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

Role of the Efflux Pumps in Antimicrobial Resistance in E. coli. Patrick Plésiat Bacteriology Department Teaching Hospital Besançon, France. ANTIBIOTIC. TARGET. Bacterial targets for antibiotics. Chromosome. Cell wall. Ribosomes. Cytoplasmic membrane.

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

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

  2. ANTIBIOTIC TARGET

  3. Bacterial targets for antibiotics Chromosome Cell wall Ribosomes Cytoplasmic membrane

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

  5. 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...

  6. 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 ?

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

  8. 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

  9. 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

  10. 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

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

  12. Fernandez-Recio J. et al. FEBS 2004, 578: 5-9

  13. Murakami S. et al. Nature 2002, 419: 587

  14. Murakami S. et al. Curr Opinion Struct. Biol. 2003, 13: 443

  15. Murakami S. et al. Curr Opinion Struct. Biol. 2003, 13: 443

  16. 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)

  17. Efflux pumps coded by mobile genetic elements Species System Family Substrates E. coli TetA/B/E MFS Tc, Min E. coli CmlAMFS Cmp E. coli Flo MFS Cmp, Flo E. coli OqxAB-TolC RNDOlaquindox, Cmp Tc: tetracycline; Min: minocycline; Cmp: chloramphenicol; Flo: florfenicol

  18. Efflux pumps of MFS, MATE, SMR, or ABC family Species System Family Substrates Genes E. coli EmrAB-TolCMFS Nal C E. coli Bcr MFS Tc, Km, Fos C E. coli MdfA MFSTc, 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

  19. 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-OprMFq, 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.

  20. SoxSR oxidative stress Rob bile salts  Porin OmpF  TolC  AcrAB EmrAB Other proteins Induction of acrAB-tolC expression tetracycline chloramphenicol salicylate-acetylsalicylate benzoate stress... MarROAB tetracycliner chloramphenicolr quinolonesr erythromycinr solvants, pine oil... Mar regulon

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

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

  23. 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

  24. 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

  25. 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 - 1625 - 64 0.2 - 1.2 Chloramphenicol 12.5 - 32100 - 512 0.8 - 2

  26. Interplays between resistance mechanisms in GNB Outer membrane permeability Other mechanisms Active efflux

  27. 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++84 gyrA (Asp87->Gly), AcrAB- 0.06 0.03 Oethinger et al. Antimicrob. Agents Chemother. 2000, 44: 10-13

  28. 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

  29. PK/PD Monte Carlo Dupont P. et al. J. Antimicrob. Chemother. 2005

  30. 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 ?

  31. 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

  32. Efflux inhibitors Phenyl-Arginyl ß N-naphtylamide

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