Translocation Studies Mid-Term Review (MTR) Meeting Marseille, France

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1 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

2 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.

3 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

4 Rationale Focus on the role of porins and efflux systems in antibiotic resistance of Gram-negative bacteria Antibiotic Uptake Efflux Antibiotic-resistant Cells

5 Permeability of Bridged Monobactams R1 O H O N N O S R2 H O O 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

6 Permeability of Bridged Monobactams R2 IC 50 β- lactamase in vitro (mm) MIC against cells in situ Effect on OMPF Conductance (from Mathias) RO OCH >64* No interaction RO CH.CONH >64* Blocking RO Cl 0.1 >64* No interaction RO S-tetrazole >64* Blocking RO H 0.1 2* No effect Carbenicillin No interaction Penicillin G Some blocking * In combination with penicillin

7 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

8 Gene Disruption Gene target: ompc tolc Baba T. et al. Molecular Systems Biology (2006)

9 β-lactams Selected Meropenem Aztreonam Cefepime Ampicillin Penicillin G Carbenicllin

10 E. coli MIC Determination (I) Strain Ampicillin Aztreonam Carbenicillin 400mM NaCl 400mM NaCl 400mM NaCl ATCC <= W W3110:: omp C W3110:: omp F W3110: ompf ompc Cefepime Meropenem Penicillin G Strain 400mM NaCl 400mM NaCl 400mM NaCl ATCC >=32 16 W <= W3110:: omp C >32 8 W3110:: omp F <=0.004 <=0.004 >32 32 W3110: ompf ompc >32 16 tolc: MIC determination is ongoing

11 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.

12 Additional Antibiotic Classes Selected Gentamicin Ciprofloxacin Chloramphenicol Erythromycin Minocycline Polymyxin B

13 E. coli MIC Determination (II) Chloramphenicol Ciprofloxacin Erythromycin Strain 400mM NaCl 400mM NaCl 400mM NaCl ATCC >32 >32 W W3110:: omp C >32 >=32 W3110:: omp F >32 32 W3110: ompf ompc >32 >32 Gentamicin Minocycline Polymyxin B Strain 400mM NaCl 400mM NaCl 400mM NaCl ATCC W W3110:: omp C W3110:: omp F W3110: ompf ompc

14 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

15 β-lactamase Inhibitors (BLI) Approach Strains with AmpC overexpressed SUSCEPTIBILITY SYNERGY PERMEABILITY OF BLI MICs β-lactams MICs β-lactams+bli IC 50 of β-lactamase in situ AmpC Contribution AmpC Inhibition Role of outer membrane & porins

16 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.

17 I) Aztreonam Penicillin G Ampicillin Carbenicillin BLI: Experiments ongoing RO or RO (BLIs) wt omp C omp F omp C omp F - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl Synergy assay II) Aztreonam Penicillin G Ampicillin Carbenicillin RO or RO (BLIs) wt ompc omp F omp C ompf - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl - NaCl + NaCl Permeability assay

18 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

19 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

20 Efflux Systems in P. aeruginosa 7 of 12 RND-pump encoding operons in P. aeruginosa have been characterized: System Substrates MexAB/ OprM * MexXY/ OprM* MexCD/ OprJ* MexEF/ OprN* MexJK/ OprM Mex(G)HI/ OpmD MexVW/ OprM β-lactams, BLI, chloramphenicol, novobiocin, macrolides, quinolones, sulfonamides, tetracyclines, trimethoprim, thiolactomycin, detergents, triclosan, aminoglycosides, β-lactams, erythromycin, fluoroquinolones, tetracyclines, β-lactams, quinolones, chloramphenicol, novobiocin, sulfonamides, tetracyclines, trimethoprim, triclosan, fluoroquinolones, tetracycline, chloramphenicol, trimethoprim, triclosan ciprofloxacin, erythromycin, tetracycline, triclosan vanadium, norfloxacin, 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.

21 Knock-out Mutants 1. Make single and multiple knock-out mutants of all major RND pumps in P. aeruginosa. Schweizer H.P. (2003) Genetics and Molecular Research 2(1): Measure susceptibility to all available antibiotics in comparison to the parent strain.

22 Disruption of various mex & opr genes Target gene A B B C genomic DNA PCR 1/2 & 3/4 Cloning vector Gene replacement vector (Gm R, SacB) antibiotic resistant & sucrose sensitive antibiotic sensitive & sucrose resistant 1 st crossingover: 2 nd crossingover: Knock-out mutant (or reversal to wt)

23 P. aeruginosa Strains Studied Strain PAO1 PAO1 mexr PAO1 oprj Affected Efflux-pump Systems Wild type MexAB-OprM overexpression (commonly found in clinical isolates) Impaired assembly of MexCD-OprJ

24 P. aeruginosa MIC Determination (I) Strain Ampicillin Aztreonam Carbenicillin Cefepime ATCC27853 > PAO1 > PAO1/ mexr > >128 8 PAO1/ oprj >128 8 >=128 4 Strain Imipenem Meropenem Penicillin G Piperacillin ATCC >128 8 PAO > PAO1/ mexr > PAO1/ oprj >128 8

25 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.

26 P. aeruginosa MIC Determination (II) Strain Chloramphenicol Ciprofloxacin Erythromycin Gentamicin ATCC27853 > > PAO1 > >32 2 PAO1/ mexr > >32 2 PAO1/ oprj > >32 2 Strain Minocycline Moxifloxacin Polymyxin B ATCC PAO PAO1/ mexr > PAO1/ oprj >=32 2 2

27 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.

28 Consortium Interactions Prof. Winterhalter Compounds sent for Conductance Measurements Summer School Participations Mircea Petrescu s Training Dr. Weingart Strains Exchange Basilea Pharmaceutica Prof. Gameiro Cephalosporins Provided for Measurements Dr. Ceccarelli & Prof. Ruggerone In silico Modeling: Porins & TolC Ph.D. Program Presentation

29 Acknowledgements UK Italy Dr. Bertoni Dr. Camara USA Prof. Schweizer All the Consortium People Basilea Dr. Shapiro Basilea Dr. Caspers