Setting-up an in-vitro model of mature biofilm of Pseudomonas

Setting-up an in-vitro model of mature biofilm of
Pseudomonas aeruginosa to evaluate the activity of antibiotics
used to treat chronic lung infection in Cystic Fibrosis patients.
Yvan Diaz Iglesias1, Muhammad-Hariri Mustafa1, Paul M. Tulkens1,
1
Françoise Van Bambeke
1Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute
Université catholique de Louvain, Brussels, Belgium
Introduction
Results
A. Kinetic of development :
40000
5
4
30000
25000
3
20000
2
15000
10000
 Biomass and viability increased over time to
reach a plateau on day 4 for both
parameters.
1
5000
Fluorescein
Crystal Violet
0
7
5
4
3
2
1
0
0
6
Fluorescence at 518nm
35000
Absorbance at 570nm
Pseudomonas aeruginosa is a major cause of chronic
pulmonary infections in Cystic Fibrosis patients
(prevalence near 75%) due to its capacity to grow as
biofilms, which are refractive to the action of antibiotics.
Appropriate models to evaluate antibiotic activity against
biofilms are therefore warranted. Existing models in
microplates take a long time (> 10 days) to reach maturity
(1).
Time (days)
Aim
B. Antibiotic activity on mature biofilms and pharmacodynamic parameters:
Activity of tobramycin
125
125
100
100
100
100
100
100
75
75
75
75
75
75
50
50
50
50
50
50
25
25
25
25
25
25
Pharmacodynamic parameters were calculated based on the
equation of the sigmoidal regression fitted of the data.
 Maximal efficacy (Emax) : reduction in viability/biomass for an
infinitely large concentration
 Relative potency (C25) : Concentration reducing of 25%
125
viability/biomass
100
100
100
100
75
75
50
50
25
25
0
4
3
2
1
0
M
-4
4
3
2
1
IC
M
-1
0
4
Crystal Violet
-1
Crystal Violet
-2
Fluorescein
-3
Fluorescein
3
25
2
50
1
50
IC
75
-3
IC
M
-4
125
% of fluorescence (FDA)
125
75
-4
4
3
2
1
0
M1
IC
125
100
-2
% of fluorescence (FDA)
125
0
Log of antibiotic concentration (mg/L)
Activity of ceftazidime
150
25
0
% of absorbance (CV)
The 5 tested antibiotics were : tobramycin [aminoglycoside],
ciprofloxacin [fluoroquinolone], meropenem, ceftazidime [βlactams], and colistin [polymyxin].
-2
Activity of meropenem
% of absorbance (CV)
The same parameters were then evaluated in 4-days old
biofilms exposed during 24h to antipseudomonal antibiotics at
concentrations ranging from 0.001 to 1000 mg/L in order to obtain
full concentration-response curves.
Log of antibiotic concentration (mg/L)
Log of antibiotic concentration (mg/L)
Plates were harvested every day and used to evaluate biofilm
biomass by crystal violet staining and total bacterial viability within
biofilm by the fluorescein diacetate assay (2).
0
0
-1
Crystal Violet
-2
Crystal Violet
-3
Fluorescein
-3
-4
4
3
2
1
0
IC
-1
Fluorescein
M
-2
-3
-4
0
0
% of absorbance (CV)
Fluorescein
Crystal Violet
% of fluorescence (FDA)
125
% of fluorescence (FDA)
125
Log of antibiotic concentration (mg/L)
Log of antibiotic concentration (mg/L)
 Antibiotic activity was concentration-dependent towards both viability and
biomass.
 Pharmacodynamic parameters towards viability:
 Emax ranged from 26% (meropenem) to 51% (colistin) reduction vs untreated
controls.
 C25 ranged from 1 to 25 time the MIC.
 Pharmacodynamic parameters towards viability:
 Emax ranged from 18% (tobramycin) to 41% (meropenem) reduction vs.
untreated controls.
 C25 were higher than 50 times the MIC.
antibiotic
Emax
MIC
C25
Viability
Biomass
µg/mL
%
%
µg/mL
x MIC
µg/mL
x MIC
Ciprofloxacin
0.06
41.07
26.39
0.35
5.85
3.38
56.30
25
Colistin
1
50.59
30.98
23.08
23.08
108.17
108.17
0
Ceftazidime
1
38.23
22.60
1.21
1.21
/
/
Meropenem
1
26.73
40.90
12.73
12.73
100.18
100.18
Tobramycin
0.25
39.31
18.79
3.44
13.76
/
/
75
4
3
2
1
0
-1
-2
-3
-4
EMax
C25
50
% of absorbance (CV)
125
0
PAO1 was used as reference strain. Biofilms were grown in
cation-adjusted Mueller-Hinton broth (CA-MHB) using 96 well cell
culture microplates (clear, TC surface treatment) for cell culture,
with daily renewal of the culture medium over 6 days.
Activity of colistin
125
% of absorbance (CV)
Methods
% of fluorescence (FDA)
Activity of ciprofloxacin
Our aim was to set up a model that reaches maturity in a
shorter time, in order to evaluate the activity of relevant
antibiotics used to treat chronic pseudomonal infections in
CF patients.
% of fluorescence (FDA)
Mailing address :
Y. Diaz Iglesias
Av. Mounier 73 (B1.73.05)
1200 Brussels, Belgium
[email protected]
Viability
Biomass
Log of antibiotic concentration (mg/L)
References
1. Tré-Hardy M. et al. Effect of antibiotic co-administration on young and
mature biofilms of cystic fibrosis clinical isolates: the importance of the
biofilm model. International Journal of Antimicrobial Agents (2009)
33:40-45. PubMed 18801647
Conclusion
2. Peeters E. et al. Comparison of multiple methods for quantification of
microbial biofilms grown in microtiter plates. Journal of Microbiological
Methods (2008) 72:157-165 PubMed 18155789
 Using coated plates and renewing the medium every day allow pseudomonal
biofilms to mature quickly in around 4 days.
YDI received a PhD scholarship from the Fonds pour la Recherche dans l’Industrie et
l’Agriculture and MHM, from the Dorctiris program of Innoviris.. This work was
supported by the Fonds de la Recherche Scientifique and the Fonds Spéciaux de
Recherche from the UCL.
 As expected, antibiotics were much less effective and potent against bacteria in
biofilms than against planktonic bacteria.
This model could be used for the screening of new anti-biofilm agents.
This poster will be made available for download after the meeting at http://www.facm.ucl.ac.be/posters.htm