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Role of the Efflux Pumps inAntimicrobial Resistance

Patrick PlésiatBacteriology Department

Teaching HospitalBesançon, France

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ANTIBIOTIC

TARGET[n]

[Cint]

affinity

3

Bacterial targets for antibiotics

Chromosome

Cell wall

Cytoplasmic membrane

Ribosomes

4

Main resistance mechanisms to drugs

ANTIBIOTIC

TARGET

Protection

Reduced affinity- mutations- recombinaisons- enzymatic modification

InactivationModification

EffluxImpermeability

SubstitutionAmplification

Cleaning

5

Drug resistance mechanisms

Drug inactivation

Target alteration

Decreased mb permeability

Active efflux

ß-lact

ams

Amino

glycos

ides

Macro

lides

Tetra

cyclin

es

Tetra

cyclin

es

Quino

lones

+++

+++

+

+

+++

+

+

+

+/-

+++

+

+

+/-

+++

+

+

+/-

++

+

+++

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Antibiotic efflux

First description60’: E. coli strains resistant to nalidixic acid (K. Arima)80’: Tet determinants (S. Levy)

Since early 90’Several hundreds of characterized or putative efflux systemsreported in the literature…

Definition of efflux systemsTransmembrane proteins able to actively transport diversesubstrate molecules from the cell interior to the external mediumPumps : functional export systems

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Drug accumulation experiments

TimeTime

Intra

cellu

lar a

ccum

ulat

ion

CCCP

ATPglucose

S

R

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Gram-negative species with known efflux systems

−Escherichia coli−Salmonella Typhimurium−Shigella dysenteriae−Klebsiella pneumoniae−Enterobacter aerogenes−Serratia marcescens−Proteus sp.−Citrobacter freundii...

−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...

−Bacteroides fragilis...

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Other bacterial species with known efflux systems

−Staphylococcus aureus−Staphylococcus sp.−Streptococcus pneumoniae−Streptococcus pyogenes−Streptococcus agalactiae−Enterococcus sp…

−Bacillus subtilis−Listeria monocytogenes−Corynebacterium sp−Lactococcus lactis−Lactobacillus brevis...

−Mycoplasma hominis...

-Mycobacterium smegmatis-Mycobacterium tuberculosis...

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Efflux mechanisms: practical implications

Do efflux systems produce clinically relevant levels of resistance ?

Does the expression of drug transporters impair the virulence ofbacterial pathogens ?

What is the prevalence of efflux systems relative to other resistancemechanisms among the clinical isolates ?

How to recognize efflux mutants in laboratory practice ?

What recommendations can be made to the physician for thetreatment of patients infected with mdr strains ?

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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 A transportertransporter protein protein

♦♦ A periplasmic A periplasmic adaptoradaptor lipoprotein lipoprotein

♦♦ A outer membrane A outer membrane channelchannel protein protein

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Energy sources

ABC transporters

– 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

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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

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Structure of drug efflux systems

H+

ATP ADP

antibiotic

MFS, SMR MATE ABC RND (MFS, ABC)

Na+

antibiotic

H+

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Efflux-based resistance in Staphylococci

Species System Family Substrates Genes Fqcyr

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.

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System NorA in System NorA in S. aureusS. aureus

ExpressionExpression–– Constitutive or Constitutive or slightly inducible slightly inducible by by FQs FQs in wild-type in wild-type strainsstrains

–– Increased Increased by mutations in by mutations in the promoter region the promoter region of of norA norA or inor inother lociother loci

SubstratesSubstrates–– Identical Identical to to those those of of pump Bmr pump Bmr in in Bacillus subtilisBacillus subtilis–– Specificity related Specificity related to C-7 to C-7 residue and hydrophobicity residue and hydrophobicity of C-8of C-8

residueresidue

InhibitorsInhibitors–– CCCP, CCCP, nigericinnigericin, , reserpinreserpin, , verapamilverapamil, , omprazoleomprazole,,

lanzoprazolelanzoprazole

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System NorA in S. aureus

Antibiotics Wild-type NorA+++ NorA-

Nalidixic acid 25 - 125 100 - >1000 ndNorfloxacin 0.8 - 1.6 50 - 80 0.2 - 0.3Ciprofloxacin 0.25 - 0.7 6 0.1 - 0.2Ofloxacin 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

(CMI µg/mL

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Active efflux

Membranepermeability

Other mechanisms

Interplays between resistance mechanisms

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Combination of mechanisms in S. aureus

Strains GyrA ParC NorA+ Cip Sparflo

1 - - - 0.5 0.1

2 - S80Y - 8 13 - E84K - 8 14 - E84K efflux 64 25 E88K S80Y - 128 326 E88K S80Y efflux >128 647 S84L E84K efflux >128 64

I. Guillemin, thesis Paris XI(CMI µg/mL

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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

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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

Gill, M. J. Antimicrob. Agents Chemother. 1999, 43: 187Gill, M. J. Antimicrob. Agents Chemother. 1999, 43: 187

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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 1I460V

S7E1 S81F K137N I460V + 32 16 32 4

Ho, P. L. J. Antimicrob. Chemother. 2001, 47: 655

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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.

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Efflux systems in E. coli

Chromosomal genes– 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)

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Efflux pumps coded by mobile genetic elements

Species System Family Substrates

E. coli TetA/B/E MFS Tc, MinE. coli CmlA MFS CmpE. coli Flo MFS Cmp, FloE. coli OqxAB-TolC RND Olaquindox, Cmp

Tig

Tc: tetracycline; Min: minocycline; Cmp: chloramphenicol; Flo: florfenicol ; Tig: tigecycline

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Efflux pumps of MFS, MATE, SMR, or ABC family

Species System Family Substrates Genes

E. coli EmrAB-TolC MFS Nal CE. coli Bcr MFS Tc, Km, Fos CE. coli MdfA MFS Tc, Rif, Cmp, Ery, Neo, Fq... CE. coli MdtG MFS Fos CE. coli MdtH MFS Fq CE. coli MdtL MFS Cmp CE. coli MdtM MFS Cmp, Fq CE. coli NorE MATE Cmp, Fq, Fos, Tmp CE. coli EmrE SMR Tc CE. coli MdtJK SMR Nal, Fos CE. 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

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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, PolyBE. coli CusAB-?2 FosE. coli MdtABC-TolC2 FqE. coli MdtEF-TolC2 Ery

P. aeruginosa MexAB-OprM1 Fq, ß-lactams1, Tc, Cmp, Nov, Ery, Fus, Tm...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; Fos: fosfomycin; Tmp: trimethoprim;3rdGC: cefepime, cefpirome. 1 expressed constitutively in wild type cells, 2 inducible expression, 3 except imipenem.

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Overexpression of acrAB and mtrCDE operons

acrRacrRacrBacrBacrAacrA

--

mutations mdrmutations mdr

MtrAMtrA

++

++MarAMarA

mtrDmtrDmtrCmtrC mtrEmtrEmtrRmtrR

--

MarRMarR__ (MppA)

SoxSSoxS SoxRSoxR__

E. coli

N. gonorrhoeae

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System AcrAB-TolC in E. coli

Antibiotics wild type AcrAB++ AcrAB-

Nalidixic acid 4 - 6 8.5 - 32 0.6Norfloxacin 0.025 - 0.1 0.3 - 1.25 ndOfloxacin 0.06 - 0.07 0.25 - 0.3 ndCiprofloxacin 0.02 0.15 nd

Ampicillin 2 - 4 5 - 6 0.6 - 2Erythromycin 128 - 256 > 512 < 2 - 8Tetracycline 1.25 - 3 5 - 16 0.25 - 0.3Chloramphenicol 4 - 7.5 10 - 28 0.6

contribution to intrinsic resistance : CMI x 2-64

acquired resistance : CMI x 2-12(CMI mg/l)

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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

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Induction of acrAB-tolC expression

tetracyclinechloramphenicol(acetyl)salicylate

benzoatestress...

marROABRob bile salts

SoxSR oxidative stress

Mar regulon :∇ Porin OmpFPorin OmpF

ΔΔ TolC TolCΔ AcrABAcrABΔ EmrAB

Δ∇Other proteins

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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

(CMI mg/l)

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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 - 4Chloramphenicol 12.5 - 32 0.8 - 2 -Erythromycin 256 64 - 128 32 - 64

Tobramycin 0.5 - 0.125Amikacin 2 - 0.5

(CMI mg/l)CMI x 2-64

CMI x 2-8

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Acquired resistance in P. aeruginosa

Antibiotics Wild type MexAB/M MexCD/J MexEF/N MexXY/M

Carbenicillin 8 - 32 64 - 256Aztreonam 2 - 4 12.5 - 32Ceftazidime 0.4 - 2 1.6 - 8Cefepime 1 3 - 4 12.5 8Cefpirom 1 - 2 4 - 8 8 - 16Imipenem 0.8 - 1 6.25 - 8Meropenem 0.2 - 0.5 0.8 - 2Ciprofloxacin 0.03 - 0.125 0.4 - 1 0.8 - 1.6 0.8 - 1.6 0.5 - 1Amikacin 2 4 - 16Tobramycin 0.25 - 0.5 1 - 2

(CMI mg/l)

Genetic events leading to increased efflux

mexZ mexYmexX

mexR mexBmexA oprM

-

-

_PA3720 PA3719PA3721

nalC

-

mdr mutations

nalB

agrZMexXY

MexAB-OprM

nalD

PA3574

-

IS

C. Vogne et al. Antimicrob. Agents Chemother. 2004, 48: 1676C. Llanes et al. Antimicrob. Agents Chemother. 2004, 48: 1797

_PA5471

+ agrW

MexXY-mediated adaptive resistance to AGs

Stain ATCC 27853 exposed to 1 MIC amikacin for 2h every 8h (Karlowsky 1994) MIC of amikacin; Bacterial killing in log10

_____________________

____

10

20

30

40

50M

IC (

mg

/ L)

_

_

_

_

_

2.5

2

1.5

1

0.5 Ino

culu

m (

log1

0 C

FU)

D. Hocquet et al. Antimicrob. Agents Chemother. 2003, 47: 1371

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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

Lomovskaya et al. Antimicrob. Agents Chemother. 1999, 43: 1340Lomovskaya et al. ICAAC Toronto 1999, abstract F-1264

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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

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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

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Efflux inhibitors

Phenyl-Arginyl ß N-naphtylamide