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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
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