Nathalie M. VandeveldeCellular and Molecular Pharmacology

Activity of antibiotics in models of naActivity of antibiotics in models of naïïve and induced biofilms of ve and induced biofilms of 

Streptococcus pneumoniae. Streptococcus pneumoniae. N.M. Vandevelde, P.M. Tulkens, F. Van BambekeN.M. Vandevelde, P.M. Tulkens, F. Van Bambeke

BIBR SYMPOSIUM

Louvain-la-Neuve

20 -

12 -

2013

20 µm

Low Temperature Scanning  Electron MicroscopyMoscoso et al,2006 J Bacteriol;188(22):7785‐95

ex: S. pneumoniae ‐

strain R6

Bacterial organized

communities :

Bacteria

+

Extracellular matrix H2

OPolysaccharides

Proteins          Extracellular DNA

on inert or living surfacesin pathological or healthy situations

Sanctuaries : protection

↔ IS + antibiotics

Biofilms 

Streptococcus pneumoniae 

Gram positive bacterium

Sepsis,Meningitis,Pneumonia,…

Sinusitis,Otitis media,Acute exacerbations of chronic bronchitis

Over 60% of bacterial infections (and up to 80% of chronic infections) are currently considered to involve microbial growth in biofilmsa

a

Moscoso et al, Int Microbiol. 2009 Jun;12(2):77‐85.

Are non antibiotic drugs responsible of changes in antibiotic activity 

against S. pneumoniae?

Pharmacodynamic studies of antibiotic activity 

Antibiotics

Interactions

S. pneumoniaeBiofilms

MOXIFLOXACIN

Fluoroquinolone  ><  DNA  replication

Naïve and induced biofilms

Are non antibiotic drugs responsible of changes in antibiotic activity 

against S. pneumoniae?

Antibiotics

Interactions

S. pneumoniaeBiofilms

Pharmacodynamic studies of antibiotic activity 

MOXIFLOXACIN

Fluoroquinolone  ><  DNA  replication

IPRATROPIUM

Bronchodilator muscarinic antagonist

Interactions

Naïve and induced biofilms

First part: 

Set up of naive and induced biofilm models 

& First pharmacodynamic studies

+ Medium caMHB + 5% LHB

+ MediumcaMHB + 5% LHB + 2% Glucose

Bacterial suspension

Fuqua and Greenberg, Nat Rev Mol Cell Biol. 2002;3(9):685‐95

up to 11 days

37°C; 5% CO2

Methodology ‐

biofilm formation and characterization

S. pneumoniaeATCC 49619

+ Medium caMHB + 5% LHB

+ MediumcaMHB + 5% LHB + 2% Glucose

Bacterial suspension

Bacterial viability within the matrix

Live↕

Dead

C‐

:Medium 

without

AB 

C+:SDS 1%m/v

C‐

C+

Biofilm thickness

Biofilm staining  with Crystal violet

0 2 4 6 8 10 120

25

50

75

100

125

150ATCC 49619 - naïve model

ATCC49619 - induced modelR6 - naïve modelR6 - induced model

Biofilm maturity (days)

Acv

(570

nm)

Measure

of the CV AbsorbanceIn situ reductionby viable cells

Results expressed in % of the Ctrl 

Measure

of the RF fluorescence(λexc 560nm; λem 590nm)

Fuqua and Greenberg, Nat Rev Mol Cell Biol. 2002;3(9):685‐95

up to 11 days

37°C; 5% CO2

Antibiotic conc. ↑

Conc. ↓C‐

C+

Methodology ‐

biofilm formation and characterization

Bacteria + 

Matrix

S. pneumoniaeATCC 49619

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

Ex: ATCC 49619 biofilms‐ ‐ : 2 days‐old ― : 11 days‐old 

Survival Biofilm mass

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

Results : First moxifloxacin pharmacodynamic studies

Matrix effect : ↓

Efficacy & Potency

Moxifloxacin

11‐days old 

2‐days old

11‐days old 

2‐days old

Data were used to fit a sigmoid function (Hill equation, slope factor set to 1) by non‐linear regression

Biofilm

modelEffect on bacterial survival Effect on biofilm thickness

Emax(% loss of viability with 95% CI)

EC50Concentration (X MIC [mg/L])

Emax(% loss of matrix with 95% CI)

EC50Concentration (X MIC [mg/L])

2 days naïve 74.07 (65.42 to 82.72) /A 56,23/A 81.25 (70.63 to 91.87) /A 0.1 /A

11 days naïve 42.18

(36.11 to 48.25) /B 3,78/B 20.87

(13.59 to 28.15) /B >104

/B

Stat. analysis: unpaired, two‐tailed t‐test for comparisons between maturity stages, values with different letters are significantly different from each other (P<0.05)

Vandevelde N.M. et al, AAC, 2014, in press.

Methodology ‐

2 biofilm models

Planctonic bacteria

Irreversible attachment

Growth and division

Signal 

molecules

Mature microcolony formation

Signal 

molecules

Adsorption

Extracellular 

polymeric 

substance

Dispersion

Planctonic bacteria

Irreversible attachment

Growth and division

Signal 

molecules

Mature microcolony formation

Signal 

molecules

Adsorption

Extracellular 

polymeric 

substance

Dispersion

Naive 

model

Induced 

model

Methodology ‐

2 biofilm models

0 2 4 6 8 10 120

50

100

150

200

250

300

350ATCC 49619 - naïve model

ATCC49619 - induced modelR6 - naïve modelR6 - induced model

Biofilm maturity (days)

Acv

(570

nm)

+ fresh medium

bacterial planktonic suspension (same bacterial count as for naive biofilms)

from a plate containing a 6 day-old naive biofilm

n

new plate

Planctonic bacteria

Signal 

molecules

Mature microcolony formation

Signal 

molecule

s

Adsorption

Extracellular 

polymeric 

substance

Dispersion

Growth and division

Irreversible attachment

***

0 2 4 6 8 10 120

50

100

150

200

250

300

350ATCC 49619 - naïve modelATCC49619 - induced model

R6 - naïve modelR6 - induced model

Biofilm maturity (days)A

cv (5

70nm

)

Naive modelInduced model

Methodology ‐

2 biofilm models

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

Ex: ATCC 49619 biofilms‐ ‐ : 2 days‐old ― : 11 days‐old 

Naive model

Induced model

Survival Biofilm thickness

Results : Moxifloxacin pharmacodynamic studies

Bacterial induction leads to :

↓ Efficacy (Emax

) on survival for 2 and 11‐days old BF

and for 2‐days old BFagainst biofim mass

Stat. analysis: unpaired, two‐tailed t‐test  Vandevelde N.M. et al, AAC, 2014, in press.

Second part: 

Modulation of moxifloxacin activity against  pneumococcal biofilms by bronchodilators

Are non antibiotic drugs responsible of changes in antibiotic activity 

against  S.pneumoniae biofilms?

ANTIBIOTICSS. pneumoniae BIOFILMS

Interactions

β‐lactams  ><

Bacterial  wall  synthesis Ex: MOXIFLOXACIN

Fluoroquinolone  ><  DNA  replication

BRONCHODILATORS (BD)

Interactions

Combinations with Bronchodilators  

PHARMACODYNAMIC  STUDIES

Ref. medium “m”

: caMHB + 5%LHB + 2% Glc

1 2Biofilm growth in different media                        Biofilm

treatment  in absence of BD

(*) Concentration in the Bronchoalveolar lavage (recovering bronchial epithelial cells)a

a

Drug Information Handbook 18th

Edition

m + IPRATROPIUM 1.45 ng/ml*

(Muscarinic Antag.)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

Naive model

Induced model

Survival Biofilm thickness

Results ‐

Combinations with Ipratropium : PD studies of MXF activity 

Ex: ATCC 49619 biofilms‐ ‐ : 2 days‐old , m― : 11 days‐old , m

‐ ‐ : 2 days‐old , m + IPR― : 11 days‐old , m + IPR

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

Naive model

Induced model

Survival Biofilm thickness

Results ‐

Combinations with Ipratropium : PD studies of MXF activity 

Ex: ATCC 49619 biofilms‐ ‐ : 2 days‐old , m― : 11 days‐old , m

‐ ‐ : 2 days‐old , m + IPR― : 11 days‐old , m + IPR

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

Naive model

Induced model

Survival Biofilm thickness

Results ‐

Combinations with Ipratropium : PD studies of MXF activity 

Ex: ATCC 49619 biofilms‐ ‐ : 2 days‐old , m― : 11 days‐old , m

‐ ‐ : 2 days‐old , m + IPR― : 11 days‐old , m + IPR

↑ Efficacy (Emax

) & Potency (EC50

)

on survival & biofilm mass 

11‐days old BF > 2‐days old BF 

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esidual matrix (of the ctrl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

-5 -4 -3 -2 -1 0 1 2 3 40

20

40

60

80

100

120

Log10 MIC broth

% R

esid

ual v

iabi

lity

(of t

he c

trl)

Naive model

Induced model

Survival Biofilm thickness

Results ‐

Combinations with Ipratropium : PD studies of MXF activity 

Ex: ATCC 49619 biofilms‐ ‐ : 2 days‐old , m― : 11 days‐old , m

‐ ‐ : 2 days‐old , m + IPR― : 11 days‐old , m + IPR

↑ Efficacy (Emax

) & Potency (EC50

)

on survival & biofilm mass 

11‐days old BF > 2‐days old BF 

Results ‐

Combinations with Ipratropium : PD studies of MXF activity 

ATCC 49619 - naive biofilm

2 6 110.1

1

10

100

1000

: reference medium: reference medium + IPRATROPIUM

******

Biofilm maturity (days)

Bio

film

thic

knes

s A

cv 5

70 n

m

ATCC 49619 - induced biofilm

2 6 110.1

1

10

100

1000

: reference medium: reference medium + IPRATROPIUM

***

***

Biofilm maturity (days)

Bio

film

thic

knes

s A

cv 5

70 n

m

Stat. analysis: unpaired, two‐tailed t‐test 

IPRATROPIUM discards biofilm 

from day 6 in the naive model from day 2 in the induced model

LogP  Ipratropium Br. : 0.21

Summary

-120

-100

-80

-60

-40

-20

0

-120

-100

-80

-60

-40

-20

0

a

b

c c

ab

c

d% m

axim

al re

duct

ion

in v

iabi

lity

(of t

he C

TRL)

% m

aximal reduction in thickness(of the C

TRL)

Emax

on survival Emax

on biofilm thickness

m

IPR 

m

IPR

Moxifloxacin maximal efficacies on bacterial survival and matrix

thickness for 11‐days old naive (open bars) and induced (squared bars) biofilms 

Stat. analysis: One way Anova with Tuckey post  test

Take home message

Old biofilm maturity stages and bacterial induction

Ipratropium, by targeting biofilm mass, highly increases antibiotic activity

on survival and matrix of residual structure

Appropriate Co‐medications

In vivo Model for Polytherapies

Matrix effect reducing antibiotic activity

Last word to you…

How biofilmsform

How resistance 

works

How resistance spreads

Last word to you…

How biofilmsform

How resistance 

works

How resistance spreads

Together, let's start the March towardsBiofilms!

All the Cellular and Molecular Pharmacology 

Research Group members and especially,Pr. Françoise Van Bambeke (promotor) & Pr. Paul M. Tulkens

Acknowlegment