Translocation Studies Mid-Term Review (MTR) Meeting Marseille, - - PowerPoint PPT Presentation

Marie Curie Actions Research Training Networks (RTN)

Translocation Studies

Mid-Term Review (MTR) Meeting Marseille, France

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

Background

Deteriorating situation regarding treatment of Gram-negative infections

• Growing concern over:

Multi-resistant Acinetobacter Multi-resistant Pseudomonas aeruginosa Carbapenem-resistant Klebsiella pneumoniae ESBL-producing K. pneumoniae and Escherichia coli

• Warnings for:

Carbapenem-resistant Enterobacter GNNFs like Burkholderia, Stenotrophomonas….

Interests & Expectations at Basilea

• Insight into the permeation of β-lactam antibiotics in

clinical isolates

• Permeation route(s) of novel compounds
• Role of porins in resistance
• Role of efflux systems in resistance

Rationale

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

Uptake Efflux

Antibiotic-resistant Cells

Permeability of Bridged Monobactams

Bridged monobactams are potent β- lactamase inhibitors When R2 = H, activity against β- lactamase in situ can be modulated by R1 When R2 = OMe or larger, all activity against β-lactamase in situ is lost, irrespective of R1 N N S R2 O O O O R1 O H H

Permeability of Bridged Monobactams

R2 IC50 β- lactamase in vitro (mM) MIC against cells in situ Effect on OMPF Conductance (from Mathias) RO 47-7303 OCH3 0.06 >64* No interaction RO 47-0243 CH.CONH2 0.05 >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

Gene target:

• ompC
• tolC

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

β-Lactams Selected

Aztreonam Cefepime Meropenem Penicillin G Ampicillin Carbenicllin

• E. coli MIC Determination (I)

Ampicillin Aztreonam Carbenicillin Strain CAMHB CAMHB 400mM NaCl CAMHB CAMHB 400mM NaCl CAMHB CAMHB 400mM NaCl

ATCC25922

8 4 0.125 <=0.06 4 4

W3110

2 0.5-1 0.06 0.008-0.016 4 2

W3110::∆omp C

4 2 0.125 0.06 16 8

W3110::∆omp F

8 4 0.125 0.06 16 16

W3110:∆ompF∆ompC

8 4 0.25 0.125 32 32 Cefepime Meropenem Penicillin G Strain CAMHB CAMHB 400mM NaCl CAMHB CAMHB 400mM NaCl CAMHB CAMHB 400mM NaCl

ATCC25922

0.03 0.008 0.06 0.03-0.125 >=32 16

W3110

0.016 <=0.002-0.004 0.008-0.016 0.008-0.016 16 4

W3110::∆omp C

0.03 0.004 0.016 0.03 >32 8

W3110::∆omp F

0.06 0.008 <=0.004 <=0.004 >32 32

W3110:∆ompF∆ompC

0.5 0.25 0.5 1 >32 16

∆tolC: MIC determination is ongoing

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

Gentamicin Minocycline Ciprofloxacin Erythromycin Polymyxin B Chloramphenicol

• E. coli MIC Determination (II)

Chloramphenicol Ciprofloxacin Erythromycin Strain CAMHB CAMHB 400mM NaCl CAMHB CAMHB 400mM NaCl CAMHB CAMHB 400mM NaCl

ATCC25922

8 2-4 0.004 0.004 >32 >32

W3110

4 1-2 0.004 0.004 16 8

W3110::∆omp C

8 4 0.004 0.004 >32 >=32

W3110::∆omp F

32 4 0.016 0.016 >32 32

W3110:∆ompF∆ompC

8 4 0.016 0.016 >32 >32 Gentamicin Minocycline Polymyxin B Strain CAMHB CAMHB 400mM NaCl CAMHB CAMHB 400mM NaCl CAMHB CAMHB 400mM NaCl

ATCC25922

0.5-2 4-8 0.5-1 0.5-1 2-4 1-4

W3110

0.5 1-2 1 1 0.5 1

W3110::∆omp C

0.25 2 2 1 1 1

W3110::∆omp F

0.25 1 4 2 1 1

W3110:∆ompF∆ompC

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

β-Lactamase Inhibitors (BLI) Approach

Strains with AmpC overexpressed

PERMEABILITY OF BLI SUSCEPTIBILITY SYNERGY

IC50 of β-lactamase in situ MICs β-lactams MICs β-lactams+BLI AmpC Contribution AmpC Inhibition Role of outer membrane & porins

BLI: β-Lactam Selection

• Overexpression of AmpC in wt and porin mutant strains
• MIC determination led to the selection of the following β-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.

BLI: Experiments ongoing

• NaCl + NaCl - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl

Aztreonam Penicillin G Ampicillin Carbenicillin RO 47-0243 or RO 44-4454 (BLIs) ∆omp F ∆omp C wt ∆omp C∆omp F

Synergy assay

I)

• NaCl + NaCl - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl

Aztreonam Penicillin G Ampicillin Carbenicillin RO 47-0243 or RO 44-4454 (BLIs) wt ∆omp C ∆omp F ∆omp C∆omp F

II)

Permeability assay

Pseudomonas aeruginosa

Ubiquitous environmental bacterium One of the top causes of opportunistic human infections bacteraemia in burn victims urinary-tract infections hospital-acquired pneumonia AIDS population predominant cause of morbidity and mortality of cystic fibrosis patients Intrinsic resistance to antibiotics and disinfectants

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

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

Efflux Systems in P. aeruginosa

System Substrates MexAB/ OprM * β-lactams, BLI, chloramphenicol, novobiocin, macrolides, quinolones, sulfonamides, tetracyclines, trimethoprim, thiolactomycin, detergents, triclosan,… MexXY/ OprM* aminoglycosides, β-lactams, erythromycin, fluoroquinolones, tetracyclines,… MexCD/ OprJ* β-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

Knock-out Mutants

http://www.pseudomonas.com

• 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

Disruption of various mex & opr genes

genomic DNA

Target gene A B B C

1 2 3 4

PCR 1/2 & 3/4

Cloning vector Gene replacement vector (GmR, SacB)

antibiotic resistant & sucrose sensitive 1st crossing-

• ver:

antibiotic sensitive & sucrose resistant 2nd crossing-

• ver:

Knock-out mutant (or reversal to wt)

• P. aeruginosa Strains Studied

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 MIC Determination (I)

Strain Ampicillin Aztreonam Carbenicillin Cefepime

ATCC27853

>128 4-8 64 2

PAO1

>128 2-8 128 2

PAO1/∆mexR

>128 32 >128 8

PAO1/∆oprJ

>128 8 >=128 4 Strain Imipenem Meropenem Penicillin G Piperacillin

ATCC27853

2 1 >128 8

PAO1

1-4 0.5-2 >128 4-8

PAO1/∆mexR

4 8-16 >128 32

PAO1/∆oprJ

4 2-4 >128 8

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

β-lactam susceptibility.

• P. aeruginosa MIC Determination (II)

Strain Chloramphenicol Ciprofloxacin Erythromycin Gentamicin

ATCC27853

>32 0.5 >32 1-2

PAO1

>32 0.125-0.25 >32 2

PAO1/∆mexR

>32 0.5-1 >32 2

PAO1/∆oprJ

>32 0.125-0.25 >32 2 Strain Minocycline Moxifloxacin Polymyxin B

ATCC27853

16 2 2

PAO1

16-32 1-2 2

PAO1/∆mexR

>32 8 2

PAO1/∆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

Acknowledgements

UK

• Dr. Camara

USA

• Prof. Schweizer

Italy

• Dr. Bertoni

Basilea

• Dr. Caspers

All the Consortium People Basilea

• Dr. Shapiro