Secondary transporters
– H + /substrate antiporters (proton motive force)
– Na + /substrate antiporters
12

PMF secondary transporters
Major Facilitator Superfamily (MFS)
– Drug efflux
♦ 12 TMS transporters
♦ 14 TMS transporters
– Active uptake/export
♦ sugars...
♦ amino acids, secondary metabolites...
Small Multidrug Resistance Family (SMR)
♦ 4 TMS transporters
Resistance/Nodulation Cell Division Family (RND)
♦ 12 TMS transporters
Multi Antimicrobial Extrusion Family (MATE)
♦ 12 TMS transporters
13

H +
Structure of drug efflux systems
Na +
antibiotic
ATP ADP
antibiotic
MFS, SMR MATE ABC RND (MFS, ABC)
H +
14

Efflux-based resistance in Staphylococci
Species System Family Substrates Genes Fqcy r
S. coagulase - MsrA ABC 14,15-M, strept.B P ++
S. aureus MsrA-like ABC 14,15-M, strept.B P +/-
S. epidermidis ErpA ABC ? 14,15-M P ?
S. aureus NorA MFS Fq, Cmp, Org. Ch ?
S. aureus QacA/B MFS Antisept. P +
S. aureus TetK MFS Tc P +++
14,15-M: 14 et 15-macrolides ; Strept.B: streptogramin B ; Fq: fluoroquinolones ; Cmp: chloramphenicol;
Organic cations.: acriflavin, cetyltrimethylammonium, Ethidium bromide, triphenylphosphonium, rhodamine ;
Antisept.: chlorhexidin, benzalkonium, cetyltrimethylammonium, pentamidine...; Tc: tetracycline.
15

Expression
System NorA in S. aureus
– Constitutive or slightly inducible by FQs in wild-type strains
– Increased by mutations in the promoter region of norA or in
other loci
Substrates
– Identical to those of pump Bmr in Bacillus subtilis
– Specificity related to C-7 residue and hydrophobicity of C-8
residue
Inhibitors
– CCCP, nigericin, nigericin,
reserpin, reserpin,
verapamil, verapamil,
omprazole, omprazole
lanzoprazole
16

System NorA in S. aureus
Antibiotics Wild-type NorA+++ NorA-
Nalidixic acid 25 - 125 100 - >1000 nd
Norfloxacin 0.8 - 1.6 50 - 80 0.2 - 0.3
Ciprofloxacin 0.25 - 0.7 6 0.1 - 0.2
Ofloxacin 0.2 - 0.5 1.5 - 3 0.4
Pefloxacin 0.5 12.5 nd
Sparfloxacin 0.1 0.2 0.1
Cetrimide 0.4 6.5 nd
Benzalkonium 1 3 nd
Ethidium bromide 5 - 6.5 25 0.5
17
(CMI µg/mL

Interplays between resistance mechanisms
Membrane
permeability
Active efflux
Other mechanisms
18

Combination of mechanisms in S. aureus
Strains GyrA ParC NorA+ Cip Sparflo
1 - - - 0.5 0.1
2 - S80Y - 8 1
3 - E84K - 8 1
4 - E84K efflux 64 2
5 E88K S80Y - 128 32
6 E88K S80Y efflux >128 64
7 S84L E84K efflux >128 64
19
(CMI µg/mL
I. Guillemin, thesis Paris XI

Efflux mechanisms in Streptococci
Species System Family Substrates Genes Fqcy
S. pyogenes MefA MFS ? 14,15-M Tn +++
S. pneumoniae MefE MFS ? 14,15-M Tn ++
S. pneumoniae ? MFS ? 14,15-M, strept.B Ch ? ?
S. pneumoniae PmrA MFS ? Cip, Nor, BET Ch ?
14,15-M: 14 et 15-macrolides ; Strept.B: streptogramin B ; Cip: ciprofloxacin; Nor: norfloxacin; BET: Ethidium
20

PmrA-mediated resistance in S. pneumoniae
Antibiotics Wild type PmrA++
Norfloxacin 2 16
Norfloxacin + reserpin 2 4
Ciprofloxacin 0.5 2
Moxifloxacin 0.12 0.12
Sparfloxacin 0.25 0.25
Acriflavin 4 16
Ethidium bromide 2 16
21
Gill, M. J. Antimicrob. Agents Chemother. 1999, 43: 187

Combinaison of mechanisms in S. pneumoniae
Strains GyrA ParC ParE Efflux Cip Levo Trova Moxi
S10B4 - - - + 1 1 0.25 0.25
S10A6 - - I460V - 1 1 0.25 0.125
S7A2 - - I460V + 2 1 0.25 0.125
S7B7 - K137N - + 2 1 0.5 0.25
S7C2 - S79F I460V + 4 2 0.5 0.25
S9E9 - K137N I460V + 16 4 0.5 0.25
S10D9 S81F K137N D435N + 16 16 1 1
I460V
S7E1 S81F K137N I460V + 32 16 32 4
22
Ho, P. L. J. Antimicrob. Chemother. 2001, 47: 655

Other Gram positives
Species System Family Substrates Genes Fqcy
B. subtilis Bmr MFS Cmp, Fq, Org. Ch ?
B. subtilis Blt MFS Cmp, Fq, Org. Ch ?
B. subtilis Bmr3 MFS Oflox, lévo, Org. Ch ? ?
Streptomyces sp Cml MFS Cmp Ch +++
Streptomyces sp Ptr MFS Pristina, Rif Ch +++
Cmp: chloramphenicol ; Fq: fluoroquinolones ; Organic cations: acriflavine, cetyltrimethylammonium,
ethidium, triphenylphosphonium, rhodamine ; Oflox.: ofloxacin ; Levo.: levofloxacin ; Pristina: pristinamycins I-
II ; Rif: rifampicin.
23

Chromosomal genes
Efflux systems in E. coli
– 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)
– Most of these intrinsic systems are not expressed in standard
laboratory growth conditions
– Spontaneous mutations may result in stable overproduction of a
single pump and resistance
Foreign genes
– Genes carried by mobile elements (plasmids, transposons)
24

25
Efflux pumps coded by mobile genetic elements
Species System Family Substrates
E. coli TetA/B/E MFS Tc, Min Tig
E. coli CmlA MFS Cmp
E. coli Flo MFS Cmp, Flo
E. coli OqxAB-TolC RND Olaquindox, Cmp
Tc: tetracycline; Min: minocycline; Cmp: chloramphenicol; Flo: florfenicol ; Tig: tigecycline

26
Efflux pumps of MFS, MATE, SMR, or ABC family
Species System Family Substrates Genes
E. coli EmrAB-TolC MFS Nal C
E. coli Bcr MFS Tc, Km, Fos C
E. coli MdfA MFS Tc, Rif, Cmp, Ery, Neo, Fq... C
E. coli MdtG MFS Fos C
E. coli MdtH MFS Fq C
E. coli MdtL MFS Cmp C
E. coli MdtM MFS Cmp, Fq C
E. coli NorE MATE Cmp, Fq, Fos, Tmp C
E. coli EmrE SMR Tc C
E. coli MdtJK SMR Nal, Fos C
E. coli MacAB-TolC ABC Ery 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-TolC 1 Fq, ß-lactams 3 , Tc, Cmp, Nov, Ery, Fus, Rif…
E. coli AcrEF-TolC 2 Fq, ß-lactams 3 , Tc, Cmp, Nov, Ery, Fus, Rif…
E. coli AcrD 2 -AcrA-TolC AGs, Ery, PolyB
E. coli CusAB-? 2 Fos
E. coli MdtABC-TolC 2 Fq
E. coli MdtEF-TolC 2 Ery
P. aeruginosa MexAB-OprM 1 Fq, ß-lactams 1 , Tc, Cmp, Nov, Ery, Fus, Tm...
N. gonorrhoeae MtrCDE 1 Tc, Cmp, ß-lactams 1 , Ery, Fus, Rif...
Fq: (fluoro)quinolones; Tc: tetracycline; Cmp: chloramphenicol; Nov: novobiocin; Ery: erythromycin; Fus:
fusidic acid; Rif: rifampicin; AGs: aminoglycosides; PolyB: polymyxin B; Fos: fosfomycin; Tmp: trimethoprim;
3 rd GC: cefepime, cefpirome. 1 expressed constitutively in wild type cells, 2 inducible expression, 3 except imipenem.
27

28
Overexpression of acrAB and mtrCDE operons
E. coli
N. gonorrhoeae
acrR
mtrR
-
-
acrA
+
+
MarA
acrB
MtrA
mtrC mtrD
mtrE
_ (MppA)
MarR
_
SoxS SoxR
mutations mdr

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
Erythromycin 128 - 256 > 512 < 2 - 8
Tetracycline 1.25 - 3 5 - 16 0.25 - 0.3
Chloramphenicol 4 - 7.5 10 - 28 0.6
contribution to intrinsic resistance : CMI x 2-64
acquired resistance : CMI x 2-12
29
(CMI mg/l)

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
gyrA (Asp87->Gly), AcrAB - 0.06 0.03
Oethinger et al. Antimicrob. Agents Chemother. 2000, 44: 10-13
30

Induction of acrAB-tolC expression
tetracycline
chloramphenicol
(acetyl)salicylate
benzoate
stress...
marROAB
Mar regulon :
∇ Porin OmpF
Δ TolC
Δ AcrAB
Δ EmrAB
Δ∇Other proteins
SoxSR oxidative stress
Rob bile salts
31

Systems MtrCDE and FarAB in N. gonorrhoeae
Antibiotics 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
contribution to intrinsic resistance : CMI x 4-64
acquired resistance : CMI x 4-8
32
(CMI mg/l)

RND efflux systems in P. aeruginosa
System Operon Substrates
MexAB-OprM mexAB,oprM FQ, ß-lactam, Tmp, Cmp, Tet, Nov, Ery...
MexXY (OprM) mexXY FQ, AG, Fep, Cpo, Tet, Ery...
MexCD-OprJ mexCD,oprJ FQ, Cpo ,Fep, Tmp, Cmp, Tet, Ery...
MexEF-OprN mexEF,oprN FQ, (Ipm), Tmp, Cmp...
MexGHI-OpmD mexGHI,opmD FQ...
MexJK (OprM) mexJK Tet, Ery...
MexVW (OprM) mexVW FQ, Cmp, Tet, Ery...
Fq: fluoroquinolones; ß-lactam (except imipenem); Tmp: trimethoprime; Cmp: chloramphenicol; Tet: tetracycline;
Nov: novobiocin; Ery: erythromycin; AG: aminoglycosides; Fep: cefepime; Cpo: cefpirome; Ipm: imipenem.
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Contribution to intrinsic resistance in P. aeruginosa
Antibiotcs Wild type MexAB/M - MexXY/M -
Norfloxacin 0.125 - 1 0.05 - 0.25 -
Ciprofloxacin 0.03 - 0.25 0.012 - 0.03 -
Carbenicillin 12.5 - 64 0.4 - 1 -
Ceftazidime 0.4 - 2 0.2 - 0.4 -
Cefepime 0.8 - 2 0.1 - 0.5 -
Meropenem 0.2 - 0.5 0.1 - 0.2 -
Tetracycline 6.25 - 16 2 2 - 4
Chloramphenicol 12.5 - 32 0.8 - 2 -
Erythromycin 256 64 - 128 32 - 64
Tobramycin 0.5 - 0.125
Amikacin 2 - 0.5
CMI x 2-64
CMI x 2-8
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(CMI mg/l)

Acquired resistance in P. aeruginosa
Antibiotics Wild type MexAB/M MexCD/J MexEF/N MexXY/M
Carbenicillin 8 - 32 64 - 256
Aztreonam 2 - 4 12.5 - 32
Ceftazidime 0.4 - 2 1.6 - 8
Cefepime 1 3 - 4 12.5 8
Cefpirom 1 - 2 4 - 8 8 - 16
Imipenem 0.8 - 1 6.25 - 8
Meropenem 0.2 - 0.5 0.8 - 2
Ciprofloxacin 0.03 - 0.125 0.4 - 1 0.8 - 1.6 0.8 - 1.6 0.5 - 1
Amikacin 2 4 - 16
Tobramycin 0.25 - 0.5 1 - 2
35
(CMI mg/l)

Genetic events leading to increased efflux
IS
mdr mutations
mexR
mexZ
PA3721
nalC
-
-
nalB
-
agrZ
PA3720 PA3719
_
-
mexA mexB oprM
+
mexX
mexY
PA5471
_
PA3574
nalD
MexXY
MexAB-OprM
agrW
C. Vogne et al. Antimicrob. Agents Chemother. 2004, 48: 1676
C. Llanes et al. Antimicrob. Agents Chemother. 2004, 48: 1797

MexXY-mediated adaptive resistance to AGs
MIC (mg / L)
50 _
_
40 _
30 _
20 _
10 _
Stain ATCC 27853 exposed to 1 MIC amikacin for 2h every 8h (Karlowsky 1994)
MIC of amikacin; Bacterial killing in log10
_
_
_
_
_
2.5
2
1.5
1
0.5
Inoculum (log10 CFU)
D. Hocquet et al. Antimicrob. Agents Chemother. 2003, 47: 1371

Target/efflux double mutants in P. aeruginosa
MIC levofloxacin (mg/L)
Target mutations Wild-type MexAB ++ MexAB ++
+ inh. 10 mg/l
Aucune 0.25 2 0.03
gyrA (Thr83->Ile) 2 8 0.5
gyrA (Thr83->Ile) + parC (Ser87->Leu) 4 32 2
gyrA (Thr83->Ile + Asp87->Tyr) + parC (Ser87->Leu) 16 128 8
38
Lomovskaya et al. Antimicrob. Agents Chemother. 1999, 43: 1340
Lomovskaya et al. ICAAC Toronto 1999, abstract F-1264

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
39

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
♦ [ 3 H] ou [ 14 C] radiolabeled or fluorescent compounds (BET,
acriflavine…)
– Sequencing of regulatory genes
40

Efflux inhibitors
Phenyl-Arginyl ß N-naphtylamide
41