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

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

Charged cyclic heptapeptide

Tripeptide tail

Fatty acid

Membrane alterations

M: Murein/PG

PM: Inner membrane

OM: Outer Membrane

These molecules interact with the negative charges of the outer membrane

Benincasa et al. Antimicrob Agents Chemother 2009, 53

No antibiotic

Polymyxin B 30µg/mL

Vaara M et al. Antimicrob Agents Chemother 1983, 24

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

Lam JS, Front Microbiol. 2011; 2

Lipid A

Core

O Specific antigen, O5

Outer membrane

Modification of lipid A

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

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

arnBCADTEF-ugd operon (arn)

arnB PA3551 arnC arnA arnT arnF ugd arnE arnD Chromosome of

reference strain PA3560

A Yan et al. J Bio Chem 2007, 282

Classic two-component regulatory systems

NH2

COOH

Cytoplasmic mbne

Periplasmic space

Cytoplasm H P

Signal

64 Response regulators 63 Histidine kinases

Outer mbne

D

Response regulator

TM1 TM2

DHp

CA

Acquired resistance to polymyxins

PhoP

PhoQ PmrB

PmrA

ParR

ParS

CprR

CprS

ColS

ColR

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.

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

Tolerance to colistin

PAO1 strain

Wild type population, MIC=2 µg/mL

Resistant adapted sub-population

Majority dies

0

2

4

6

8

10

12

0 1 2 3 4 5 6 7 8

Log(U

FC

/mL

)

Time (h)

2 µg/mL

4 µg/mL

8 µg/mL

16 µg/mL

32 µg/mL

Tolerance to colistin ex-vivo/ in vivo

CF sputum

Mutant PAO1 arn::luxCDABE

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

0

2

4

6

8

0 4 8

log(U

FC

/mL

)

Time (h)

0 µg/mL

8 µg/mL

16 µg/mL

Murine model of acute P. aeruginosa pneumonia

Colistin concentrations after nebulization

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.

Serum Epithelial lining fluid

Athanassa ZE et al. Intensive care Med. 2012, 38

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

Pasteur Institute, Paris (France) Unité Défense Inné et inflammation

- Lhousseine Touqui - Michel Chignard

Braunschweig (Germany) - Suzanne Hauβler - Agatha Bielecka

MDR, XDR and PDR

Souche clinique XDR Wild type strain

Magiorakos AP et al. Clin Microbiol Infect 2012; 18

FEP PIP TZP CTX

TCC TIC CAZ MEM

IMP GM TM ATM

CST CIP AN

Polymyxins remain the last hope