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Impact of efflux mechanisms on susceptibility and resistance to beta-lactam antibiotics in Pseudomonas aeruginosa : beyond the usual concepts Houssein Abdel Aziz Chalhoub Promoter: Prof. Franoise Van Bambeke Cellular and Molecular Pharmacology

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Impact of efflux mechanisms on susceptibility and resistance to beta-lactam antibiotics in Pseudomonas aeruginosa: beyond the usual concepts

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Houssein Abdel Aziz Chalhoub Promoter: Prof. Françoise Van Bambeke Cellular and Molecular Pharmacology Louvain Drug Research Institute Université catholique de Louvain, Brussels, Belgium

22 December 2016

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Bacteria & Humans

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Bacteria: sp spread wide idely ly in in th the en envir ironment…

Picture of Everest from : Luca Galuzzi

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Bacteria: sp spread wide idely ly in in th the en envir ironment…

http://www.earthtimes.org/scitech/billion-years-old-bacterial-enzyme/1578/

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“They are so small that you can’t seen them without a microscope, but they are there…”

http://dlb-network.com/photographyomn/pseudomonas-aeruginosa-gram-stain

Bacteria

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Bacterial infections

Modeling Pseudomonas aeruginosa pathogenesis in plant hosts : doi:10.1038/nprot.2008.224.

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Antibiotic resistance: post-antibiotic era …

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http://davidjernigan.blogspot.be/2016/08/preparing-body-for-probiotic-success_2.html

Antibiotic resistance: microbial war…

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Antibiotic resistance: revenge of the microbes

Clinical use

Penicillin 1943

Sir Alexander Fleming

Alfred Eisenstaedt—Time & Life Pictures/Getty Images

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Antibiotic resistance: revenge of the microbes

Clinical use RESISTANCE

Penicillin 1943 1945 (2 years)

Sir Alexander Fleming

Alfred Eisenstaedt—Time & Life Pictures/Getty Images

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Antibiotic resistance: revenge of the microbes

Clinical use RESISTANCE

Penicillin 1943 1945 (2 years) Vancomycin 1972 1988 (16 years)

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Antibiotic resistance: revenge of the microbes

Clinical use RESISTANCE

Penicillin 1943 1945 (2 years) Vancomycin 1972 1988 (16 years) Imipenem 1985 1998 (13 years)

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Antibiotic resistance: revenge of the microbes

Clinical use RESISTANCE

Penicillin 1943 1945 (2 years) Vancomycin 1972 1988 (16 years) Imipenem 1985 1998 (13 years) Daptomycin 2003 2004 (1 year)

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Antibiotic resistance: revenge of the microbes

Clinical use RESISTANCE

Penicillin 1943 1945 (2 years) Vancomycin 1972 1988 (16 years) Imipenem 1985 1998 (13 years) Daptomycin 2003 2004 (1 year) Ceftaroline 2010 2011 (1 year)

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Clinical use RESISTANCE

Penicillin 1943 1945 (2 years) Vancomycin 1972 1988 (16 years) Imipenem 1985 1998 (13 years) Daptomycin 2003 2004 (1 year) Ceftaroline 2010 2011 (1 year)

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Antibiotic resistance: revenge of the microbes

http://xombit.com/2013/02/nadal-magnus-demoledor-liftado

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Pseudomonas aeruginosa

http://edition.cnn.com/2015/02/25/health/deadly-bacteria-doctors-offices/ U.S. Centers for Disease Control and Prevention - Medical Illustrator - James Archer

1-5 µm

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Pseudomonas aeruginosa: moving by the use of flagella…

From: https://www.youtube.com/watch?v=a_5FToP_mMY A clip from the NOVA production, "Judgment Day."

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Pseudomonas aeruginosa: takes advantage of a sick person…

http://www.healthcarea2z.org/word

HIV

Cancer patients receiving chemotherapy Severe burns Invasive surgery

Low immune function

Cystic Fibrosis

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http://www.bbc.com/news/health-32755065 realheroesbecomeangels.blogspot.com

Build-up

  • f thick mucus

Opportunistic pathogens: Lung infections in Cystic Fibrosis patients

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Pseudomonas aeruginosa:

Lung infections in Cystic Fibrosis patients

http://www.bbc.com/news/health-32755065

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Classes of antipseudomonal antibiotics

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β-lactams

Picture from: https://en.wikipedia.org/wiki/Intravenous_therapy

Aminoglycosides

Polymyxins

Quinolones

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Classes of antipseudomonal antibiotics

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Pictures from: http://www.realme.fr/720-porta-neb-compresseur-nebuliseur-pneumatique-d-aerosoltherapie.html http://www.inhalation.se/explore/preciseinhale-and-you/

Aminoglycosides (Tobramycin)

Polymyxins

(colistin)

Quinolones

(Levofloxacin) Aztreonam

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Classes of antipseudomonal antibiotics

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Picture from: http://www.dailymail.co.uk/health/article-3430291

Quinolones

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Pseudomonas aeruginosa:

Lung infections in Cystic Fibrosis patients

http://www.bbc.com/news/health-32755065

Multidrug-Resistant

Pseudomonas aeruginosa

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Classes of antipseudomonal antibiotics

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β-lactams

Penicillins: Ticarcillin / clavulanic acid Piperacillin / Tazobactam Carbapenems: Meropenem Imipenem Cephalosporins: Ceftazidime, cefepime Monobactam: Aztreonam

Picture from: https://en.wikipedia.org/wiki/Intravenous_therapy

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  • P. aeruginosa and resistance mechanisms to β-lactam antibiotics

 Downregulating / mutating porins [OprD]

β-lactams (imipenem, meropenem…)

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Adapted from Lister et al. Clin. Microbiol. Rev. 2009; 22: 582-610 http://www.bellybelly.com.au/baby/baby-hunger-cues/ http://thestir.cafemom.com/big_kid/181864/expired_medicine_dates_still_safe

Antibiotic Outer membrane Cell membrane

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  • P. aeruginosa and resistance mechanisms to β-lactam antibiotics

OprM MexA MexB 27

Active efflux [MexAB-OprM, MexXY-OprM, MexEF-OprN, MexCD-OprJ]

β-lactams (ticarcillin, meropenem…)

Adapted from: Lister et al. Clin. Microbiol. Rev. 2009; 22: 582-610. Clin Microbiol Rev. 2006 Apr;19(2):382-402.

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Extended-Spectrum-β-Lactamases: ceftazidime Cephalosporinase AmpC: ceftazidime Carbapenemase: meropenem, ceftazidime

Adapted from Lister et al. Clin. Microbiol. Rev. 2009; 22: 582-610 28

β-lactamases: antibiotic degradation

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  • P. aeruginosa and resistance mechanisms to β-lactam antibiotics

β-lactamases (cephalosporinases AmpC-type, carbapenemases, ESBLs...)

OprM MexA MexB

 Downregulating / mutating porins [OprD]

29 Antibiotic

Active efflux [MexAB-OprM, MexXY-OprM, MexEF-OprN, MexCD-OprJ]

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Impact of efflux mechanisms on susceptibility and resistance to beta-lactam antibiotics in Pseudomonas aeruginosa: beyond the usual concepts

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Hussein Abdel Aziz Chalhoub Promoter: Prof. Françoise Van Bambeke Cellular and Molecular Pharmacology Louvain Drug Research Institute Université catholique de Louvain, Brussels, Belgium

22 December 2016

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1. Activity of beta-lactam antibiotics against Pseudomonas aeruginosa from patients with cystic fibrosis 2. Role of efflux versus other resistance mechanisms against:

  • a. Meropenem (carbapenems)
  • b. Ceftazidime / avibactam (new β-lactamases inhibitor)
  • c. Temocillin (old beta-lactam antibiotic)

Main objectives of the thesis

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  • International collection of P. aeruginosa from patients with cystic fibrosis chronically

exposed to different antipseudomonal agents.  Bacterial isolates: 333 clinically relevant isolates were obtained from:  Dr Michael Tunney (Queen’s University of Belfast, UK): n=99;  Drs Anne Vergison / Olivier Denis (Hôpital Erasme, Brussels, Belgium): n=88;  Prof. Patrick Plésiat (CHRU Besançon, Besançon, France): n=80;  Prof. Barbara Kahl (University of Münster, Münster, Germany): n=68.

Source of isolates

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Pictures from: http://www.bbc.com/news/health-32755065 http://www.mirror.co.uk/news/technology-science/science/uk-scientists-developing-cystic-fibrosis-5992360

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Antibacterial activity of beta-lactams against P. aeruginosa from cystic fibrosis patients

Ticarcillin Piperacillin + Tazobactam Ceftazidime Imipenem Meropenem

Breakpoint (mg/L)

16 16 8 8 8

Related publication: Mustafa, Chalhoub et al, Antimicrob Agents Chemother. 2016; 60:6735–41.

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Antibacterial activity of beta-lactams against P. aeruginosa from cystic fibrosis patients

Ticarcillin Piperacillin + Tazobactam Ceftazidime Imipenem Meropenem

Breakpoint (mg/L)

16 16 8 8 8

  • A microorganism is defined as susceptible or resistant by a

level of antimicrobial activity associated with a high likelihood of therapeutic success or therapeutic failure, respectively.

Related publication: Mustafa, Chalhoub et al, Antimicrob Agents Chemother. 2016; 60:6735–41.

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Antibacterial activity of beta-lactams against P. aeruginosa from cystic fibrosis patients

Ticarcillin

Breakpoint (mg/L)

16

% Susceptible

18

% Resistant

82

MIC90 (mg/L)

>512

1 2 4 8 16 32 64 128 256 512 1024 2048 25 50 75 100 > 512

P.aeruginosa (n = 333)

MIC90 Ticarcillin

MICs(mg/L)

% of isolates (cumulative) Related publication: Mustafa, Chalhoub et al, Antimicrob Agents Chemother. 2016; 60:6735–41.

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Antibacterial activity of beta-lactams against P. aeruginosa from cystic fibrosis patients

Ticarcillin Piperacillin + Tazobactam Ceftazidime Imipenem Meropenem

Breakpoint (mg/L)

16 16 8 8 8

% Susceptible

18 32 36 51 49

% Resistant

82 68 64 49 51

MIC90 (mg/L)

>512 512 512 32 16

  • Summary. Imipenem and meropenem were the most

active antibiotics…

Related publication: Mustafa, Chalhoub et al, Antimicrob Agents Chemother. 2016; 60:6735–41.

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Meropenem (β-lactam)

  • Stable to AmpC and ESBL but not carbapenemases.

Carbapenemases

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High-level resistance to meroperem

Pseudomonas (cystic fibrosis patients) Meropenem MIC = 64-128 mg/L

Related publications: Chalhoub et al, Intern. J. Antimicrob. Ag. in press

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Pseudomonas (cystic fibrosis patients) Meropenem MIC = 64-128 mg/L Pseudomonas (Hospital acquired pneumonia) Meropenem MIC = 128 mg/L

High-level resistance to meroperem

Comparison with Pseudomonas from hospital acquired pneumonia

Related publications: Chalhoub et al, Intern. J. Antimicrob. Ag. in press

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Pseudomonas (cystic fibrosis patients) Meropenem MIC = 64-128 mg/L

High-level resistance to meroperem

Comparison with Pseudomonas from hospital acquired pneumonia

Cystic fibrosis Hospital acquired pneumonia AmpC-type cephalosporinases

X

ESBL(s) Carbapenemase(s)

X

Pseudomonas (Hospital acquired pneumonia) Meropenem MIC = 128 mg/L

Related publications: Chalhoub et al, Intern. J. Antimicrob. Ag. in press

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Adapted from Jason Sello, Brown university, 2011

Efflux inhibitor

PAβN; Phenylalanine- arginine β- naphthylamide

Meropenem + efflux inhibitor in Pseudomonas

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Pseudomonas (cystic fibrosis patients) Meropenem MIC = 64-128 mg/L Pseudomonas (Hospital acquired pneumonia) Meropenem MIC = 128 mg/L

  • PA

 N + PA  N

  • PA

 N + PA  N 2 4 8 1 6 3 2 6 4 1 2 8 2 5 6 s t r a i n s f r

  • m

C F p a t i e n t s c a r b a p e n e m a s e s ( - ) s t r a i n s f r

  • m

P n e u m

  • n

i a p a t i e n t s C a r b a p e n e m a s e s ( + )

M E M

High-level resistance to meroperem

Comparison with Pseudomonas from hospital acquired pneumonia

Related publications: Chalhoub et al, Intern. J. Antimicrob. Ag. in press

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Pseudomonas (Hospital acquired pneumonia) Meropenem MIC = 128 mg/L

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Pseudomonas (cystic fibrosis patients) Meropenem MIC = 64-128 mg/L

  • PAN

+ PAN

  • PAN

+ PAN 2 4 8 16 32 64 128 256

strains from CF patients carbapenemases (-) strains from HAP patients Carbapenemases (+)

MEM MIC (mg/L) MexAB-OprM MexXY-OprM MexCD-OprJ MexAB-OprM MexXY-OprM MexCD-OprJ

High-level resistance to meroperem

Comparison with Pseudomonas from hospital acquired pneumonia

Related publications: Chalhoub et al, Intern. J. Antimicrob. Ag. in press

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Pseudomonas (Hospital acquired pneumonia) Pseudomonas (cystic fibrosis patients)

+

  • clinical isolates
  • Sequencing OprD genes confirmed these results (premature stop mutations)

OprD porin High-level resistance to meroperem

Comparison with Pseudomonas from hospital acquired pneumonia

Related publications: Chalhoub et al, Intern. J. Antimicrob. Ag. in press

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Impact of efflux mechanisms on susceptibility and resistance to beta-lactam antibiotics in Pseudomonas aeruginosa: beyond the usual concepts

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1- [Efflux + / porin -] are enough to confer high- level resistance to meropenem.

Related publications: Chalhoub et al, Intern. J. Antimicrob. Ag. in press

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High-level resistance to ceftazidime

Cystic fibrosis Hospital acquired pneumonia AmpC-type cephalosporinases

X

ESBL(s) Carbapenemase(s)

X

Related publication: Chalhoub et al, J Antimicrob Chemother. 2015;70:1596-8.

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AmpC cephalosporinases inhibitor: avibactam

Related publication: Chalhoub et al, J Antimicrob Chemother. 2015;70:1596-8.

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Ceftazidime/avibactam activity in the whole collection

  • f isolates (n = 333)
  • Susceptible isolates to ceftazidime

increased from 36% to 76 % after addition

  • f avibactam [4mg/L].

Promising results for cystic fibrosis patients

Related publication: Chalhoub et al, J Antimicrob Chemother. 2015;70:1596-8.

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24 % resistance to this new combination !?

Ceftazidime/avibactam activity in the whole collection

  • f isolates (n = 333)

Related publication: Chalhoub et al, J Antimicrob Chemother. 2015;70:1596-8.

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The mechanism of resistance to ceftazidime/avibactam combination

Control strains (induced with sub-MIC imipenem) MIC (mg/L) Ceftazidime Reference strain, PAO1 32 PAO1 overproducing MexXY-OprM 32 PAO1 overproducing MexCD-OprJ 32

(Prepared for submission as a correspondence to the Journal of Antimicrobial Chemotherapy) Chalhoub et al., Poor membrane permeability impedes avibactam activity in Pseudomonas aeruginosa

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The mechanism of resistance to ceftazidime/avibactam combination

Control strains (induced with sub-MIC imipenem) MIC (mg/L) Ceftazidime Ceftazidime+Avibactam Reference strain, PAO1 32 0.125 PAO1 overproducing MexXY-OprM 32 0.125 PAO1 overproducing MexCD-OprJ 32 0.125

(Prepared for submission as a correspondence to the Journal of Antimicrobial Chemotherapy) Chalhoub et al., Poor membrane permeability impedes avibactam activity in Pseudomonas aeruginosa

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The mechanism of resistance to ceftazidime/avibactam combination

Control strains (induced with sub-MIC imipenem) MIC (mg/L) Ceftazidime Ceftazidime+Avibactam Reference strain, PAO1 32 0.125 PAO1 overproducing MexXY-OprM 32 0.125 PAO1 overproducing MexCD-OprJ 32 0.125 PAO1 overproducing MexAB-OprM 32 4 PAO1 overproducing MexEF-OprN 32 2 PAOD1OprD-mutant 32 4

(Prepared for submission as a correspondence to the Journal of Antimicrobial Chemotherapy) Chalhoub et al., Poor membrane permeability impedes avibactam activity in Pseudomonas aeruginosa

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OprD loss

MexAB-OprM, MexEF-OprN efflux systems

  • verexpression

Cephalosporinases

The mechanism of resistance to ceftazidime/avibactam combination

Adapted from Lister et al. Clin. Microbiol. Rev. 2009; 22: 582-610

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Impact of efflux mechanisms on susceptibility and resistance to beta-lactam antibiotics in Pseudomonas aeruginosa: beyond the usual concepts

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1- [Efflux + / porin -] are enough to confer high-level resistance to meropenem 2- [Efflux + / porin -] are pre-existing as resistance mechanisms to avibactam, which is not used yet in CF patients !!

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Impact of efflux mechanisms on susceptibility and resistance to beta-lactam antibiotics in Pseudomonas aeruginosa: beyond the usual concepts

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Is there any antibiotic resistant to beta-lactamases, and not affected by efflux systems and OprD porin downregulation ??

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Impact of efflux mechanisms on susceptibility and resistance to beta-lactam antibiotics in Pseudomonas aeruginosa: beyond the usual concepts

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Temocillin (β-lactam)

  • Derived from the class of carboxypenicillins (ticarcillin, carbenicillin)

57 57

  • Withdrawn due to lack of activity against wild-type strains of

Pseudomonas aeruginosa, Gram-positive organisms and anaerobes.

  • Not used against Pseudomonas.

by Beecham Pharmaceuticals in the 1989s

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Temocillin H2O+ β-lactamases Temocillin

Temocillin: stable against β-lactamases

Born in the 1989’s (UK)

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Temocillin versus ticarcillin in the active site of β-lactamases

Adapted from:

  • Cubist pharmaceuticals
  • A Matagne et al. - Biochem J. 1993 Aug 1; 293(Pt 3): 607–611.

W1: water molecule

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Temocillin

Temocillin & Efflux in P. aeruginosa

Δ mexAB

Temocillin

In 2012 FACM Group, LDRI, Belgium

60

Isogenic strains of PA MIC (mg/L) temocillin ticarcillin Wild type PAO1 512 64 PAO1 delta mexAB

2 1

Susceptible

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Temocillin

Susceptible

Temocillin & Efflux in P. aeruginosa

Δ mexAB

In 2012 FACM Group, LDRI, Belgium

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Natural mutations of these efflux pumps can be observed in isolates from patients with cystic fibrosis !!!

Related publication : Buyck et al. J.

  • Antimicrob. Chemother. 2012; 67: 771-5.

Picture from: http://www.bbc.com/news/health-32755065

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Temocillin considered as inactive against Pseudomonas showed surprising activity !!!

TIC Temocillin TZP CAZ Meropenem Ceftazidime +avibactam

Breakpoint

16

16

16 8 8 8 MIC90

>512

>512

512 512 16 64 % Susceptible

18

29#

32 36 49 76 % Resistant

82

71

68 64 51 24

# proposed breakpoint for temocillin in Belgium: 16 mg/L

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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Temocillin + efflux inhibitor in Pseudomonas

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  • Activity of temocillin was improved in the

presence of the efflux inhibitor

  • % susceptibility increased from ~30 to 55%

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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N-Phenyl-1- naphthylamine

Efflux speed

  • Activity of temocillin was improved

in the presence of the efflux pump inhibitor.

  • Lowest efflux speed for isolates

with temocillin MICs < 128mg/L.

Speed of efflu lux by y MexAB-OprM pumps

Temocillin

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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Sequencing mexA/B genes in P. aeruginosa from cystic fibrosis patients

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MexA-Linker

MexB- exporter protein

OprM-gated porin

http://learn.genetics.utah.edu/content/cells/organelles/ https://genedoe.wordpress.com/tag/dna/

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Sequencing mexA/B genes in P. aeruginosa from cystic fibrosis patients

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Major mutations

  • DNA Fragment Deletions
  • Stop mutations

Minor mutations

  • Synonymous mutations
  • Missense mutations

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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truncated/aberrant mexA truncated/aberrant mexB missense mutations in mexA missense mutations in mexB missense mutations mexA+mexB synonymous mutations WT 2 4 8 16 32 64 128 256 512 1024 2048

(e) MIC vs. mutations Temocillin MIC (mg/L)

a a a a b,c c b,c t r u n c a t e d / a b e r r a n t m e x A t r u n c a t e d / a b e r r a n t m e x B m i s s e n s e m u t a t i

  • n

s i n m e x A m i s s e n s e m u t a t i

  • n

s i n m e x B m i s s e n s e m u t a t i

  • n

s m e x A + m e x B s y n

  • n

y m

  • u

s m u t a t i

  • n

s W T

  • 0.8
  • 0.6
  • 0.4
  • 0.2

0.0

(f) NPN efflux vs. mutations

a a b b a,b,c c b,c

Vmax (fluo units/sec)

Mutations in mexA/B genes in P. aeruginosa as a function of temocillin activity

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Efflux speed

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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MexB mutant (truncated + aberrant protein) Temocillin MIC = 8 mg/L MexB mutant (AA substitutions) Temocillin MIC = 256 mg/L MexB mutant (AA substitutions) Temocillin MIC = 512 mg/L Front view Front view Front view

Modelisation mexB mutations in the same clone isolated from 3 German patients

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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Truncated MexB and low efflux speed but high temocillin MIC !

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  • Truncated MexB (M720_Q1046del): temocillin MIC = 128 mg/L

Strains

MIC (mg/L) Temocillin Ticarcillin Wild type delta mexB

2 1

Clinical isolate delta mexB

128 32 Wild type 512 128

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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Influx of temocillin inside Pseudomonas ??

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OprD family, 19 members

  • f carboxylate channels

Porin (Front view) Porin (Extracellular side)

http://sbcb.bioch.ox.ac.uk/memprotmd/beta/protein/pdbid/3sys

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Temocillin & the 19 Gates (OprD porin family) of P. aeruginosa

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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Outer membrane carboxylate channels OccD/K in P. aeruginosa

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  • prD-single mutant of P. aeruginosa
  • OprD porin is not utilized by temocillin !

MICs (mg/L) IPM MEM Temocillin Strains 1 0.37 256

Wild type PA

4 3 256 PA::oprD

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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Blocking OccK1 in occK2-single mutant of P. aeruginosa

  • OccK1/OccK2 porins are involved in the

uptake of temocillin

MICs (mg/L) IPM MEM Temocillin Strains 1 0.37 256

Wild type PA

1 0.37 786 PA::occK2/1

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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TMO IPM MEM MICs (mg/L)

40 1.5 1 Empty vector 40 0.225 0.225 +OprD 20 1.5 1 +OccK1 22 1.5 1 +OccK2

++ OprD

bla OprD

++ Occk1

bla Occk1

++ Occk2

bla Occk2

Overexpression of OccK1 or OccK2 in porin-deficient E.coli

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TMO IPM MEM MICs (mg/L)

40 1.5 1 Empty vector 40 0.225 0.225 +OprD 20 1.5 1 +OccK1 22 1.5 1 +OccK2

++ OprD

bla OprD

++ Occk1

bla Occk1

++ Occk2

bla Occk2

Overexpression of OccK1 or OccK2 in porin-deficient E.coli

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SLIDE 77

Niels Bagge et al. Antimicrob. Agents Chemother. 2004;48:1175-1187

Exopolysaccharides (alginate) from P. aeruginosa

Mol Microbiol. 2012 May;84(4):595-607.

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Exo xopolysaccharides (alg lgin inate-like) ??

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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J Antimicrob Chemother. 1991 Mar;27(3):285-93. The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis- derived mucin and Pseudomonas aeruginosa alginate. Bolister N1, Basker M, Hodges NA, Marriott C.

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Exopolysaccharide abundance in cultures

  • f clinical isolates as a function of temocillin MICs

2 4 8 16 32 64 128 256 512 1024 2 4 6 8 10

temocillin MICs (mg/L) CFW fluorescence (ratio to PAO1 value)

0.08 0.05 <0.001 0.002 0.09 0.02 0.07 0.07 0.13 0.17

not sequenced missense mutation in mexB truncated/aberrant mexB missense mutation in mexA truncated/aberrant mexA synonymous mutations missense mutation in mexA and mexB

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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 Temocillin resistance is mainly due to active MexAB efflux system with participation of exopolysaccharides production and/or OccK1/OccK2-porin loss !

OccK1/OccK2 porins Exopolysaccharides

MexAB-OprM

Summary (temocillin in cystic fibrosis)

Related publication: Chalhoub et al, Nature Sci. Reports. in press.

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Impact of efflux mechanisms on susceptibility and resistance to beta-lactam antibiotics in Pseudomonas aeruginosa: beyond the usual concepts

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1- [Efflux + / porin -] are enough to confer high-level resistance to meropenem 2- [Efflux + / porin -] are pre-existing as resistance mechanisms to avibactam, which is not used yet in CF patients !! 3- Mutations in MexAB-OprM efflux pumps appear as a mechanism of restored susceptibility to temocillin in Pseudomonas !!!

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 Efflux is a key mechanism of resistance for Pseudomonas from CF patients.  Porins are important players if associated with efflux.  Bacterial outer membrane is sufficiently strong barrier against β- lactams.

Conclusion

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 Several paralogous efflux and porin genes to accommodate and extrude different substrates.  Pseudomonas invades different territories with enough fitness and adaptability.

Conclusion

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 Temocillin should be included in CF antimicrobial susceptibility testing.  Temocillin could be also used as carbapenem sparing agent against Pseudomonas with OprD porin mutations !

Conclusion

http://www.bbc.com/news/health-32755065

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 Check the prevalence of mutations in mexA/mexB genes in Pseudomonas from a recent CF population (2016/2017).  Determine whether mexA/mexB mutations in CF are beneficial for the activity of temocillin only or could also affect other substrates.

General perspectives (research)

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 Develop fast techniques to screen epidemic and risky clones.  Prevent intra- and inter-hospital dissemination

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epidemic Pseudomonas clones.  Establishment of early appropriate and targeted antimicrobial therapies.

Perspectives (management of CF infections)

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Acknowledgments

Cystic Fibrosis centres

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Dr Hector Rodriguez-Villalobos Professor Mathias Winterhalter

Acknowledgments

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Welcome

Acknowledgments

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Financial support

Acknowledgments

Associated to the Région Wallonne project