2 - BACTERIOWEB
2 - BACTERIOWEB
2 - BACTERIOWEB
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Role of the Efflux Pumps in<br />
Antimicrobial Resistance<br />
Patrick Plésiat<br />
Bacteriology Department<br />
Teaching Hospital<br />
Besançon, France<br />
1
ANTIBIOTIC<br />
[C int ]<br />
TARGET<br />
[n]<br />
affinity<br />
2
Cell wall<br />
Bacterial targets for antibiotics<br />
Chromosome<br />
Cytoplasmic membrane<br />
Ribosomes<br />
3
Main resistance mechanisms to drugs<br />
Inactivation<br />
Modification<br />
Cleaning<br />
Protection<br />
ANTIBIOTIC<br />
TARGET<br />
Substitution<br />
Amplification<br />
Efflux<br />
Impermeability<br />
Reduced affinity<br />
- mutations<br />
- recombinaisons<br />
- enzymatic modification<br />
4
Drug inactivation<br />
Target alteration<br />
Decreased mb permeability<br />
Active efflux<br />
Drug resistance mechanisms<br />
+++<br />
+++<br />
+<br />
+<br />
ß-lactams<br />
+++<br />
+<br />
+<br />
+<br />
Aminoglycosides<br />
Quinolones<br />
+/-<br />
+++<br />
+<br />
+<br />
+/-<br />
+++<br />
+<br />
+<br />
Macrolides<br />
+/-<br />
++<br />
+<br />
+++<br />
5<br />
Tetracyclines<br />
Tetracyclines
First description<br />
Antibiotic efflux<br />
60’: E. coli strains resistant to nalidixic acid (K. Arima)<br />
80’: Tet determinants (S. Levy)<br />
Since early 90’<br />
Several hundreds of characterized or putative efflux systems<br />
reported in the literature…<br />
Definition of efflux systems<br />
Transmembrane proteins able to actively transport diverse<br />
substrate molecules from the cell interior to the external medium<br />
Pumps : functional export systems<br />
6
Intracellular accumulation<br />
Drug accumulation experiments<br />
S<br />
R<br />
CCCP<br />
Time<br />
ATP<br />
glucose<br />
7
8<br />
Gram-negative species with known efflux systems<br />
−Escherichia coli<br />
−Salmonella Typhimurium<br />
−Shigella dysenteriae<br />
−Klebsiella pneumoniae<br />
−Enterobacter aerogenes<br />
−Serratia marcescens<br />
−Proteus sp.<br />
−Citrobacter freundii...<br />
−Bacteroides fragilis...<br />
−Pseudomonas aeruginosa<br />
−Pseudomonas putida<br />
−Burkholderia cepacia<br />
−Burkholderia pseudomallei<br />
−Stenotrophomonas maltophilia<br />
−Alcaligenes eutrophus<br />
−Acinetobacter baumannii...<br />
−Neisseria gonorrhoeae<br />
−Haemophilus influenzae<br />
−Campylobacter coli, jejuni<br />
−Helicobacter pylori<br />
−Vibrio parahaemolyticus<br />
−Vibrio cholerae<br />
−Yersinia pestis...
9<br />
Other bacterial species with known efflux systems<br />
−Staphylococcus aureus<br />
−Staphylococcus sp.<br />
−Streptococcus pneumoniae<br />
−Streptococcus pyogenes<br />
−Streptococcus agalactiae<br />
−Enterococcus sp…<br />
−Mycoplasma hominis...<br />
−Bacillus subtilis<br />
−Listeria monocytogenes<br />
−Corynebacterium sp<br />
−Lactococcus lactis<br />
−Lactobacillus brevis...<br />
-Mycobacterium smegmatis<br />
-Mycobacterium tuberculosis...
Efflux mechanisms: practical implications<br />
Do efflux systems produce clinically relevant levels of resistance ?<br />
Does the expression of drug transporters impair the virulence of<br />
bacterial pathogens ?<br />
What is the prevalence of efflux systems relative to other resistance<br />
mechanisms among the clinical isolates ?<br />
How to recognize efflux mutants in laboratory practice ?<br />
What recommendations can be made to the physician for the<br />
treatment of patients infected with mdr strains ?<br />
10
Structure of bacterial efflux systems<br />
One component systems<br />
– Mostly in Gram positive species (except Tet...)<br />
– A single transporter protein in the cytoplasmic membrane<br />
– Determines the substrate specificity and resistance<br />
Three component (tripartite) systems<br />
– Exclusively in Gram negative species (GNB)<br />
♦ A transporter protein<br />
♦ A periplasmic adaptor lipoprotein<br />
♦ A outer membrane channel protein<br />
11
ABC transporters<br />
Energy sources<br />
– ATP binding cassette pumps<br />
– Hydrolysis of ATP into ADP + Pi<br />
– Mostly in Gram positive species<br />
Secondary transporters<br />
– H + /substrate antiporters (proton motive force)<br />
– Na + /substrate antiporters<br />
12
PMF secondary transporters<br />
Major Facilitator Superfamily (MFS)<br />
– Drug efflux<br />
♦ 12 TMS transporters<br />
♦ 14 TMS transporters<br />
– Active uptake/export<br />
♦ sugars...<br />
♦ amino acids, secondary metabolites...<br />
Small Multidrug Resistance Family (SMR)<br />
♦ 4 TMS transporters<br />
Resistance/Nodulation Cell Division Family (RND)<br />
♦ 12 TMS transporters<br />
Multi Antimicrobial Extrusion Family (MATE)<br />
♦ 12 TMS transporters<br />
13
H +<br />
Structure of drug efflux systems<br />
Na +<br />
antibiotic<br />
ATP ADP<br />
antibiotic<br />
MFS, SMR MATE ABC RND (MFS, ABC)<br />
H +<br />
14
Efflux-based resistance in Staphylococci<br />
Species System Family Substrates Genes Fqcy r<br />
S. coagulase - MsrA ABC 14,15-M, strept.B P ++<br />
S. aureus MsrA-like ABC 14,15-M, strept.B P +/-<br />
S. epidermidis ErpA ABC ? 14,15-M P ?<br />
S. aureus NorA MFS Fq, Cmp, Org. Ch ?<br />
S. aureus QacA/B MFS Antisept. P +<br />
S. aureus TetK MFS Tc P +++<br />
14,15-M: 14 et 15-macrolides ; Strept.B: streptogramin B ; Fq: fluoroquinolones ; Cmp: chloramphenicol;<br />
Organic cations.: acriflavin, cetyltrimethylammonium, Ethidium bromide, triphenylphosphonium, rhodamine ;<br />
Antisept.: chlorhexidin, benzalkonium, cetyltrimethylammonium, pentamidine...; Tc: tetracycline.<br />
15
Expression<br />
System NorA in S. aureus<br />
– Constitutive or slightly inducible by FQs in wild-type strains<br />
– Increased by mutations in the promoter region of norA or in<br />
other loci<br />
Substrates<br />
– Identical to those of pump Bmr in Bacillus subtilis<br />
– Specificity related to C-7 residue and hydrophobicity of C-8<br />
residue<br />
Inhibitors<br />
– CCCP, nigericin, nigericin,<br />
reserpin, reserpin,<br />
verapamil, verapamil,<br />
omprazole, omprazole<br />
lanzoprazole<br />
16
System NorA in S. aureus<br />
Antibiotics Wild-type NorA+++ NorA-<br />
Nalidixic acid 25 - 125 100 - >1000 nd<br />
Norfloxacin 0.8 - 1.6 50 - 80 0.2 - 0.3<br />
Ciprofloxacin 0.25 - 0.7 6 0.1 - 0.2<br />
Ofloxacin 0.2 - 0.5 1.5 - 3 0.4<br />
Pefloxacin 0.5 12.5 nd<br />
Sparfloxacin 0.1 0.2 0.1<br />
Cetrimide 0.4 6.5 nd<br />
Benzalkonium 1 3 nd<br />
Ethidium bromide 5 - 6.5 25 0.5<br />
17<br />
(CMI µg/mL
Interplays between resistance mechanisms<br />
Membrane<br />
permeability<br />
Active efflux<br />
Other mechanisms<br />
18
Combination of mechanisms in S. aureus<br />
Strains GyrA ParC NorA+ Cip Sparflo<br />
1 - - - 0.5 0.1<br />
2 - S80Y - 8 1<br />
3 - E84K - 8 1<br />
4 - E84K efflux 64 2<br />
5 E88K S80Y - 128 32<br />
6 E88K S80Y efflux >128 64<br />
7 S84L E84K efflux >128 64<br />
19<br />
(CMI µg/mL<br />
I. Guillemin, thesis Paris XI
Efflux mechanisms in Streptococci<br />
Species System Family Substrates Genes Fqcy<br />
S. pyogenes MefA MFS ? 14,15-M Tn +++<br />
S. pneumoniae MefE MFS ? 14,15-M Tn ++<br />
S. pneumoniae ? MFS ? 14,15-M, strept.B Ch ? ?<br />
S. pneumoniae PmrA MFS ? Cip, Nor, BET Ch ?<br />
14,15-M: 14 et 15-macrolides ; Strept.B: streptogramin B ; Cip: ciprofloxacin; Nor: norfloxacin; BET: Ethidium<br />
20
PmrA-mediated resistance in S. pneumoniae<br />
Antibiotics Wild type PmrA++<br />
Norfloxacin 2 16<br />
Norfloxacin + reserpin 2 4<br />
Ciprofloxacin 0.5 2<br />
Moxifloxacin 0.12 0.12<br />
Sparfloxacin 0.25 0.25<br />
Acriflavin 4 16<br />
Ethidium bromide 2 16<br />
21<br />
Gill, M. J. Antimicrob. Agents Chemother. 1999, 43: 187
Combinaison of mechanisms in S. pneumoniae<br />
Strains GyrA ParC ParE Efflux Cip Levo Trova Moxi<br />
S10B4 - - - + 1 1 0.25 0.25<br />
S10A6 - - I460V - 1 1 0.25 0.125<br />
S7A2 - - I460V + 2 1 0.25 0.125<br />
S7B7 - K137N - + 2 1 0.5 0.25<br />
S7C2 - S79F I460V + 4 2 0.5 0.25<br />
S9E9 - K137N I460V + 16 4 0.5 0.25<br />
S10D9 S81F K137N D435N + 16 16 1 1<br />
I460V<br />
S7E1 S81F K137N I460V + 32 16 32 4<br />
22<br />
Ho, P. L. J. Antimicrob. Chemother. 2001, 47: 655
Other Gram positives<br />
Species System Family Substrates Genes Fqcy<br />
B. subtilis Bmr MFS Cmp, Fq, Org. Ch ?<br />
B. subtilis Blt MFS Cmp, Fq, Org. Ch ?<br />
B. subtilis Bmr3 MFS Oflox, lévo, Org. Ch ? ?<br />
Streptomyces sp Cml MFS Cmp Ch +++<br />
Streptomyces sp Ptr MFS Pristina, Rif Ch +++<br />
Cmp: chloramphenicol ; Fq: fluoroquinolones ; Organic cations: acriflavine, cetyltrimethylammonium,<br />
ethidium, triphenylphosphonium, rhodamine ; Oflox.: ofloxacin ; Levo.: levofloxacin ; Pristina: pristinamycins I-<br />
II ; Rif: rifampicin.<br />
23
Chromosomal genes<br />
Efflux systems in E. coli<br />
– 37 putative drug transporters: 19 MFS, 3 SMR, 7 RND, 7 ABC,<br />
1 MATE<br />
– 20 pumps are able to transport toxic/antibiotic molecules<br />
– 15-17 pumps may provide with some resistance to antibiotics when<br />
overproduced from cloned genes (Nishino K et al. J. Bacteriol. 2001)<br />
– Most of these intrinsic systems are not expressed in standard<br />
laboratory growth conditions<br />
– Spontaneous mutations may result in stable overproduction of a<br />
single pump and resistance<br />
Foreign genes<br />
– Genes carried by mobile elements (plasmids, transposons)<br />
24
25<br />
Efflux pumps coded by mobile genetic elements<br />
Species System Family Substrates<br />
E. coli TetA/B/E MFS Tc, Min Tig<br />
E. coli CmlA MFS Cmp<br />
E. coli Flo MFS Cmp, Flo<br />
E. coli OqxAB-TolC RND Olaquindox, Cmp<br />
Tc: tetracycline; Min: minocycline; Cmp: chloramphenicol; Flo: florfenicol ; Tig: tigecycline
26<br />
Efflux pumps of MFS, MATE, SMR, or ABC family<br />
Species System Family Substrates Genes<br />
E. coli EmrAB-TolC MFS Nal C<br />
E. coli Bcr MFS Tc, Km, Fos C<br />
E. coli MdfA MFS Tc, Rif, Cmp, Ery, Neo, Fq... C<br />
E. coli MdtG MFS Fos C<br />
E. coli MdtH MFS Fq C<br />
E. coli MdtL MFS Cmp C<br />
E. coli MdtM MFS Cmp, Fq C<br />
E. coli NorE MATE Cmp, Fq, Fos, Tmp C<br />
E. coli EmrE SMR Tc C<br />
E. coli MdtJK SMR Nal, Fos C<br />
E. coli MacAB-TolC ABC Ery C<br />
Nal: nalidixic acid; Tc: tetracycline + glycylcyclines; Km: kanamycin; Fos: fosfomycin; Rif: rifampicin;<br />
Cmp: chloramphenicol; Ery: erythromycin; Neo: neomycin; Fq: fluoroquinolones; Tmp: trimethoprim
Efflux pumps of the RND family<br />
Bacteria System Substrates<br />
E. coli AcrAB-TolC 1 Fq, ß-lactams 3 , Tc, Cmp, Nov, Ery, Fus, Rif…<br />
E. coli AcrEF-TolC 2 Fq, ß-lactams 3 , Tc, Cmp, Nov, Ery, Fus, Rif…<br />
E. coli AcrD 2 -AcrA-TolC AGs, Ery, PolyB<br />
E. coli CusAB-? 2 Fos<br />
E. coli MdtABC-TolC 2 Fq<br />
E. coli MdtEF-TolC 2 Ery<br />
P. aeruginosa MexAB-OprM 1 Fq, ß-lactams 1 , Tc, Cmp, Nov, Ery, Fus, Tm...<br />
N. gonorrhoeae MtrCDE 1 Tc, Cmp, ß-lactams 1 , Ery, Fus, Rif...<br />
Fq: (fluoro)quinolones; Tc: tetracycline; Cmp: chloramphenicol; Nov: novobiocin; Ery: erythromycin; Fus:<br />
fusidic acid; Rif: rifampicin; AGs: aminoglycosides; PolyB: polymyxin B; Fos: fosfomycin; Tmp: trimethoprim;<br />
3 rd GC: cefepime, cefpirome. 1 expressed constitutively in wild type cells, 2 inducible expression, 3 except imipenem.<br />
27
28<br />
Overexpression of acrAB and mtrCDE operons<br />
E. coli<br />
N. gonorrhoeae<br />
acrR<br />
mtrR<br />
-<br />
-<br />
acrA<br />
+<br />
+<br />
MarA<br />
acrB<br />
MtrA<br />
mtrC mtrD<br />
mtrE<br />
_ (MppA)<br />
MarR<br />
_<br />
SoxS SoxR<br />
mutations mdr
System AcrAB-TolC in E. coli<br />
Antibiotics wild type AcrAB ++ AcrAB -<br />
Nalidixic acid 4 - 6 8.5 - 32 0.6<br />
Norfloxacin 0.025 - 0.1 0.3 - 1.25 nd<br />
Ofloxacin 0.06 - 0.07 0.25 - 0.3 nd<br />
Ciprofloxacin 0.02 0.15 nd<br />
Ampicillin 2 - 4 5 - 6 0.6 - 2<br />
Erythromycin 128 - 256 > 512 < 2 - 8<br />
Tetracycline 1.25 - 3 5 - 16 0.25 - 0.3<br />
Chloramphenicol 4 - 7.5 10 - 28 0.6<br />
contribution to intrinsic resistance : CMI x 2-64<br />
acquired resistance : CMI x 2-12<br />
29<br />
(CMI mg/l)
Efflux/target double mutants of E. coli<br />
Genotype/Phenotype Oflo Cipro<br />
wild type AG100 0.03 ≤0.015<br />
AcrAB ++ 0.125 0.06<br />
gyrA (Asp87->Gly) 0.25 0.25<br />
gyrA (Asp87->Gly; Ser83->Leu) 4 2<br />
gyrA (Asp87->Gly), AcrAB ++ 8 4<br />
gyrA (Asp87->Gly), AcrAB - 0.06 0.03<br />
Oethinger et al. Antimicrob. Agents Chemother. 2000, 44: 10-13<br />
30
Induction of acrAB-tolC expression<br />
tetracycline<br />
chloramphenicol<br />
(acetyl)salicylate<br />
benzoate<br />
stress...<br />
marROAB<br />
Mar regulon :<br />
∇ Porin OmpF<br />
Δ TolC<br />
Δ AcrAB<br />
Δ EmrAB<br />
Δ∇Other proteins<br />
SoxSR oxidative stress<br />
Rob bile salts<br />
31
Systems MtrCDE and FarAB in N. gonorrhoeae<br />
Antibiotics wild type CDE ++ CDE - FarAB -<br />
Penicillin G 0.008 0.032 0.008 nd<br />
Erythromycin 0.25 1 - 2 0.06 0.25<br />
Tetracycline 0.25 0.5 nd nd<br />
Rifampicin 0.06 0.25 0.015 nd<br />
Linoleic acid 1600 nd 25 - 50 50<br />
Palmitic acid 100 nd 12.5 12.5<br />
contribution to intrinsic resistance : CMI x 4-64<br />
acquired resistance : CMI x 4-8<br />
32<br />
(CMI mg/l)
RND efflux systems in P. aeruginosa<br />
System Operon Substrates<br />
MexAB-OprM mexAB,oprM FQ, ß-lactam, Tmp, Cmp, Tet, Nov, Ery...<br />
MexXY (OprM) mexXY FQ, AG, Fep, Cpo, Tet, Ery...<br />
MexCD-OprJ mexCD,oprJ FQ, Cpo ,Fep, Tmp, Cmp, Tet, Ery...<br />
MexEF-OprN mexEF,oprN FQ, (Ipm), Tmp, Cmp...<br />
MexGHI-OpmD mexGHI,opmD FQ...<br />
MexJK (OprM) mexJK Tet, Ery...<br />
MexVW (OprM) mexVW FQ, Cmp, Tet, Ery...<br />
Fq: fluoroquinolones; ß-lactam (except imipenem); Tmp: trimethoprime; Cmp: chloramphenicol; Tet: tetracycline;<br />
Nov: novobiocin; Ery: erythromycin; AG: aminoglycosides; Fep: cefepime; Cpo: cefpirome; Ipm: imipenem.<br />
33
Contribution to intrinsic resistance in P. aeruginosa<br />
Antibiotcs Wild type MexAB/M - MexXY/M -<br />
Norfloxacin 0.125 - 1 0.05 - 0.25 -<br />
Ciprofloxacin 0.03 - 0.25 0.012 - 0.03 -<br />
Carbenicillin 12.5 - 64 0.4 - 1 -<br />
Ceftazidime 0.4 - 2 0.2 - 0.4 -<br />
Cefepime 0.8 - 2 0.1 - 0.5 -<br />
Meropenem 0.2 - 0.5 0.1 - 0.2 -<br />
Tetracycline 6.25 - 16 2 2 - 4<br />
Chloramphenicol 12.5 - 32 0.8 - 2 -<br />
Erythromycin 256 64 - 128 32 - 64<br />
Tobramycin 0.5 - 0.125<br />
Amikacin 2 - 0.5<br />
CMI x 2-64<br />
CMI x 2-8<br />
34<br />
(CMI mg/l)
Acquired resistance in P. aeruginosa<br />
Antibiotics Wild type MexAB/M MexCD/J MexEF/N MexXY/M<br />
Carbenicillin 8 - 32 64 - 256<br />
Aztreonam 2 - 4 12.5 - 32<br />
Ceftazidime 0.4 - 2 1.6 - 8<br />
Cefepime 1 3 - 4 12.5 8<br />
Cefpirom 1 - 2 4 - 8 8 - 16<br />
Imipenem 0.8 - 1 6.25 - 8<br />
Meropenem 0.2 - 0.5 0.8 - 2<br />
Ciprofloxacin 0.03 - 0.125 0.4 - 1 0.8 - 1.6 0.8 - 1.6 0.5 - 1<br />
Amikacin 2 4 - 16<br />
Tobramycin 0.25 - 0.5 1 - 2<br />
35<br />
(CMI mg/l)
Genetic events leading to increased efflux<br />
IS<br />
mdr mutations<br />
mexR<br />
mexZ<br />
PA3721<br />
nalC<br />
-<br />
-<br />
nalB<br />
-<br />
agrZ<br />
PA3720 PA3719<br />
_<br />
-<br />
mexA mexB oprM<br />
+<br />
mexX<br />
mexY<br />
PA5471<br />
_<br />
PA3574<br />
nalD<br />
MexXY<br />
MexAB-OprM<br />
agrW<br />
C. Vogne et al. Antimicrob. Agents Chemother. 2004, 48: 1676<br />
C. Llanes et al. Antimicrob. Agents Chemother. 2004, 48: 1797
MexXY-mediated adaptive resistance to AGs<br />
MIC (mg / L)<br />
50 _<br />
_<br />
40 _<br />
30 _<br />
20 _<br />
10 _<br />
Stain ATCC 27853 exposed to 1 MIC amikacin for 2h every 8h (Karlowsky 1994)<br />
MIC of amikacin; Bacterial killing in log10<br />
_<br />
_<br />
_<br />
_<br />
_<br />
2.5<br />
2<br />
1.5<br />
1<br />
0.5<br />
Inoculum (log10 CFU)<br />
D. Hocquet et al. Antimicrob. Agents Chemother. 2003, 47: 1371
Target/efflux double mutants in P. aeruginosa<br />
MIC levofloxacin (mg/L)<br />
Target mutations Wild-type MexAB ++ MexAB ++<br />
+ inh. 10 mg/l<br />
Aucune 0.25 2 0.03<br />
gyrA (Thr83->Ile) 2 8 0.5<br />
gyrA (Thr83->Ile) + parC (Ser87->Leu) 4 32 2<br />
gyrA (Thr83->Ile + Asp87->Tyr) + parC (Ser87->Leu) 16 128 8<br />
38<br />
Lomovskaya et al. Antimicrob. Agents Chemother. 1999, 43: 1340<br />
Lomovskaya et al. ICAAC Toronto 1999, abstract F-1264
Therapeutic implications of efflux systems<br />
Resistance levels conferred by intrinsic pumps<br />
– Low to moderate drug resistance (MIC x 2 - 16)<br />
– Clinical significance<br />
♦ Lack of clinical data !<br />
♦ Poor response to treatment when the concentrations of<br />
antibiotics are low at the infection site (insufficient dosage,<br />
inappropriate drug, abcess...)<br />
♦ Increased emergence of target mutants ?<br />
Emergence of efflux mutants under treatment<br />
– Cross resistance to structurally unrelated molecules<br />
– Role of fluoroquinolones<br />
39
How to characterize efflux mechanisms<br />
Plasmid or transposon encoded efflux systems<br />
– Multiresistance phenotype<br />
– Detection of efflux gene(s): PCR, nucleic probes<br />
Upregulation of intrinsic efflux systems<br />
– Protein levels<br />
♦ Western blotting of membrane extracts with specific antibodies<br />
– mRNA levels<br />
♦ Northern blot, MacroArray, MicroArray<br />
♦ Real Time RT-PCR (Light Cycler, Taq Man, I Cycler…)<br />
– Intracellular accumulation of antibiotics<br />
♦ [ 3 H] ou [ 14 C] radiolabeled or fluorescent compounds (BET,<br />
acriflavine…)<br />
– Sequencing of regulatory genes<br />
40
Efflux inhibitors<br />
Phenyl-Arginyl ß N-naphtylamide<br />
41