Translocation StudiesMid-Term Review (MTR) Meeting

Marseille, France

F. Vidal-Aroca, M.G.P. Page and J. Dreier

Marie Curie ActionsResearch Training Networks (RTN)

Deteriorating situation regarding treatment of Gram-negative infections

• Growing concern over:Multi-resistant AcinetobacterMulti-resistant Pseudomonas aeruginosaCarbapenem-resistant Klebsiella pneumoniaeESBL-producing K. pneumoniae and Escherichia coli

• Warnings for:Carbapenem-resistant EnterobacterGNNFs like Burkholderia, Stenotrophomonas….

Background

Interests & Expectations at Basilea

• Insight into the permeation of -lactam antibiotics in clinical isolates

• Permeation route(s) of novel compounds

• Role of porins in resistance

• Role of efflux systems in resistance

Rationale

Antibiotic

Uptake

Efflux

Antibiotic-resistant Cells

Focus on the role of porins and efflux systems in antibiotic resistance of Gram-negative bacteria

N

N

S

R2

OO

O

O

R1

O

HH

Bridged monobactams are potent -lactamase inhibitors

When R2 = H, activity against -lactamase in situ can be modulated by R1

When R2 = OMe or larger, all activity against -lactamase in situ is lost, irrespective of R1

Permeability of Bridged Monobactams

Permeability of Bridged Monobactams

R2

IC50 -lactamase

in vitro (mM)

MIC

against cells

in situ

Effect on OMPF

Conductance

(from Mathias)

RO 47-7303 OCH30.06 >64* No interaction

RO 47-0243 CH.CONH20.05 >64* Blocking

RO 46-9392 Cl 0.1 >64* No interaction

RO 46-8377 S-tetrazole 0.009 >64* Blocking

RO 44-4454 H 0.1 2* No effect

Carbenicillin - - 4 No interaction

Penicillin G - - 16 Some blocking

* In combination with penicillin

Permeability of Bridged Monobactams

• Is the lack of correlation due to OmpC?

Knock-out strain with only OmpF

• Need more data points to look for trends

Select more penicillins with a spread of MIC

values

Select more bridged monobactams

• Are the conditions appropriate?

low pH, high salt vs neutral pH, isotonic

Gene Disruption

Baba T. et al. Molecular Systems Biology (2006)

Gene target:

• ompC

• tolC

MeropenemAztreonam

Cefepime

Penicillin G

Ampicillin Carbenicllin

-Lactams Selected

E. coli MIC Determination (I)

tolC: MIC determination is ongoing

Ampicillin Aztreonam Carbenicillin

Strain CAMHBCAMHB

400mM NaClCAMHB

CAMHB 400mM NaCl

CAMHBCAMHB

400mM NaCl

ATCC25922 8 4 0.125 <=0.06 4 4W3110 2 0.5-1 0.06 0.008-0.016 4 2W3110::ompC 4 2 0.125 0.06 16 8W3110::omp F 8 4 0.125 0.06 16 16W3110:ompFompC 8 4 0.25 0.125 32 32

Cefepime Meropenem Penicillin G

Strain CAMHBCAMHB

400mM NaClCAMHB

CAMHB 400mM NaCl

CAMHBCAMHB

400mM NaCl

ATCC25922 0.03 0.008 0.06 0.03-0.125 >=32 16W3110 0.016 <=0.002-0.004 0.008-0.016 0.008-0.016 16 4W3110::ompC 0.03 0.004 0.016 0.03 >32 8W3110::omp F 0.06 0.008 <=0.004 <=0.004 >32 32W3110:ompFompC 0.5 0.25 0.5 1 >32 16

Conclusions

1. Equal contribution of OmpC and OmpF to aztreonam, ampicillin and carbenicillin susceptibilities.

2. Strong influence of the double K.O. mutant on cefepime and meropenem susceptibilities.

3. Weak influence of the single K.O. mutants on cefepime and meropenem susceptibilities.

4. High osmolarity potentiates the effect of cefepime > penicillin G > others.

Additional Antibiotic Classes Selected

Ciprofloxacin Gentamicin Minocycline

Chloramphenicol Erythromycin Polymyxin B

E. coli MIC Determination (II)

Chloramphenicol Ciprofloxacin Erythromycin

Strain CAMHBCAMHB

400mM NaClCAMHB

CAMHB 400mM NaCl

CAMHBCAMHB

400mM NaCl

ATCC25922 8 2-4 0.004 0.004 >32 >32W3110 4 1-2 0.004 0.004 16 8W3110::ompC 8 4 0.004 0.004 >32 >=32W3110::omp F 32 4 0.016 0.016 >32 32W3110:ompFompC 8 4 0.016 0.016 >32 >32

Gentamicin Minocycline Polymyxin B

Strain CAMHBCAMHB

400mM NaClCAMHB

CAMHB 400mM NaCl

CAMHBCAMHB

400mM NaCl

ATCC25922 0.5-2 4-8 0.5-1 0.5-1 2-4 1-4W3110 0.5 1-2 1 1 0.5 1W3110::ompC 0.25 2 2 1 1 1W3110::omp F 0.25 1 4 2 1 1W3110:ompFompC 0.5 2 0.5 0.5 1 0.5

Conclusions

1. The susceptibility of E. coli to gentamicin or polymyxin B is NOT influenced by OmpF and OmpC porins.

2. Minocycline susceptibility is increased in the double K.O. mutant.

3. Deletion of OmpF slightly decreases the ciprofloxacin susceptibility.

4. Chloramphenicol and erythromycin data need to be confirmed

-Lactamase Inhibitors (BLI) Approach

Strains with AmpC overexpressed

MICs -lactams MICs -lactams+BLIIC50 of -lactamase

in situ

AmpC Contribution AmpC Inhibition Role of outer membrane & porins

SUSCEPTIBILITY SYNERGY PERMEABILITY OF BLI

BLI: -Lactam Selection

• Overexpression of AmpC in wt and porin mutant strains

• MIC determination led to the selection of the following -lactams antibiotics for synergy test: aztreonam, penicillin G, ampicillin and carbenicillin

• The bacteria growth was not affected at concentrations of 256 mg/l of bridged monobactans tested

• The overexpression of AmpC does not influence the MIC of other classes of antibiotics.

- NaCl + NaCl - NaCl + NaCl - NaCl + NaCl - NaCl + NaClAztreonamPenicillin GAmpicillinCarbenicillin

RO 47-0243 or RO 44-4454 (BLIs)omp Fomp Cwt omp Comp F

- NaCl + NaCl - NaCl + NaCl - NaCl + NaCl - NaCl + NaClAztreonamPenicillin GAmpicillinCarbenicillin

RO 47-0243 or RO 44-4454 (BLIs)wt omp C omp F omp Comp F

BLI: Experiments ongoing

Synergy assay

I)

II)

Permeability assay

Pseudomonas aeruginosa

Ubiquitous environmental bacterium

One of the top causes of opportunistic human infections

bacteraemia in burn victimsurinary-tract infectionshospital-acquired pneumoniaAIDS populationpredominant cause of morbidity and mortality of cystic fibrosis

patients

Intrinsic resistance to antibiotics and disinfectants

Piddock L.J.V. Clin Microbiol Rev, Apr. 2006, p. 382–402

Efflux Pumps

Resistance Mechanism

1- Enzymatic inactivation

2- Alternative metabolic pathways

3- Reduced uptake

4- Alteration of the target site

5- Membrane bound efflux pumps

Efflux Systems in P. aeruginosa

System Substrates

MexAB/ OprM * -lactams, BLI, chloramphenicol, novobiocin, macrolides, quinolones, sulfonamides, tetracyclines, trimethoprim, thiolactomycin, detergents, triclosan,…

MexXY/ OprM* aminoglycosides, -lactams, erythromycin, fluoroquinolones, tetracyclines,…

MexCD/ OprJ* -lactams, quinolones, chloramphenicol, novobiocin, sulfonamides, tetracyclines, trimethoprim, triclosan,…

MexEF/ OprN* fluoroquinolones, tetracycline, chloramphenicol, trimethoprim, triclosan…

MexJK/ OprM ciprofloxacin, erythromycin, tetracycline, triclosan

Mex(G)HI/ OpmD vanadium, norfloxacin,…

MexVW/ OprM chloramphenicol, erythromycin, fluoroquinolones, tetracycline,…

Other clinically relevant bacteria with RND efflux systems:A. baumannii, B. cepacia, B. pseudomallei, S. maltophilia, N. gonorrhoeae, N. meningitidis, S. marcescens, E. coli, S. enterica, E. aerogenes, K. pneumoniae, K. oxytoca, C. jejuni, P. mirabilis, H. influenzae.

7 of 12 RND-pump encoding operons in P. aeruginosa have been characterized:

* major contributors to MDR

http://www.pseudomonas.com

Knock-out Mutants

1. Make single and multiple knock-out mutants of all major RND pumps in P. aeruginosa.

2. Measure susceptibility to all available antibiotics in comparison to the parent strain.Schweizer H.P. (2003) Genetics and Molecular Research 2(1):48-62

Target gene

A B B C1 2 3 4

genomic DNA

PCR 1/2 & 3/4

Cloning vector

Gene replacement vector (GmR, SacB)

1st crossing-over:

antibiotic resistant & sucrose sensitive

2nd crossing-over:

antibiotic sensitive & sucrose resistant

Knock-out mutant (or reversal to wt)

Disruption of various mex & opr genes

Strain Affected Efflux-pump Systems

PAO1 Wild type

PAO1 mexR MexAB-OprM overexpression

(commonly found in clinical isolates)

PAO1 oprJ Impaired assembly of MexCD-OprJ

P. aeruginosa Strains Studied

Strain Ampicillin Aztreonam Carbenicillin Cefepime

ATCC27853 >128 4-8 64 2PAO1 >128 2-8 128 2PAO1/mexR >128 32 >128 8PAO1/oprJ >128 8 >=128 4

Strain Imipenem Meropenem Penicillin G Piperacillin

ATCC27853 2 1 >128 8PAO1 1-4 0.5-2 >128 4-8PAO1/mexR 4 8-16 >128 32PAO1/oprJ 4 2-4 >128 8

P. aeruginosa MIC Determination (I)

Conclusions

1. Wild type efflux-pump expression caused high resistance to ampicillin, carbenicillin and penicillin G.

2. The mexAB-oprM system strongly contributed to resistance towards piperacillin, aztreonam, meropenem and cefepime.

3. No contribution of the main pumps to imipenem resistance was observed.

4. The oprJ K.O. mutant did not cause significant differences in -lactam susceptibility.

Strain Chloramphenicol Ciprofloxacin Erythromycin Gentamicin

ATCC27853 >32 0.5 >32 1-2PAO1 >32 0.125-0.25 >32 2PAO1/mexR >32 0.5-1 >32 2PAO1/oprJ >32 0.125-0.25 >32 2

P. aeruginosa MIC Determination (II)

Strain Minocycline Moxifloxacin Polymyxin B

ATCC27853 16 2 2PAO1 16-32 1-2 2PAO1/mexR >32 8 2PAO1/oprJ >=32 2 2

Conclusions

1. Wild type efflux-pump expression caused high resistance to chloramphenicol, erythromycin and minocycline.

2. The mexAB-oprM system strongly contributed to resistance towards ciprofloxacin and moxifloxacin.

3. No contribution of the main pumps to gentamicin and polymyxin B resistance was observed.

4. The oprJ K.O. mutant did not cause significant differences in susceptibility to the tested compounds.

Consortium Interactions

Prof. Winterhalter

•Compounds sent for Conductance Measurements

•Summer School Participations

•Mircea Petrescu’s Training

Dr. Weingart

•Strains Exchange

Prof. Gameiro

•Cephalosporins Provided for

Measurements

Basilea Pharmaceutica

Dr. Ceccarelli & Prof. Ruggerone

•In silico Modeling:

Porins & TolC

•Ph.D. Program Presentation

Italy

Dr. Bertoni

UK

Dr. CamaraUSA

Prof. Schweizer

Acknowledgements

Basilea

Dr. Caspers

Basilea

Dr. Shapiro

All the Consortium

People