E. coli - BACTERIOWEB

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

ANTIBIOTIC
TARGET

Cell wall
Bacterial targets for antibiotics
Chromosome
Cytoplasmic membrane
Ribosomes

Main resistance mechanisms to drugs
Inactivation
Modification
Protection
ANTIBIOTIC
TARGET
Substitution
Amplification
Efflux
Impermeability
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
R
CCCP
Time
ATP
glucose

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

Antiporters
Energy sources
– 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
♦ 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

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

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 plasmid genes (Nishino K et al. J. Bacteriol. 2001)
– Upregulation of a single pump results 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 Family Substrates
E. coli TetA/B/E MFS Tc, Min
E. coli CmlA MFS Cmp
E. coli Flo MFS Cmp, Flo
E. coli OqxAB RND Olaquindox
Tc: tetracycline; Min: minocycline; Cmp: chloramphenicol; Flo: florfenicol

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; 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; 3 rd GC: cefepime, cefpirome.
1 expressed constitutively in wild type cells, 2 inducible expression, 3 except imipenem.

Induction of acrAB-tolC expression
tetracycline
chloramphenicol
salicylate-acetylsalicylate
benzoate
stress...
MarRAB
∇ Porin OmpF
∆ TolC
∆ AcrAB
∆ EmrAB
∆∇Other proteins
Mar regulon
SoxSR oxidative stress
Rob bile salts
tetracycline r
chloramphenicol r
quinolones r
erythromycin r
solvants, pine oil...

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

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

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

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
Active efflux
Other mechanisms

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

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 gain of efflux mutants under treatment
– Cross resistance to structurally unrelated molecules
– Role of fluoroquinolones

Drug total daily dosage
(mg)
PK/PD Monte Carlo
Treatment MIC (mg/L) Target Attainment Rate (%)
unitary dose interval
(hours)
Cmax/MIC > 10 AUC/MIC > 125
Ciproflox. 1200 8 0.12 66 87
0.25 6 7
1600 6 0.12 66 90
0.25 5 12
2400 8 0.12 98 100
0.25 60 85
0.5 4.2 3.7
Levoflox. 500 24 0.5 70 40
1 4 3
1000 12 0.5 72 72
1 4 5

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

Efflux inhibitors
Phenyl-Arginyl ß N-naphtylamide