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 Main resistance mechanisms to drugs

Inactivation Modification ANTIBIOTIC

Efflux Impermeability Protection

TARGET

Reduced affinity Substitution - mutations Amplification - recombinaisons - enzymatic modification Gram-negative species with known efflux systems

Escherichia coli Pseudomonas aeruginosa

Salmonella Typhimurium Pseudomonas putida Burkholderia cepacia Shigella dysenteriae Burkholderia pseudomallei Klebsiella pneumoniae Stenotrophomonas maltophilia Enterobacter aerogenes Alcaligenes eutrophus... Serratia marcescens

Proteus vulgaris Haemophilus influenzae

Citrobacter freundii... Campylobacter jejuni Helicobacter pylori Vibrio parahaemolyticus Vibrio cholerae Bacteroides fragilis... Neisseria gonorrhoeae... 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

S

ATP glucose

R

Intracellular accumulation CCCP

Time 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 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

antibiotic

antibiotic H+ Na+ H+

ATP ADP

MFS, SMR MATE ABC RND, MFS, ABC 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, 1MATE – 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, 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 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; 3rdGC: cefepime, cefpirome. 1 expressed constitutively in wild type cells, 2 inducible expression, 3 except imipenem. Induction of acrAB-tolC expression

tetracycline chloramphenicol SoxSR oxidative stress salicylate-acetylsalicylate MarROAB benzoate Rob bile salts stress...

 Porin OmpF tetracycliner  TolC chloramphenicolr  AcrAB quinolonesr EmrAB erythromycinr Other proteins solvants, pine oil... Mar regulon Overexpression of acrAB and mtrCDE operons

_ (MppA) MarA MarR + _ - SoxS SoxR acrA acrB acrR

MtrA + - mtrCmtrD mtrE mtrR

mutations mdr *

* * * * *

*

* * * *

Webber M. et al. Antimicrob. Agents Chemother. 2001, 45: 1550 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 Interplays between resistance mechanisms in GNB

Outer membrane Other mechanisms permeability

Active efflux 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) 42 gyrA (Asp87->Gly), AcrAB++ 84 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 unitary dose interval Cmax/MIC > 10 AUC/MIC > 125 (mg) (hours)

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

14 3 1000 12 0.5 72 72 14 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 3 14  [ H] ou [ C] radiolabeled or fluorescent compounds (BET, acriflavine…) – Sequencing of regulatory genes Efflux inhibitors

Phenyl-Arginyl ß N-naphtylamide