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

Transcription

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 Cell wall Chromosome Ribosomes Cytoplasmic membrane

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

5 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...

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 Intracellular accumulation S R CCCP ATP glucose 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 H + Na + antibiotic H + ATP ADP MFS, SMR MATE ABC RND, MFS, ABC

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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 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)

17 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

18 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

19 Efflux pumps of the RND family Bacteria System Substrates E. coli AcrAB-TolC 1 Fq, ß-lactams 3, Tc, Cmp, Nov, Ery, Fus, Rif E. coli AcrEF-TolC 2 Fq, ß-lactams 3, Tc, Cmp, Nov, Ery, Fus, Rif E. coli AcrD 2 -AcrA-TolC AGs, Ery, PolyB E. coli CusAB-? 2 Fos E. coli MdtABC-TolC 2 Fq E. coli MdtEF-TolC 2 Ery P. aeruginosa MexAB-OprM 1 Fq, ß-lactams 1, Tc, Cmp, Nov, Ery, Fus, Tm... P. aeruginosa MexCD-OprJ 2 Fq, 3 rd GC, Tc, Cmp, Ery, Tmp P. aeruginosa MexEF-OprN 2 Fq, Cmp, Tmp P. aeruginosa MexXY 2 -OprM Fq, AGs, 3 rd GC, Ery, Tc N. gonorrhoeae MtrCDE 1 Tc, Cmp, ß-lactams 1, 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.

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

21 mutations mdr Overexpression of acrab and mtrcde operons acrr - + acra MarA acrb _ (MppA) MarR SoxS _ SoxR MtrA mtrr - + mtrc mtrd mtre

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23 Systems MtrCDE and FarAB in N. gonorrhoeae Antibiotics wild type CDE ++ CDE - FarAB - Penicillin G nd Erythromycin Tetracycline nd nd Rifampicin nd Linoleic acid 1600 nd Palmitic acid 100 nd

24 System AcrAB-TolC in E. coli Antibiotics wild type AcrAB ++ AcrAB - Nalidixic acid Norfloxacin nd Ofloxacin nd Ciprofloxacin nd Ampicillin Erythromycin > 512 < 2-8 Tetracycline Chloramphenicol

25 System MexAB-OprM in P. aeruginosa Antibiotics wild type MexAB ++ MexAB - Norfloxacin Ofloxacin Ciprofloxacin Carbenicillin Aztreonam Ceftazidime Cefepime Meropenem Tetracycline Chloramphenicol

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 AG AcrAB gyra (Asp87->Gly) gyra (Asp87->Gly; Ser83->Leu) 4 2 gyra (Asp87->Gly), AcrAB gyra (Asp87->Gly), AcrAB

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

29 PK/PD Monte Carlo Drug Treatment MIC (mg/l) Target Attainment Rate (%) total daily dosage (mg) unitary dose interval (hours) Cmax/MIC > 10 AUC/MIC > 125 Ciproflox Levoflox

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

32 Efflux inhibitors Phenyl-Arginyl ß N-naphtylamide