Role of the Efflux Pumps in Antimicrobial Resistance in E. coli

Patrick PlésiatBacteriology Department

Teaching HospitalBesançon, France

ANTIBIOTIC

TARGET

Bacterial targets for antibiotics

Chromosome

Cell wall

Cytoplasmic membrane

Ribosomes

Main resistance mechanisms to drugs

ANTIBIOTIC

TARGET

Protection

Reduced affinity- mutations- recombinaisons- enzymatic modification

InactivationModification

EffluxImpermeability

SubstitutionAmplification

Gram-negative species with known efflux systems

Escherichia coliSalmonella TyphimuriumShigella dysenteriaeKlebsiella pneumoniaeEnterobacter aerogenesSerratia marcescensProteus vulgarisCitrobacter freundii...

Pseudomonas aeruginosaPseudomonas putidaBurkholderia cepaciaBurkholderia pseudomalleiStenotrophomonas maltophiliaAlcaligenes eutrophus...

Haemophilus influenzaeCampylobacter jejuniHelicobacter pyloriVibrio parahaemolyticusVibrio choleraeNeisseria gonorrhoeae...

Bacteroides fragilis...

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 ?

Drug accumulation experiments

TimeTime

Intr

acel

lula

r ac

cum

ulat

ion

CCCP

ATPglucose

S

R

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

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 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

Structure of drug efflux systems

H+

ATP ADP

antibiotic

MFS, SMR MATE ABC RND, MFS, ABC

Na+

antibiotic

H+

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 Family Substrates

E. coli TetA/B/E MFS Tc, MinE. 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

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-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 rdGC: cefepime, cefpirome. 1 expressed constitutively in wild type cells, 2 inducible expression, 3 except imipenem.

Induction of acrAB-tolC expression

tetracyclinechloramphenicol

salicylate-acetylsalicylatebenzoate

stress...

tetracycliner

chloramphenicolr

quinolonesr

erythromycinr

solvants, pine oil...

MarROABRob bile salts

SoxSR oxidative stress

AcrABAcrAB

EmrAB

Porin OmpFPorin OmpF

TolCTolC

Other proteins

Mar regulon

Overexpression of acrAB and mtrCDE operons

mtrDmtrDmtrCmtrC mtrEmtrE

mtrRmtrR

acrRacrR

acrBacrBacrAacrA--

mutations mdrmutations mdr

MtrAMtrA

++

++

MarAMarA

--

MarRMarR__ (MppA)

SoxSSoxS SoxRSoxR__

Webber M. et al. Antimicrob. Agents Chemother. 2001, 45: 1550

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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

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

Active efflux

Outer membranepermeability

Other mechanisms

Interplays between resistance mechanisms in GNB

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

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

Treatment MIC (mg/L) Target Attainment Rate (%)Drug total daily dosage

(mg)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

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