14-2-2012 Tours-INRA 1

Antibiotic efflux: from discovery to Antibiotic efflux: from discovery to epidemiological, clinical, and veterinary impactepidemiological, clinical, and veterinary impact

Françoise

Van Bambeke & Paul M. Tulkens

Pharmacologie cellulaire et moléculaireLouvain Drug Research

InstituteUniversité

catholique de Louvain, Brussels, Belgium

14-2-2012 Tours-INRA 2

DiscoveryDiscovery

and and significancesignificance

……..

14-2-2012 Tours-INRA 3

Chemotherapeutic

agents exert

toxic

effects on specific

target

cells

antifungals

anticancer

agents

antibiotics

14-2-2012 Tours-INRA 4

Chemotherapeutic

agents exert

toxic

effects on specific

target

cells

How can

these

drugsreach

their

target

inside

the cells

?

nucleic

acids

ribosomes

enzymes

Van Bambeke

et al. (2000) Biochem. Pharmacol. 60:457-70

14-2-2012 Tours-INRA 5

Reaching

an intracellular

target

…

polar drug

lipophilic

drug

physico-chemical properties are inadequate for reaching an intracellular target !

Van Bambeke

et al. (2000) Biochem. Pharmacol. 60:457-70

14-2-2012 Tours-INRA 6

Reaching

an intracellular

target

…

amphipathic

drug

most drugs are amphipathic

by design,to be able to cross membrane barriers !

Van Bambeke

et al. (2000) Biochem. Pharmacol. 60:457-70

14-2-2012 Tours-INRA 7

Intracellular

chemotherapeutic

agents

But a diffusible compound may have

potentially harmful effects !Van Bambeke

et al. (2000) Biochem. Pharmacol. 60:457-70

14-2-2012 Tours-INRA 8

Why

efflux transporters

?

Extrusion by efflux pumps

Van Bambeke

et al. (2000) Biochem. Pharmacol. 60:457-70

14-2-2012 Tours-INRA 9

Why

efflux transporters

?

Extrusion by efflux pumps

general mean of protectionagainst cell invasion by diffusible molecules

Van Bambeke

et al. (2000) Biochem. Pharmacol. 60:457-70

14-2-2012 Tours-INRA 10

Most antibiotics

are amphiphilic

!

macrolide tetracycline

fluoroquinolone

rifampicin

sulfamide

lincosamide

+

+

++

+

+cationic amphiphiles

Van Bambeke

et al. (2000) Biochem. Pharmacol. 60:457-70

14-2-2012 Tours-INRA 11

Most antibiotics

are amphiphilic

!

fluoroquinolone

-lactam

fusidic

acid

-

-

-

anionic

amphiphiles

Van Bambeke

et al. (2000) Biochem. Pharmacol. 60:457-70

14-2-2012 Tours-INRA 12

Antibiotic

efflux transporters

are ubiquituous

spectrumprokaryotes

narrow

broad

MATE SMR

MFS

ABC

Gram (+) Gram (-)

RND

distribution

eukaryotes

Mesaros

et al. (2005) Louvain médical. 124:308-20

14-2-2012 Tours-INRA 13

Antibiotics

as substrates

of efflux pumps

Antibiotic bacteria

fungi

superiorclass Gram (+) Gram(-)

eucaryotes

-lactamsfusidic

acidmacrolidesstreptograminstetracyclinesaminoglycosideschloramphenicolrifamycinssulfamidestrimethoprimfluoroquinolones

Van Bambeke

et al. (2000) Biochem. Pharmacol. 60:457-70

14-2-2012 Tours-INRA 14

Antibiotic

efflux and resistance: time line …

tetracy

clines

1980

1990

2000

macro

lides

fluoroq

uinolo

nes

Beta-la

ctame

s

amino

glycos

ides

chlora

mphen

icol

rifamp

inoxa

zolidin

ones

14-2-2012 Tours-INRA 15

Efflux as a mechanism

of export in antibiotic

producers

spectrum

narrow

broad

specific

for the produced

antibiotic

ABC MFS

Ptr

of S. pristinaespiralis pristinamycinLmrA

of S. lincolnensis lincomycinRifP

of A. mediterranei rifampicin

OleB

or C of S. antibioticus oleandomycinSmB

of S. ambofaciens macrolidesTlrc

of S. fradiae tylosin

14-2-2012 Tours-INRA 16

Efflux as a mechanism

of resistance in Gram-positive bacteria

spectrum

narrow

broad

specific

for one (or a few) families

of drugs

ABC MFS

NorA

of S. aureus FQ,Tet, chlMefE

of S. pneumoniae MLPmrA

of S. pneumoniae FQMefA

of S. pyogenes ML

PatA/PatB

of S. pneumoniae FQ, chlMsrA

of S. epidermidis erythromycin

14-2-2012 Tours-INRA 17

FQ efflux pumps

in S. pneumoniae

Terry et al., Nature Reviews Microbiology

2005; 3:

566-572

Primary

transporters«

ATP-Binding

Cassette

»

Secondary

transporters(Proton motive force)

PmrAGill et al, AAC 1999; 43:187-9

PatA/PatBMarrer et al, AAC 2006; 50:685-93

14-2-2012 Tours-INRA 18

Efflux as a mechanism

of resistance in Gram-negative

bacteria

spectrum

narrow

broad

broad

spectrum, conferring

cross-resistance

MFS

RNDMexAB-OprM

of P. aeruginosa -lac, FQ,Tet, ML, chl, rif, sulfAcrAB-TolC

of E. coli -lac,

FQ,Tet, ML, chl, rif, sulf

TetA

of E. coliTetMsrA

of S. epidermidis erythromycin

specific

for one (or a few) families

of drugs

14-2-2012 Tours-INRA 19

Efflux and resistance in P. aeruginosa

Mesaros

et al. (2005) Louvain médical. 124:308-20

MexB

MexA

OprM

MexY

MexX

OprM

MexD

MexC

OprJ

MexF

MexE

OprN

Constitutive

basal expressionoverexpressed

upon

induction

No basal expression;expression

upon

induction

14-2-2012 Tours-INRA 20

RoleRole

of of antibioticantibiotic

efflux efflux in in epidemiologyepidemiology

and and resistanceresistance

……..

14-2-2012 Tours-INRA 21

Does

efflux mean

«

resistance

»

vs. epidemiological

breakpoints

?

MICs

vs breakpoints

for 183 S. pneumoniae

isolated

from

CAP

Lismond

et al., JAC (2011) 66:948-951

•

Efflux (+) strains

consideredas susceptible

•

FQ with

high

intrinsic

activity

can

be

substrates

for efflux !

14-2-2012 Tours-INRA 22

Does

efflux mean

«

resistance

»

vs. epidemiological

breakpoints

?

MICs

vs EUCAST breakpoints

for 109 P. aeruginosa

without

or with

efflux mechanisms,

isolated

from

ICU patients (VAP)

Riou

et al, ECCMID 2010

meropenem

mexA < 2 mexA 20.03125

0.06250.125

0.250.5

1248

163264

128256

mexA expression level

amikacin

mexX < 5 mexX 50.25

0.51248

163264

128256

mexX expression level

MIC

pip-tazo

mexA < 2 mexA 20.03125

0.06250.125

0.250.5

1248

163264

128256

mexA expression level

MexXsubs

tr.

MexAsubs

tr.

MexAsubs

tr.

14-2-2012 Tours-INRA 23

Efflux and intrinsic

resistance

Is Pseudomonas

«

intrinsically

»

resistant

to temocillin

?

Buyck

et al, J. Antimicrob. Chemother. (2012) 67(3):771-5

MexAB-OprM

mutants are highly

susceptible!

14-2-2012 Tours-INRA 24

Efflux and intrinsic

resistance

Is Pseudomonas

«

intrinsically

»

resistant

to macrolides ?

Buyck

et al, ICAAC (2011)

0.25 0.5 1 2 4 8 16 32 64 128

256

512

1024

0

25

50

75

100MHB-OprM(+)MHB-OprM(-)

MIC (mg/L)

cum

ulat

ive

perc

enta

ge

Major role

of constitutively-expressed

transporters!

14-2-2012 Tours-INRA 25

RoleRole

of efflux of efflux pumpspumps

in the in the clinicsclinics

……

14-2-2012 Tours-INRA 26

FQ resistance

by efflux in S. pneumoniae is

mediated

by PatA/PatB

FQstrains

CIP MXF-Res +Res -R +R

49619 1 0.5 0.125 0.125

patA 0.5 0.5 0.125 0.125

patB 0.5 0.5 0.125 0.125

pmrA 1 0.5 0.125 0.125

SP335 32 2 0.5 0.25

patA 1 0.5 0.125 0.125

patB 1 0.5 0.125 0.125

pmrA 8 0.5 0.25 0.125

CIP affected

but not MXF !

14-2-2012 Tours-INRA 27

FQ induce

efflux, whether

substrates

or not

Both

CIP and MXF are inducers

!

El Garch

et al., JAC

(2010)

65:2076-82

14-2-2012 Tours-INRA 28

Selection

of resistance

in S. pneumoniae by subMIC

concentrations of FQ

ciprofloxacin moxifloxacin

Avrain

et al, JAC (2007) 60:965-972

no efflux

basal efflux

Ciprofloxacin

selects for resistance

by efflux

14-2-2012 Tours-INRA 29

Selection

of resistance

in S. pneumoniae by subMIC

concentrations of FQ

moxifloxacin

Avrain

et al, JAC (2007) 60:965-972

no efflux

basal efflux

Moxifloxacin

selects for resistance

by target

mutation(topoisomerases)

GyrA

(Ser81Phe)ParC

(Ser79Tyr)

ParE

(Ile460Val)ParE

(Arg447Cys)

14-2-2012 Tours-INRA 30

Efflux in

S. pneumoniae: is

it

important in the clinics

?

Suspected

efflux based

on phenotypic

analysis

(CIP MIC +/-

reserpine)

CAP BPCO0

20

40

60

80

100 1 2 2

reserpine effect on MIC (x dilutions)

origin of isolates

% s

trai

ns

Lismond

& Degives, unpublished

acute pathology

«

one shot

»antibiotic

exposure

chronic

pathology

repetitiveantibiotic

exposures

183 strains 107 strains

14-2-2012 Tours-INRA 31

Efflux in

P. aeruginosa: is it important in the clinics ?

Prevalence of MexA

and MexX

overexpressers

in 62 phylogentically-related pairs of P. aeruginosa

isolated from ICU patients (VAP)

DAY x (%)

38.71%

22.58%

20.97%

17.74%

DAY 0 (%)

66.13%

12.90%

11.29%

9.68%

MexA-/MexX-

MexA+/MexX-

MexX+/MexA-

MexA+/MexX+

Riou

et al, ECCMID 2010

14-2-2012 Tours-INRA 32

Efflux selection in

P. aeruginosa

during treatment

Riou

et al, ECCMID 2010

Antibiotic no. patients

Piperacillin-tazobactam

(TZP) 26Amikacin

(AMK) 22Meropenem

(MEM) 20Cefepime

(CEF) 19Ciprofloxacin (CIP) 6

Antipseudomonal

antibiotics received by the patients during

treatment

DAY 0 DAY X05

1015

202530

3540

455055

60

0

234

1

nb effluxsystems

num

ber o

f str

ains

global influence of treatment

number of efflux systems detected at day 0 and day X

69% com

binations

14-2-2012 Tours-INRA 33

And in And in veterinaryveterinary

practice ?practice ?

14-2-2012 Tours-INRA 34

Efflux and resistance

Efflux involved

in intrinsic

resistance

and low

level

resistance

14-2-2012 Tours-INRA 35

Risks

of cross-contamination!

Efflux system

14-2-2012 Tours-INRA 36

Antibiotics

as inhibitors

of efflux

Tetracyclines

as inhibitors

of efflux

14-2-2012 Tours-INRA 37

Efflux and Efflux and bacteriabacteria

in in ourour

team team ……

www.facm.ucl.ac.be

GRAM(+)

GRAM(-)

L. Avrain M. Riou J. Buyck A. Anantharajah

F. el Garch C. Vallet A. Lismond F. Degives

V. Mohymont

14-2-2012 Tours-INRA 38

Les aventures dLes aventures d’’un Tourangeauun Tourangeau àà

Bruxelles Bruxelles ……

Appliqué

à

suivre les travaux pratiques des étudiants …

… et à

tester la gastronomie locale !

Antibiotic efflux: from discovery to �epidemiological, clinical, and veterinary impactDiscovery and significance ….Chemotherapeutic agents exert toxic effects�on specific target cellsChemotherapeutic agents exert toxic effects�on specific target cellsReaching an intracellular target …Reaching an intracellular target …Intracellular chemotherapeutic agentsWhy efflux transporters ?Why efflux transporters ?Most antibiotics are amphiphilic !Most antibiotics are amphiphilic !Antibiotic efflux transporters are ubiquituousAntibiotics as substrates of efflux pumpsAntibiotic efflux and resistance: time line …Efflux as a mechanism of export �in antibiotic producersEfflux as a mechanism of resistance�in Gram-positive bacteriaFQ efflux pumps in S. pneumoniaeEfflux as a mechanism of resistance �in Gram-negative bacteriaEfflux and resistance in P. aeruginosaRole of antibiotic efflux �in epidemiology and resistance ….Does efflux mean « resistance » vs. epidemiological breakpoints ?Does efflux mean « resistance » vs. epidemiological breakpoints ?Efflux and intrinsic resistanceEfflux and intrinsic resistanceRole of efflux pumps in the clinics …FQ resistance by efflux in S. pneumoniae �is mediated by PatA/PatB FQ induce efflux, whether substrates or not Selection of resistance in S. pneumoniae �by subMIC concentrations of FQSelection of resistance in S. pneumoniae �by subMIC concentrations of FQEfflux in S. pneumoniae: �is it important in the clinics ?Efflux in P. aeruginosa:�is it important in the clinics ?Efflux selection in P. aeruginosa during treatmentAnd in veterinary practice ?Efflux and resistanceRisks of cross-contamination!Antibiotics as inhibitors of effluxEfflux and bacteria in our team … Les aventures d’un Tourangeau�à Bruxelles …