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Mario Vaneechoutte & Pieter Deschaght Current developments in anti-biofilm strategies and (assessing their efficacy with an ex vivo sputum) biofilm models EYIM Session Microbiology 25 April 2012 Paris, France. Many novel antibacterial/anti-biofilm

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Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

Mario Vaneechoutte & Pieter Deschaght

Current developments in anti-biofilm strategies

and

(assessing their efficacy with an ex vivo sputum)

biofilm models

EYIM

Session Microbiology

25 April 2012

Paris, France


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

Many novel antibacterial/anti-biofilm

treatment opportunities for chronic infections

are being developed

Clinical trial with patients: cumbersome

Chronic infection/Biofilm models to test/predict activity


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

1. quorum sensing inhibitors: e.g., furanones, garlick (allicine)

2. antisense RNA strategies to block bacterial transcription and translation

3. antiserum against DNA-binding protein IHF to degrade matrix structure

4. D-amino acids to replace D-ala to degrade matrix structure

5. bacteriophages, with polysaccharide depolymerases to degrade matrix structure

6. iron chelators: e.g., desferoxamine, lactoferrine, conalbumin

7. nitric oxide (NO), toxic to mucoid strains

8. itaconate to block the glyoxylate shunt: waken up persister cells

9. antibiotics combined with the above strategies/compounds

Activity testing/predicting models


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo activity

of anti-biofilm treatments

Which model has the highest predictive power

regarding the biofilm eradication succes in the patient?

1. Diffusion antibiogram, starting from planktonic cells

2. Microtiter plate (peg) / glass biofilm susceptibility testing

3. Rotating wall vessel biofilms - Flow cell biofilms

4. Artificial sputum culture with porcine/bovine mucus and herring DNA

5. Co-culture models of ∆F508 cell lines and P. aeruginosa

6. Animal infection models

7. Ex vivo biofilm sputum model

Patient


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

1. quorum sensing inhibitors: e.g., furanones, garlick (allicine)

JAC 53: 1054-1061

2004

Res. Microbiol. 160: 144-151.

2009


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

2. antisense strategies to block bacterial transcription and translation

Hu et al. 2011. World J Microbiol Biotechnol. DOI 10.1007/s11274-011-0658-x

MotA, a cytoplasmic membrane protein

generates the force necessary to drive the flagellum

is one of the key regulation factors in the initial period of biofilm formation.

Inhibition of P. aeruginosa biofilm formation by

the cell-penetrating peptide (KFF)3K + anti-motA-Peptide Nucleic Acid (PNA)

Biofilm formation

No treatment

1 µM (KFF)3K-PNA

5 µM (KFF)3K-PNA

10 µM (KFF)3K-PNA


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

  • 3. antiserum against DNA-binding protein IHF

  • Extracellular DNA (eDNA) is a key component of EPS in many pathogenic biofilms.

  • Whitchurch et al. 2002. Extracellular DNA required for bacterial biofilm formation.

  • Science 295: 1487 pulmozyme (rh DNAse)

  • Goodman et al. 2011. Mucosal Immunol 4: 625-637.

  • DNABII family of proteins have strong structural influence on intracellular DNA.

  • DNABII is also critical for the integrity of the EPS matrix of biofilms that contain eDNA.

  • In vitro:

  • DNABII rapidly disrupts the biofilm EPS formed by multiple human pathogens in vitro.

  • Synergism with otherwise ineffective traditional antimicrobial approaches in vitro.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

3. antiserum against DNA-binding protein IHF

Goodman et al. 2011. Mucosal Immunol 4: 625-637.

Viable planktonic bacteria released from

a nontypeable Haemophilus influenzae (NTHI) biofilm

after treatment with anti-DNAIIB (= anti-IHF)


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

4. D-amino acids (D-AAs)

Kolodkin-Gal et al. 2010. Science 328: 627-629.

Bacillus subtilis

D-AAs: D-tyrosine, D-leucine, D-tryptophan, and D-methionine

inhibit biofilm formation + degrade biofilm.

In contrast, the corresponding L-isomers were inert in the biofilm-inhibition assay.

Individual D-AAs varied in their activity:

D-tyrosine was more effective (at 3 µM) than D-methionine (at 2 mM)

Mixture of the 4 D-AAs was most potent: 10 nM

Bacteria produce D-amino acids (D-AAs) in stationary phase/mature biofilm

 D-AAs replace D-ala in cell wall, anchor for TasA fibers (Bacillus subtilis)

 TasA can no longer bind to cell wall

[Biofilm matrix = EPS + amyloid fibers composed of the protein TasA]

 Biofilm disruption (see also our results with the EVSM)


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

5. bacteriophages

Hughes et al. 1998a. J Appl Microbiol 85: 583-590.

Hughes et al. 1998.b. Microbiol 144: 3039–3047

Lytic zone

EPS degradation zone


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

5. bacteriophages

Sutherland et al. 2004. FEMS Microbiol. 232: 1-6.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

5. bacteriophages

Glonti et al. 2010. J Appl Microbiol 108: 695-702.

Khawaldeh et al. 2011. J Med Microbiol 60: 1697-1700.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

6. iron chelators: desferoxamine, lactoferrin, conalbumin, EDTA, EGTA

Moreau-Marquis et al. 2008. Am J Physiol Lung Cell Mol Physiol 295: L25–L37

Iron in

CF lung bronchoalveolar lavage (BAL) fluid, CF sputum: 8 µM

BAL isolated from healthy patients: 0.018 µM

due to intrinsic iron sequestration problem of ∆F508 CFTR cells

O'May et al. 2009. J Med Microbiol 58:765-773.

"In addition, clinical strains responded differently to different chelators."

Musk & Hergenrother. 2008. J Appl Microbiol 105: 380-388.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

6. iron chelators: e.g., desferoxamine, lactoferrine, conalbumin

Moreau-Marquis et al. 2009. Am J Respir Cell Mol Biol 41: 305-313.

Culture of P. aeruginosa biofilm, during 6 hours, on ∆F508 airway cells

Live/Dead staining + CLSM.

No treatment Desferoxamine

400 µg/ml (DFO)

See also our results with EVSM

Tobramycine Tobramycine

1000 µg/ml + DFO


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

7. nitric oxide

Yoon et al. 2006. J Clin Invest 116: 436-446.

at pH 6.5, 15 mM NO2– kills mucA mutant P. aeruginosa in CF airway conditions after 16 days

has no adverse effects on cultured human airway epithelia in vitro.

In this study, we believe that we have discovered the Achilles’ heel

of the formidable mucoid form of P. aeruginosa,

which could lead to improved treatment for CF airway disease.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

P. aeruginosa is capable of robust anaerobic growth by respiration

using nitrate (NO3–) or nitrite (NO2–) as terminal electron acceptors.

7. nitric oxide

CF ASL and sputum concentrations of NO3– and NO2–: up to 600 μM

final electron acceptors for anaerobic respiration and growth by P. aeruginosa

P. aeruginosa uses NAR and NIR to reduce NO3– to NO2– to NO

increased levels of NO, a toxic intermediate of NO3– and NO2– reduction

synthesis of protective NO reductase (NOR) by P. aeruginosa.

NAR NIR NOR NOS

Leukocyte attacks + leukocyte killing by P. aeruginosa rhamnolipids

ROS

mucA mutations

alginate production

mucoid conversion

NOR

sensitivity to HNO2

NO3- NO2- NO N2O N2

Mucoid strains are

most sensitive to HNO2


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

8. blocking the glyoxylate shunt with itaconate

Persister cells use the glyoxylate shunt instead of the Krebs cycle

Krebs cycle

 reducing agents: NADH, FADH2

 18 ATP

 rapid growth

 ROS

 Oxydative stress

 Bactericidal


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

8. blocking the glyoxylate shunt with itaconate

Lindsey et al. 2008. Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase. Microbiol 154: 1616-1627.

Persister cells

switch off Krebs cycle

switch to glyoxylate shunt

 low NADH/FADH2 production

 low ATP production

 slow growth (dormancy)

 intrinsic AB resistance

 low ROS production

 high resistance to killing

Isocitrate lyase is absent in man

 good antimicrobial target

inhibition by itaconate:

itaconate


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

I. Novel anti- (Pseudomonas) biofilm strategies

9. novel antibiotic formulations and combinations

AB + AB: Tré-Hardy et al. 2009. Int J Antimicrob Agents 34: 370-374.

AB + AMP: Nagant et al. 2010. Appl Microbiol Biotechnol 88: 251-263

AB+ Phage: Comeau et al. 2008. PLoS ONE 2(8): e799.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo activity

of anti-biofilm treatments

Which model has the highest predictive power

regarding the biofilm eradication succes in the patient?

1. Diffusion antibiogram, starting from planktonic cells

2. Microtiter plate (peg) biofilm susceptibility testing

3. Rotating wall vessel biofilms - Flow cell biofilms

4. Artificial sputum culture with bovine mucus

5. Co-culture models of CF cell lines and P. aeruginosa

6. Animal infection models

7. Ex vivo biofilm sputum model

Patient


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

  • II. II. Models for predicting the in vivo efficacy

  • of anti-biofilm treatments

  • Diffusion antibiogram for P. aeruginosa, cultured from sputum of CF patients:

  • = starting from planktonic cells: Foweraker et al. (2005)

  • irreproducible within and between labs

  • even same colony morphology yields different susceptibility patterns

  • limited correlation between susceptibility and clinical outcome

Foweraker et al. 2005. JAC 55: 921-927.

Figure 1


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

2. Microtiter biofilm-based susceptibility testing:

Tré-Hardy et al. 2009. Int J Antimicrob Agents 33: 40-45.

Observations:

strong differences between planktonic cells and biofilm grown cells

strong differences between young and mature biofilms


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

2. Microtiter biofilm-based susceptibility testing


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

2. Microtiter biofilm-based susceptibility testing

Moskowitz et al. 2011. Ped Pulmonol 46: 184-192.

Set up: 39 participants.

Treated with 14-day courses of two antibiotics,

that were selected on basis of diffusion antibiogram (planktonic cells)

or on basis of microtiter biofilm susceptibility testing results

Conclusions: In this pilot study,

antibiotic regimens based on biofilm testing

did not differ significantly from

regimens based on conventional testing

in terms of microbiological and clinical responses.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

3. Rotating wall vessel technology: low shear

Crabbé et al. 2009. Environm Microbiol


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

4. Artificial sputum medium

10 mg/ml porcine stomach mucin

1.4 mg/ml herring sperm DNA


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

5. Co-cell culture with Pseudomonas aeruginosa

Observations:

biofilm formation on lung epithelial cell culture vs biofilm on abiotic surfaces (glass):

1500-fold more production of biofilm

25-fold increase of resistance to tobramycin

Limitations:

Long term infection difficult: cells rapidly killed by P. aeruginosa

No mucus compound

No human immunity compound

Limited complexity of microflora: 1 species, 1 strain (PAO1)

Many parameters, such as coating, cell line, cell maturity, buffer, ... influence outcome

Moreau-Marquis et al. 2008. Am J Physiol Lung Cell Mol Physiol 295: L25-L37.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

6. Animal infection models: CFTR knockouts of mouse, rat, pig

Limitations:

Expensive, cumbersome, ethical considerations

And still: Limited chronic colonization (artificial: sea weed alginate beads)

No human cells, mucus, immune compounds

No original biofilm


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

In summary: Whatever modification to the susceptibility testing biofilm model:

planktonic growth - biofilm

young biofilm - mature biofilm

plastic biofilm - cell line associated biofilm

young cell lines - mature cell lines

(nonchronic) animal infection models

All have their merits, but

 very different predictions about biofilm formation and

biofilm susceptibility

Which one predicts most reliably the susceptibility

of the P. aeruginosa biofilm in the patient?


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

7. Ex vivo sputum biofilm model

Limitations of current biofilm models:

Each model or variation of parameters leads to strongly different predictions

about biofilm formation and biofilm susceptibility

Original biofilm structure (mucus associated microcolonies) as in patient is absent

Multiple genotypes and phenotypes of P. aeruginosa are absent (usually PAO1)

Extracellular human DNA is absent

Mucus from patient is absent

Leukocytes, cytokines of patient are absent


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

Time for a different approach?

1. Add the anti-Pseudomonas- anti-biofilm treatment (at break point concentration)

directly to P. aeruginosa colonized sputum of CF patients

= address the original biofilm in the original patient environment

2. Monitor the effect of the treatment on the P. aeruginosa load

in comparison with the P. aeruginosa load of untreated sputum

7. the Ex Vivo Sputum Biofilm Model (EVSM)


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

7. Ex vivo sputum biofilm model

Is using sputum a valid approach

for testing susceptibility

of P. aeruginosa biofilms in the CF airways?

This depends on the localisation of the chronic biofilm colonisation/infection

1. At the epithelium of lungs?

group of Gerald Pier

Foweraker. 2009. Recent advances in cystic fibrosis. Brit Med Bull 89: 93-110.

or?

2. In the lumen of the conductive airways, within the mucus layer?  sputum


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

7. Ex vivo sputum biofilm model

Where is the chronic biofilm colonisation/infection located?

Percentage of bacteria

at distances of 5-17 and 2-5 µm

from epithelial surface

of lungs from 9 CF patients

MAMs: mucus associated microcolonies


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

7. Ex vivo sputum biofilm model

Bjarnsholt et al. 2009. Ped Pulmonol 44: 547-558.

Expectorated sputum contains the persistent biofilm fraction from CF airways


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

Results with ex vivo biofilm model

(culture based analysis)

Results are very patient dependent  Need for personalized approach

Strains isolated from sputum and recultured (red) are rapidly killed

Same strains in original sputum associated biofilm (purple) are not

DFO is not very effective in original sputum biofilm

Airway model of Moreau-Marquis et al. (2009)

Tobra + EDTA can eradicate all cultivable biofilm cells in some patients


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

7. Ex vivo sputum biofilm model

Results with ex vivo biofilm model

(culture based analysis)

Effects of Tobra (400 vs 1000 µg/ml) and D-amino acids (3 µM)

added to patient sputum colonized with P. aeruginosa


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

7. Ex vivo sputum biofilm model

Methods to assess treatment efficacy

A. Troublesome approaches:    1. Culture: only cultivable cells are assessed, not dormant biofilm part. Workload high.

    2. DNA-qPCR: also dead cells are assessed  Treatment effects are not observable.

    3. Reverse transcription qPCR: cumbersome:

- RNA instability

- different transcription levels of different genes

in biofilm-associated dormant and planktonic cells.

    4. Life/Dead staining: too much interference of free (leukocyte) DNA in sputum

B. Possible approaches:    1. FISH  biofilm structure is assessed. Quantification troublesome?

2. PMA + DNA-qPCR: All living cells but no dead cells are quantified.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

7. Ex vivo sputum biofilm model

PMA + DNA-qPCR: All living cells and no dead cells are quantified

Nocker A, Cheung CY, Kamper AK. 2006.  Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Meth 67: 310-320.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

7. Ex vivo sputum biofilm model

Advantages of using colonized sputum

Readily available in large quantities

Personalized information

Other treatments of patient present (mucolytics, potentiators, correctors)

Differences in genetic CFTR background of CF patients present

Differences in modifier genes & immune respons of CF patients present

Differences in status of colonisation (recent, long-term) present

Most probably: highest predictive power regarding treatment success in patient.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

II. Models for predicting the in vivo efficacy

of anti-biofilm treatments

7. Ex vivo sputum biofilm model

Limitations of the ex vivo sputum model

Unequal distribution of colonisation?

When obtained after physiotherapy, MAM distribution turns out to be fairly even

The EVSM is not suited

to find out how reduced P. aeruginosa /bacterial colonisation affects patient health.

to assess the side effects of antibacterial treatments on the airway epithelium:

 ex vivo primary cell lines might be most informative and most personalized.

to assess CFTR corrector and potentiator effects

 ex vivo primary cell lines might be most informative and most personalized.


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

Special thanks to

Pieter Deschaght & Leen Van Simaey (LBR)

the sputum donors

the nursing staff of MucoGent, University Hospital Gent, Belgium

Linda Mahieu, Marleen Vanderkerken and Ann Raman

MucoVereniging België

The ex vivo sputum model

Slides available at: http://users.ugent.be/~mvaneech/LBR.htm

[email protected]


Mario vaneechoutte pieter deschaght current developments in anti biofilm strategies and

http://users.ugent.be/~mvaneech/LBR.htm


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