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

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Role of the Efflux Pumps in Antimicrobial
Resistance in E. coli
Patrick Plésiat Bacteriology Department Teaching
Hospital Besançon, France
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ANTIBIOTIC
TARGET
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Bacterial targets for antibiotics
Chromosome
Cell wall
Ribosomes
Cytoplasmic membrane
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Main resistance mechanisms to drugs
Inactivation Modification
ANTIBIOTIC
Efflux Impermeability
Protection
TARGET
Reduced affinity - mutations - recombinaisons -
enzymatic modification
Substitution Amplification
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Gram-negative species with known efflux systems

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

• Pseudomonas aeruginosa
• Pseudomonas putida
• Burkholderia cepacia
• Burkholderia pseudomallei
• Stenotrophomonas maltophilia
• Alcaligenes eutrophus...

• Haemophilus influenzae
• Campylobacter jejuni
• Helicobacter pylori
• Vibrio parahaemolyticus
• Vibrio cholerae
• Neisseria gonorrhoeae...

• Bacteroides fragilis...

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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 ?

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Drug accumulation experiments
S
ATP glucose
Intracellular accumulation
R
CCCP
Time
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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 transporter protein
• A periplasmic adaptor lipoprotein
• A outer membrane channel protein

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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

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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

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Structure of drug efflux systems
antibiotic
antibiotic
H
Na
H
ATP
ADP
RND, MFS, ABC
MFS, SMR
MATE
ABC
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Fernandez-Recio J. et al. FEBS 2004, 578 5-9
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Murakami S. et al. Nature 2002, 419 587
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Murakami S. et al. Curr Opinion Struct. Biol.
2003, 13 443
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Murakami S. et al. Curr Opinion Struct. Biol.
2003, 13 443
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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)

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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-TolC RND Olaquindox,
Cmp Tc tetracycline Min
minocycline Cmp chloramphenicol Flo
florfenicol
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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 Cm
p, 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
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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, 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.
  1 expressed constitutively in wild type cells, 2
  inducible expression, 3 except imipenem.

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Induction of acrAB-tolC expression
tetracycline chloramphenicol salicylate-acetylsali
cylate benzoate stress...
MarROAB
tetracycliner chloramphenicolr quinolonesr erythro
mycinr solvants, pine oil...
Mar regulon
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Overexpression of acrAB and mtrCDE operons
_
(MppA)
MarA
MarR
_

SoxS
SoxR
-
acrB
acrA
acrR
MtrA

-
mtrR
mutations mdr
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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
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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 gt 512 lt 2
- 8 Tetracycline 1.25 - 3 5 - 16 0.25 -
0.3 Chloramphenicol 4 - 7.5 10 - 28 0.6
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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
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Interplays between resistance mechanisms in GNB
Outer membrane permeability
Other mechanisms
Active efflux
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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-gtGly) 0.25 0.25
• gyrA (Asp87-gtGly Ser83-gtLeu) 4 2
• gyrA (Asp87-gtGly), AcrAB 8 4
• gyrA (Asp87-gtGly), AcrAB- 0.06 0.03

Oethinger et al. Antimicrob. Agents Chemother.
2000, 44 10-13
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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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PK/PD Monte Carlo
Dupont P. et al. J. Antimicrob. Chemother. 2005
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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 ?

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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