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

Permeability of Bridged Monobactams IC 50 β - MIC Effect on OMPF lactamase against Conductance R2 in vitro cells (from Mathias) (mM) in situ RO 47-7303 0.06 >64* No interaction OCH 3 RO 47-0243 0.05 >64* Blocking CH.CONH 2 RO 46-9392 0.1 >64* No interaction Cl RO 46-8377 0.009 >64* Blocking S-tetrazole RO 44-4454 0.1 2* No effect H 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: • omp C • tol C Baba T. et al. Molecular Systems Biology (2006)

β -Lactams Selected Meropenem Penicillin G Aztreonam Carbenicllin Ampicillin Cefepime

E. coli MIC Determination (I) Ampicillin Aztreonam Carbenicillin CAMHB CAMHB CAMHB Strain CAMHB CAMHB CAMHB 400mM NaCl 400mM NaCl 400mM NaCl 8 4 0.125 4 4 ATCC25922 <=0.06 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 CAMHB CAMHB CAMHB Strain CAMHB CAMHB CAMHB 400mM NaCl 400mM NaCl 400mM NaCl ATCC25922 0.03 0.008 0.06 0.03-0.125 >=32 16 0.016 <=0.002-0.004 0.008-0.016 0.008-0.016 16 4 W3110 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 ∆ tol C: 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 Ciprofloxacin Minocycline Polymyxin B Erythromycin Chloramphenicol

E. coli MIC Determination (II) Chloramphenicol Ciprofloxacin Erythromycin CAMHB CAMHB CAMHB Strain CAMHB CAMHB CAMHB 400mM NaCl 400mM NaCl 400mM NaCl 8 2-4 0.004 0.004 >32 >32 ATCC25922 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 CAMHB CAMHB CAMHB Strain CAMHB CAMHB CAMHB 400mM NaCl 400mM NaCl 400mM NaCl ATCC25922 0.5-2 4-8 0.5-1 0.5-1 2-4 1-4 0.5 1-2 1 1 0.5 1 W3110 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 SUSCEPTIBILITY SYNERGY PERMEABILITY OF BLI IC 50 of β -lactamase MICs β -lactams MICs β -lactams+BLI in situ 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 I) RO 47-0243 or RO 44-4454 (BLIs) ∆ omp C ∆ omp F ∆ omp C ∆ omp F wt - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl Aztreonam Penicillin G Synergy assay Ampicillin Carbenicillin II) RO 47-0243 or RO 44-4454 (BLIs) ∆ omp C ∆ omp F ∆ omp C ∆ omp F wt - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl Aztreonam Penicillin G Permeability assay Ampicillin Carbenicillin

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 7 of 12 RND-pump encoding operons in P. aeruginosa have been characterized: System Substrates β -lactams, BLI, chloramphenicol, novobiocin, macrolides, quinolones, MexAB/ OprM * sulfonamides, tetracyclines, trimethoprim, thiolactomycin, detergents, triclosan,… aminoglycosides, β -lactams, erythromycin, fluoroquinolones, MexXY/ OprM* tetracyclines,… β -lactams, quinolones, chloramphenicol, novobiocin, sulfonamides, MexCD/ OprJ* 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,… * major contributors to MDR 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 .

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 Schweizer H.P. (2003) Genetics and Molecular Research 2(1):48-62 the parent strain.

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 (Gm R , SacB) 1 st crossing- antibiotic resistant & sucrose over: sensitive 2 nd crossing- antibiotic sensitive & sucrose resistant over: Knock-out mutant (or reversal to wt)

P. aeruginosa Strains Studied Strain Affected Efflux-pump Systems PAO1 Wild type PAO1 ∆ mex R MexAB-OprM overexpression (commonly found in clinical isolates) PAO1 ∆ opr J Impaired assembly of MexCD-OprJ

P. aeruginosa MIC Determination (I) Strain Ampicillin Aztreonam Carbenicillin Cefepime >128 4-8 64 2 ATCC27853 >128 2-8 128 2 PAO1 PAO1/ ∆ mexR >128 32 >128 8 PAO1/ ∆ oprJ >128 8 >=128 4 Strain Imipenem Meropenem Penicillin G Piperacillin 2 1 >128 8 ATCC27853 1-4 0.5-2 >128 4-8 PAO1 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 mex AB- opr M 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 opr J K.O. mutant did not cause significant differences in β -lactam susceptibility.