beta-lactam antibiotics in pseudomonas aeruginosa · 1. activity of beta-lactam antibiotics against...

Impact of efflux mechanisms on susceptibility and resistance to

beta-lactam antibiotics in Pseudomonas aeruginosa

beyond the usual concepts

1

Houssein Abdel Aziz Chalhoub

Promoter Prof Franccediloise Van Bambeke

Cellular and Molecular Pharmacology

Louvain Drug Research Institute

Universiteacute catholique de Louvain Brussels Belgium

22 December 2016

2

Bacteria amp Humans

3

Bacteria spread widely in the environmenthellip

Picture of Everest from Luca Galuzzi

4


httpwwwearthtimesorgscitechbillion-years-old-bacterial-enzyme1578

5

ldquoThey are so small that you canrsquot seen them without a microscope but they are therehelliprdquo

httpdlb-networkcomphotographyomnpseudomonas-aeruginosa-gram-stain

Bacteria

6

Bacterial infections

Modeling Pseudomonas aeruginosa pathogenesis

in plant hosts doi101038nprot2008224

7

Antibiotic resistance post-antibiotic era hellip

8httpdavidjerniganblogspotbe201608preparing-body-for-probiotic-success_2html

Antibiotic resistance microbial warhellip

9

Antibiotic resistance revenge of the microbes

Clinical use

Penicillin 1943

Sir Alexander FlemingAlfred EisenstaedtmdashTime amp Life PicturesGetty Images

10


Clinical use RESISTANCE

Penicillin 1943 1945 (2 years)


11




Vancomycin 1972 1988 (16 years)

12





Imipenem 1985 1998 (13 years)

13






Daptomycin 2003 2004 (1 year)

14







Ceftaroline 2010 2011 (1 year)







15


httpxombitcom201302nadal-magnus-demoledor-liftado

16

Pseudomonas aeruginosa

httpeditioncnncom20150225healthdeadly-bacteria-doctors-offices

US Centers for Disease Control and Prevention - Medical Illustrator - James Archer

1-5 microm

17

Pseudomonas aeruginosa moving by the use of flagellahellip

Fromhttpswwwyoutubecomwatchv=a_5FToP_mMYA clip from the NOVA production Judgment Day

18

Pseudomonas aeruginosa takes advantage of a sick personhellip

httpwwwhealthcarea2zorgword

HIV Cancer patients receiving

chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19

httpwwwbbccomnewshealth-32755065

realheroesbecomeangelsblogspotcom

Build-up of thick mucus

Opportunistic pathogens

Lung infections in Cystic Fibrosis patients

20




Classes of antipseudomonal antibiotics

21

β-lactams

Picture from httpsenwikipediaorgwikiIntravenous_therapy

Aminoglycosides

PolymyxinsQuinolones


22Pictures from httpwwwrealmefr720-porta-neb-compresseur-nebuliseur-pneumatique-d-aerosoltherapiehtml

httpwwwinhalationseexplorepreciseinhale-and-you

Aminoglycosides(Tobramycin)

Polymyxins(colistin)

Quinolones(Levofloxacin)

Aztreonam


23Picture from httpwwwdailymailcoukhealtharticle-3430291

Quinolones

24




Multidrug-ResistantPseudomonas aeruginosa


25

β-lactams

PenicillinsTicarcillin clavulanic acidPiperacillin Tazobactam

Carbapenems MeropenemImipenem

Cephalosporins Ceftazidime cefepime

Monobactam Aztreonam


P aeruginosa and resistance mechanisms to β-lactam antibiotics

Downregulating mutating porins [OprD]β-lactams (imipenem meropenemhellip)

26Adapted from Lister et al Clin Microbiol Rev 2009 22 582-610httpwwwbellybellycomaubabybaby-hunger-cueshttpthestircafemomcombig_kid181864expired_medicine_dates_still_safe

Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27

Active efflux [MexAB-OprM MexXY-OprM MexEF-OprN MexCD-OprJ]β-lactams (ticarcillin meropenemhellip)

Adapted from Lister et al Clin Microbiol Rev 2009 22 582-610Clin Microbiol Rev 2006 Apr19(2)382-402

Extended-Spectrum-β-Lactamases ceftazidime

Cephalosporinase AmpC ceftazidime

Carbapenemase meropenem ceftazidime

Adapted from Lister et al Clin Microbiol Rev 2009 22 582-610 28

β-lactamases antibiotic degradation


β-lactamases (cephalosporinases AmpC-typecarbapenemases ESBLs)

OprM

MexA

MexB

Downregulating mutating porins [OprD]

29

Antibiotic

Active efflux [MexAB-OprM MexXY-OprM MexEF-OprN MexCD-OprJ]




30

Hussein Abdel Aziz Chalhoub





22 December 2016

1 Activity of beta-lactam antibiotics against Pseudomonas

aeruginosa from patients with cystic fibrosis

2 Role of efflux versus other resistance mechanisms against

a Meropenem (carbapenems)

b Ceftazidime avibactam (new β-lactamases inhibitor)

c Temocillin (old beta-lactam antibiotic)

Main objectives of the thesis

31

International collection of P aeruginosa from patients with cystic fibrosis chronically

exposed to different antipseudomonal agents

Bacterial isolates 333 clinically relevant isolates were obtained from

Dr Michael Tunney (Queenrsquos University of Belfast UK) n=99

Drs Anne Vergison Olivier Denis (Hocircpital Erasme Brussels Belgium) n=88

Prof Patrick Pleacutesiat (CHRU Besanccedilon Besanccedilon France) n=80

Prof Barbara Kahl (University of Muumlnster Muumlnster Germany) n=68

Source of isolates

32Pictures from httpwwwbbccomnewshealth-32755065httpwwwmirrorcouknewstechnology-sciencescienceuk-scientists-developing-cystic-fibrosis-5992360

33

Antibacterial activity of beta-lactams against P aeruginosa

from cystic fibrosis patients

TicarcillinPiperacillin + Tazobactam Ceftazidime Imipenem Meropenem

Breakpoint (mgL)

16 16 8 8 8

Related publication Mustafa Chalhoub et al Antimicrob Agents Chemother 2016 606735ndash41

34




Breakpoint (mgL)

16 16 8 8 8

A microorganism is defined as susceptible or resistant by alevel of antimicrobial activity associated with a highlikelihood of therapeutic success or therapeutic failurerespectively


35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2

Paeruginosa (n = 333)

MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8

Susceptible 18 32 36 51 49

Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16

Summary Imipenem and meropenem were the most active antibioticshellip


37

Meropenem

(β-lactam)

Stable to AmpC and ESBL but not carbapenemases

Carbapenemases

38

High-level resistance to meroperem

Pseudomonas(cystic fibrosis patients)

Meropenem MIC = 64-128 mgL

Related publications Chalhoub et al Intern J Antimicrob Ag in press

39



Pseudomonas (Hospital acquired pneumonia)

Meropenem MIC = 128 mgL


Comparison with Pseudomonas from hospital acquired pneumonia


40





Cystic fibrosis Hospital acquired pneumonia

AmpC-type cephalosporinases XESBL(s)

Carbapenemase(s) X




Adapted from Jason Sello Brown university 2011

Efflux inhibitorPAβN

Phenylalanine-

arginine β-

naphthylamide

Meropenem + efflux inhibitor in Pseudomonas

41

42





- PA N + PA N - PA N + PA N

2

4

8

1 6

3 2

6 4

1 2 8

2 5 6

s t r a i n s f r o m

C F p a t i e n t s

c a r b a p e n e m a s e s ( - )


P n e u m o n i a p a t i e n t s

C a r b a p e n e m a s e s ( + )

ME

M

MI

C

(m

g

L)






43



- PAN + PAN - PAN + PAN

2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44



+ - clinical isolates

Sequencing OprD genes confirmed these results (premature stop mutations)

OprD porin







45

1- [Efflux + porin -] are enough to confer high-

level resistance to meropenem


46

High-level resistance to ceftazidime



Carbapenemase(s) X

Related publication Chalhoub et al J Antimicrob Chemother 2015701596-8

47

AmpC cephalosporinases

inhibitor avibactam


48

Ceftazidimeavibactam activity in the whole collection

of isolates (n = 333)

Susceptible isolates to ceftazidime

increased from 36 to 76 after addition

of avibactam [4mgL]

Promising results

for cystic fibrosis patients


49

24 resistance

to this new combination




50

The mechanism of resistance to ceftazidimeavibactam

combination

Control strains

(induced with sub-MIC imipenem)

MIC (mgL)

Ceftazidime

Reference strain PAO1 32

PAO1 overproducing MexXY-OprM 32

PAO1 overproducing MexCD-OprJ 32

(Prepared for submission as a correspondence to the Journal of Antimicrobial Chemotherapy)

Chalhoub et al Poor membrane permeability impedes avibactam activity in Pseudomonas aeruginosa

51


combination

Control strains


MIC (mgL)

Ceftazidime Ceftazidime+Avibactam

Reference strain PAO1 32 0125

PAO1 overproducing MexXY-OprM 32 0125

PAO1 overproducing MexCD-OprJ 32 0125



52


combination

Control strains


MIC (mgL)





PAO1 overproducing MexAB-OprM 32 4

PAO1 overproducing MexEF-OprN 32 2

PAOD1 OprD-mutant 32 4



53

OprD loss MexAB-OprM

MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination

Adapted from Lister et al Clin Microbiol Rev 2009 22 582-610




54

1- [Efflux + porin -] are enough to confer high-level resistance to meropenem

2- [Efflux + porin -] are pre-existing as resistance mechanismsto avibactam which is not used yet in CF patients




55

Is there any antibiotic resistant to beta-lactamases and not affected by efflux systems and OprD porin downregulation




56

Temocillin (β-lactam)

Derived from the class of carboxypenicillins (ticarcillin carbenicillin)

5757

Withdrawn due to lack of activity against wild-type strains of

Pseudomonas aeruginosa Gram-positive organisms and anaerobes

Not used against Pseudomonas

by Beecham Pharmaceuticals in the 1989s

58

Temocillin

H2O+ β-lactamases

Temocillin

Temocillin stable against β-lactamases

Born in the 1989rsquos (UK)

59

Temocillin versus ticarcillin

in the active site of β-lactamases

Adapted from bull Cubist pharmaceuticalsbull A Matagne et al - Biochem J 1993 Aug 1 293(Pt 3) 607ndash611

W1 water molecule

Temocillin

Temocillin amp Efflux in P aeruginosa

Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60

Isogenic strains of PA

MIC (mgL)

temocillin ticarcillin

Wild type PAO1 512 64

PAO1 delta mexAB 2 1

Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61

Natural mutations of these efflux

pumps can be observed

in isolates from patients with

cystic fibrosis

Related publication Buyck et al J

Antimicrob Chemother 2012 67 771-5

Picture from httpwwwbbccomnewshealth-32755065

62

Temocillin considered as inactive against Pseudomonas

showed surprising activity

TIC Temocillin TZP CAZ MeropenemCeftazidime+avibactam

Breakpoint 16 16 16 8 8 8

MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24

proposed breakpoint for temocillin in Belgium 16 mgL

Related publication Chalhoub et al Nature Sci Reports in press

Temocillin + efflux inhibitor in Pseudomonas

63

Activity of temocillin was improved in the

presence of the efflux inhibitor

susceptibility increased from ~30 to 55


64

N-Phenyl-1-

naphthylamine

Efflux speed

Activity of temocillin was improved in the presence of the efflux pump inhibitor

Lowest efflux speed for isolates

with temocillin MICs lt 128mgL

Speed of efflux by MexAB-OprM pumps

Temocillin


Sequencing mexAB genes in P aeruginosa


65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin

httplearngeneticsutaheducontentcellsorganelleshttpsgenedoewordpresscomtagdna



66

Major mutations

-DNA Fragment Deletions

-Stop mutations

Minor mutations

-Synonymous mutations

-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048

(e) MIC vs mutations

Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00

(f) NPN efflux vs mutations

a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)

Mutations in mexAB genes in P aeruginosa as a function of

temocillin activity

67

Efflux speed


68

MexB mutant (truncated + aberrant protein)Temocillin MIC = 8 mgL

MexB mutant (AA substitutions)Temocillin MIC = 256 mgL


Front view Front view Front view

Modelisation mexB mutations in the same

clone isolated from 3 German patients


Truncated MexB and low efflux speed but high temocillin MIC

69

Truncated MexB (M720_Q1046del) temocillin MIC = 128 mgL

StrainsMIC (mgL)

Temocillin Ticarcillin

Wild typedelta mexB

2 1

Clinical isolatedelta mexB

128 32

Wild type 512 128


Influx of temocillin inside Pseudomonas

70

OprD family 19 members

of carboxylate channels

Porin(Front view)

Porin(Extracellular side)

httpsbcbbiochoxacukmemprotmdbetaproteinpdbid3sys

71

Temocillin amp the 19 Gates (OprD porin family) of P aeruginosa


72

Outer membrane carboxylate channels OccDK in P aeruginosa

73

oprD-single mutant of P aeruginosa

OprD porin is not utilized by temocillin

MICs (mgL)

IPMMEMTemocillinStrains

1037256Wild type PA

43256PAoprD


74

Blocking OccK1 in occK2-single mutant of P aeruginosa

OccK1OccK2 porins are involved in the

uptake of temocillin

MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2

Overexpression of OccK1 or OccK2 in porin-deficient Ecoli

76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


Niels Bagge et al Antimicrob Agents Chemother

2004481175-1187

Exopolysaccharides (alginate) from P aeruginosa

Mol Microbiol 2012 May84(4)595-607

78

Exopolysaccharides (alginate-like)


79

J Antimicrob Chemother 1991 Mar27(3)285-93The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginateBolister N1 Basker M Hodges NA Marriott C

80

Exopolysaccharide abundance in cultures

of clinical isolates as a function of temocillin MICs

2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10

temocillin MICs (mgL)

CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced

missense mutation in mexBtruncatedaberrant mexB

missense mutation in mexAtruncatedaberrant mexA

synonymous mutations missense mutation

in mexA and mexB


81

Temocillin resistance is mainly due to active MexAB efflux system

with participation of exopolysaccharides production andor

OccK1OccK2-porin loss

OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM

Summary (temocillin in cystic fibrosis)





82


2- [Efflux + porin -] are pre-existing as resistance mechanisms to avibactam which is not used yet in CF patients

3- Mutations in MexAB-OprM efflux pumps appear as a mechanism of restored susceptibility to temocillin in Pseudomonas

83

Efflux is a key mechanism of resistance for Pseudomonas from CF

patients

Porins are important players if associated with efflux

Bacterial outer membrane is sufficiently strong barrier against β-

lactams

Conclusion

84

Several paralogous efflux and porin genes to accommodate and

extrude different substrates

Pseudomonas invades different territories with enough fitness

and adaptability

Conclusion

85

Temocillin should be included in CF antimicrobial susceptibility testing

Temocillin could be also used as carbapenem sparing agent against

Pseudomonas with OprD porin mutations

Conclusion


86

Check the prevalence of mutations in mexAmexB genes in

Pseudomonas from a recent CF population (20162017)

Determine whether mexAmexB mutations in CF are beneficial for

the activity of temocillin only or could also affect other substrates

General perspectives (research)

87

Develop fast techniques to screen epidemic and risky clones

Prevent intra- and inter-hospital dissemination of epidemic

Pseudomonas clones

Establishment of early appropriate and targeted antimicrobial

therapies

Perspectives (management of CF infections)

88

Acknowledgments

Cystic Fibrosis centres

89

Dr Hector Rodriguez-VillalobosProfessor Mathias Winterhalter

Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments

Associated to the ReacutegionWallonne project

2

Bacteria amp Humans

3



4



5



Bacteria

6




7




9


Clinical use

Penicillin 1943


10





11





12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


3



4



5



Bacteria

6




7




9


Clinical use

Penicillin 1943


10





11





12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


4



5



Bacteria

6




7




9


Clinical use

Penicillin 1943


10





11





12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


5



Bacteria

6




7




9


Clinical use

Penicillin 1943


10





11





12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


6




7




9


Clinical use

Penicillin 1943


10





11





12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


7




9


Clinical use

Penicillin 1943


10





11





12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments




9


Clinical use

Penicillin 1943


10





11





12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


10





11





12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


11





12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


12






13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


13







14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


14














15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments








15



16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


16




1-5 microm

17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


17



18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


18




chemotherapy

Severe burns

Invasive surgery

Low immune function

Cystic Fibrosis

19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


19






20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


20





21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments



21

β-lactams


Aminoglycosides








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments








Aztreonam



Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments




Quinolones

24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


24






25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments



25

β-lactams









Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments





Antibiotic

Outer membrane

Cell membrane


OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments



OprM

MexA

MexB

27










OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments









OprM

MexA

MexB


29

Antibiotic





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments





30






22 December 2016








31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments









31








Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments









Source of isolates


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


33




Breakpoint (mgL)

16 16 8 8 8


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


34




Breakpoint (mgL)

16 16 8 8 8



35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


35



Ticarcillin

Breakpoint (mgL)

16

Susceptible 18

Resistant 82

MIC90 (mgL) gt5121 2 4 8 16 32 64 128

256

512

1024

2048

0

25

50

75

100

gt 51

2


MIC90

Ticarcillin

MICs(mgL)

o

f is

ola

tes

(cum

ula

tive

)


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


36




Breakpoint (mgL)

16 16 8 8 8


Resistant 82 68 64 49 51

MIC90 (mgL) gt512 512 512 32 16



37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


37

Meropenem

(β-lactam)


Carbapenemases

38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


38





39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


39








40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


40







Carbapenemase(s) X






Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments




Phenylalanine-

arginine β-

naphthylamide


41

42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


42






2

4

8

1 6

3 2

6 4

1 2 8

2 5 6


C F p a t i e n t s





ME

M

MI

C

(m

g

L)






43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments




43




2

4

8

16

32

64

128

256

strains from

CF patients

carbapenemases (-)

strains from

HAP patients

Carbapenemases (+)

ME

M M

IC (

mg

L)

MexAB-OprM

MexXY-OprM

MexCD-OprJ

MexAB-OprM

MexXY-OprM

MexCD-OprJ




44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


44





OprD porin







45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments





45




46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


46




Carbapenemase(s) X


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


47


inhibitor avibactam


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


48





of avibactam [4mgL]

Promising results



49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


49

24 resistance





50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


50


combination

Control strains


MIC (mgL)

Ceftazidime






51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


51


combination

Control strains


MIC (mgL)







52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


52


combination

Control strains


MIC (mgL)










53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


53


MexEF-OprN

efflux systems

overexpression

Cephalosporinases


combination





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments





54






55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments





55





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments





56



5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments




5757





58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


58

Temocillin

H2O+ β-lactamases

Temocillin



59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


59




W1 water molecule

Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


Temocillin


Δ mexABTemocillin

In 2012

FACM Group

LDRI Belgium

60


MIC (mgL)




Susceptible

Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


Temocillin

Susceptible


Δ mexAB

In 2012

FACM Group

LDRI Belgium

61




cystic fibrosis




62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


62





MIC90 gt512 gt512 512 512 16 64

Susceptible

18 29 32 36 49 76

Resistant 82 71 68 64 51 24




63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments



63





64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


64

N-Phenyl-1-

naphthylamine

Efflux speed





Temocillin




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments




65

MexA-Linker

MexB-

exporter

protein

OprM-gated porin




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments




66

Major mutations


-Stop mutations

Minor mutations


-Missense mutations


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

2

4

8

16

32

64

128

256

512

1024

2048


Te

mo

cillin

MIC

(m

gL

)

a a a abc c bc

trunca

teda

berra

nt mex

A

trunca

teda

berra

nt mex

B

mis

sense

muta

tions

in m

exA

mis

sense

muta

tions

in m

exB

mis

sense

muta

tions

mex

A+m

exB

synonym

ous m

utatio

nsW

T

-08

-06

-04

-02

00


a a b babc c bc

Vm

ax (

flu

o u

nit

ss

ec)


temocillin activity

67

Efflux speed


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


68









69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments



69


StrainsMIC (mgL)


Wild typedelta mexB

2 1


128 32

Wild type 512 128



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments



70



Porin(Front view)



71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


71



72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


72


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


73



MICs (mgL)


1037256Wild type PA

43256PAoprD


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


74




MICs (mgL)


1037256Wild type PA

1037786PAoccK21


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


75

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


76

TMOIPMMEMMICs (mgL)

40151Empty

vector

4002250225+OprD

20151+OccK1

22151+OccK2

++ OprD

bla

OprD

++ Occk1

bla

Occk1

++ Occk2

bla

Occk2



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments



2004481175-1187



78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


78



79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


79


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


80



2 4 8 16 32 64 128

256

512

1024

0

2

4

6

8

10


CF

W f

luo

res

ce

nce

(rati

o t

o P

AO

1 v

alu

e)

008 005 lt0001 0002 009 002 007 007013 017

not sequenced




in mexA and mexB


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


81




OccK1OccK2porins

Exopolysaccharides

Mex

AB

-Op

rM






82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments





82




83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


83


patients



lactams

Conclusion

84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


84




and adaptability

Conclusion

85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


85




Conclusion


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


86






87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


87



Pseudomonas clones


therapies


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


88

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


89


Acknowledgments

90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


90

Welcome

Acknowledgments

91

Financial support

Acknowledgments


91

Financial support

Acknowledgments


beta-lactam antibiotics in pseudomonas aeruginosa · 1. activity of beta-lactam antibiotics against...

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