Mécanismes d adaptation et de Résistance de Pseudomonas aeruginosa à la colistine dans la mucoviscidose Jeannot K, Nogues A, Dutruel T, Plésiat P National Reference Centre for Antibiotic Resistance Department of Bacteriology, University Hospital of Besançon, France Société de Médecine de Franche-Comté 14 oct. 2013
Polymyxins AntiMicrobial Peptides family Charged cyclic heptapeptide Cyclic Polycationic lipopeptides Polymyxin E (colistin) and polymyxin B Colistin is produced by Paenibacillus polymyxa colistinus IV and nebulization Colistin Methane-Sulfonate (CMS) Acting as a prodrug Without antimicrobial activity Hydrolyzed in colistin Tripeptide tail Fatty acid
Membrane alterations OM: Outer Membrane M: Murein/PG PM: Inner membrane Benincasa et al. Antimicrob Agents Chemother 2009, 53 No antibiotic Polymyxin B 30µg/mL Vaara M et al. Antimicrob Agents Chemother 1983, 24 These molecules interact with the negative charges of the outer membrane
Acquired Resistance to colistin Report of SENTRY Antimicrobial Surveillance Program (2006-2009) 0.4% of P. aeruginosa and 0.9% of Acinetobacter spp are resistant to colistin Gales AC et al. J Antimicrob Chemother. 2011, 9 Emergence of strains resistant to colistin In CF patients Low to high resistance levels (8 to 512 mg/l) Miller AK et al. Antimicrob Agents Chemother. 2011, 12. Moskowitz SM et al. Antimicrob Agents Chemother. 2011, 2. Gutu AD et al. Antimicrob Agents Chemother. 2013, 5. In non CF patients Low to moderate resistance levels (4 to 64 mg/l) Landman D et al. J Antimicrob Chemother. 2005, 55 Wang CY et al. Clin Microbiol Infect. 2006, 12 Falagas ME et al. J Antimicrob Chemother. 2007, 67 Falagas ME et al. J Med Microbiol. 2008, 55 Abraham N et al. FEMS Microbiol Lett. 2009, 298 Schurek KN et al. Antimicrob Agents Chemother. 2009, 10 Barrow K et al. Antimicrob Agents Chemother. 2009, 12 Muller C et al. Antimicrob Agents Chemother. 2011, 3
LPS modification O Specific antigen, O5 Modification of lipid A Outer membrane Core Lipid A Lam JS, Front Microbiol. 2011; 2 Addition of 4-amino-4- deoxy-l-arabinose (Ara4N) to the 1 and 4 phosphate groups Ernst RK et al. J. Infect. Dis. 2007, 196
Resistance to colistin Chromosome of reference strain PA3551 arnb arnc arna arnd arnt arne arnf ugd PA3560 arnbcadtef-ugd operon (arn) Encoded by arnbcadtef-ugd operon Responsible for synthesis and transport of Ara4N through the inner membrane Expression of arn operon is dependent on two-component systems A Yan et al. J Bio Chem 2007, 282
Classic two-component regulatory systems Signal Outer mbne Periplasmic space 64 Response regulators 63 Histidine kinases Cytoplasmic mbne TM1 TM2 NH 2 Cytoplasm DHp H P Response regulator CA COOH D
Acquired resistance to polymyxins PhoQ PmrB ParS CprS ColS PhoP PmrA ParR CprR ColR 10 CF clinical strains isolated from chronically colistin treated CF patients - MIC to colistin >512 μg/ml Miller AK et al. Antimicrob Agents Chemother. 2011, 12. CF clinical strain isolated from chronically colistin treated CF patients - MIC to colistin 4 μg/ml Muller C et al. Antimicrob Agents Chemother. 2011, 3 11 CF clinical strains isolated from chronically colistin treated CF patients - MIC to colistin from 256 to >512 μg/ml Moskowitz SM et al. Antimicrob Agents Chemother. 2011, 2.
Log(UFC/mL) Tolerance to colistin PAO1 strain 12 10 8 6 4 2 2 µg/ml 4 µg/ml 8 µg/ml 16 µg/ml 32 µg/ml 0 0 1 2 3 4 5 6 7 8 Time (h) Wild type population, MIC=2 µg/ml Majority dies Resistant adapted sub-population
log(ufc/ml) Tolerance to colistin ex-vivo/ in vivo Group Age Sexe Infection by P.aeruginosa I 9-22 H/F No II 23/63 H/F No III 19-51 H Yes IV 18-50 F Yes CF sputum Mutant PAO1 arn::luxcdabe 8 6 4 2 0 µg/ml 8 µg/ml 16 µg/ml Murine model of acute P. aeruginosa pneumonia 0 0 Time (h) 4 8
Colistin concentrations after nebulization Serum Epithelial lining fluid Athanassa ZE et al. Intensive care Med. 2012, 38 20 patients with ventilator-associated pneumonia (VAP) Gram negative bacteria only susceptible to polymyxins CMS administered at a dose of 80 mg (1 MUI) every 8 h for 7 days.
Conclusions Acquired resistance of clinical strains of P. aeruginosa to polymyxins is associated with mutations in proteins PmrB, PhoQ, ParS and/or ParR Resistance levels in CF strains (4 to 128 µg/ml) Additional arn-independent mechanisms likely contribute to elevated colistin resistance in CF strains Tolerance to polymyxins is a drug-induced, arn-independent, reversible adaptive process that helps P. aeruginosa to survive clinically relevant concentrations of drugs (up to 8 µg/ml) Pathogenicity of resistant strains in non CF and CF patients remains to be established.
Acknowledgements NRC & Research group - Patrick Plésiat - Meryem Berrazeg - Charlotte Richardot - Damien Fournier - Catherine Llanes - Caroline Brechet - Marjorie Robert-Nicoud - Amélie Mille - Paulo Juarez - Aurélie Noguès University of Poitiers (France) Pharmacology of Antimicrobial Agents Inserm U-1070 - William Couet Braunschweig (Germany) - Suzanne Hauβler - Agatha Bielecka Pasteur Institute, Paris (France) Unité Défense Inné et inflammation - Lhousseine Touqui - Michel Chignard
MDR, XDR and PDR FEP PIP TZP CTX TIC TCC CAZ MEM IMP GM TM ATM CST CIP AN Wild type strain Souche clinique XDR Polymyxins remain the last hope Magiorakos AP et al. Clin Microbiol Infect 2012; 18