Benjamin Taylor1, 2, David Williams1 & Jon Nosworthy3. 1Cardiff University School of Dentistry 2Knowledge Transfer Partnership Associate 3Advanced Medical Solutions plc

1. MODELLING WOUND BIOFILMS IN A
THERMO-REVERSIBLE MATRIX WITH
FLUORESCENT MARKERS
Benjamin Taylor1, 2, David Williams1 & Jon Nosworthy3.
1Cardiff University School of Dentistry
2Knowledge Transfer Partnership Associate
3Advanced Medical Solutions plc

2. Fluorescent probes
Today ‘off the shelf’ molecular markers are readily
available to assess bacterial viability.
SYTO®9 (Life Technologies Ltd, Paisley, UK) is a
fluorescent marker which binds nucleic acid
within live cells where it fluoresces green (light
wavelength 500 nm in live microorganisms) when
excited by light at 480nm.
What is a thermo-reversible matrix?
Poloxamer (Poloxamer 407: Sigma-Aldrich
company Ltd, Dorset, UK) is an inert powdered
polymer which can be dissolved into standard
bacteriological culture broth such as Muller-
Hinton or Tryptone Soya Broth.
The dissolved Poloxamer forms a semi-firm gel
matrix at temperatures > ≈ 15oC and liquefies < ≈
15oC.
It has been suggested that organisms inoculated
and suspended in a Poloxamer matrix form
microcolonies with a biofilm phenotype (Gilbert
et al., 1998; Clutterbuck et al., 2007; Yamanda et
al., 2011).
Aims:
• Develop a simple fluorescent in vitro model
able to support bacterial cells as a biofilm
phenotype.
• Use the test model to examine potential
antibiofilm compounds for antibiofilm activity
using fluorescence as an indicator.
Introduction
Image 1: Confocal image of P. aeruginosa stained
with SYTO®9 fluorescing at 500 nm.

3. Preparing Poloxamer biofilms
A B
C
D
1 2
A) 1) A standardised culture of P. aeruginosa (ATCC 9027) and 2)
Tryptone Soya Broth (TSB) + 30 % Poloxamer was prepared.
B) A sample of P. aeruginosa was inoculated into cooled (<15oC) TSB +
Poloxamer and thoroughly homogenised.
C) A 250-µL sample was then added to 1.5ml centrifuge tubes, avoiding
the formation of air bubbles.
D) All centrifuge tubes were incubated (with lids open) at 35oC for 24
hours in 60% relative humidity to prevent drying.

4. Extracting biofilm cells from Poloxamer
E F G
H
E) The ‘biofilm tubes’ were removed from the incubator and
‘flash cooled’ at -70oC for 2-3 minutes to liquefy the poloxamer.
F) A 500-µL volume of chilled (5-6oC) sodium chloride peptone
broth was added and homogenised into each biofilm tube.
G) All tubes were centrifuged at 3000 g for 5 minutes.
H) The supernatant was discarded and procedure F, G and H
repeated if necessary.

5. Testing novel antibiofilm compounds against extracted cells
I J K
L
M
I) A 100-µL dilution of antibiofilm compound was
added to extracted cells for a given time.
J) The antibiofilm compounds were washed out
with neutralisers.
K) Cells were re-suspended in equal volumes of
buffer and SYTO 9® live fluorescent stain.
L) 100-µL samples were dispensed into a clear-
bottom black 96-well plate and incubated at 21oC
for 15 minutes in the dark.
M) Fluorescence and data was captured by
excitation/emission scanning at 480/500 nm
wavelengths .
Note: Steps D to M were repeated for P.
aeruginosa grown in TSB as a planktonic control.

6. Results
Anti-biofilm compound A
50 100 150 200 250 300 350 400 450 500 550
-50
-40
-30
-20
-10
0
10
20
30
Time (seconds)
%changeRFU*
(emissionat500nm)
Figure 1. Kill rates of P. aeruginosa when exposed to antibiofilm compound A.
Black line = biofilm control (TSB + 30% poloxamer + Sterile H2O). Blue line = biofilm test (TSB + 30%
poloxamer + compound A). Green line = planktonic control (TSB + Sterile H2O). Red line = planktonic
test (TSB + compound A). Error bars show ± SEM (n=14; 2 biofilm/planktonic samples read at 7
different spatial points). *Relative Florescent Units.

7. Anti-biofilm compound B
50 100 150 200 250 300 350 400 450 500 550
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
10
20
Time (seconds)
%changeRFU*
(emissionat500nm)
Figure 2. Kill rates of P. aeruginosa when exposed to antibiofilm compound B.
Black line = biofilm control (TSB + 30% poloxamer + Sterile H2O). Blue line = biofilm test (TSB + 30%
poloxamer + compound B). Green line = planktonic control (TSB + Sterile H2O). Red line = planktonic
test (TSB + compound B). Error bars show ± SEM (n=14; 2 biofilm/planktonic samples read at 7
different spatial points). *Relative Florescent Units.

8. Anti-biofilm compound C
50 100 150 200 250 300 350 400 450 500 550
-80
-70
-60
-50
-40
-30
-20
-10
0
10
Time (seconds)
%changeRFU*
(emissionat500nm)
Figure 3. Kill rates of P. aeruginosa when exposed to antibiofilm compound C.
Black line = biofilm control (TSB + 30% poloxamer + Sterile H2O). Blue line = biofilm test (TSB +
30% poloxamer + compound C). Green line = planktonic control (TSB + Sterile H2O). Red line =
planktonic test (TSB + compound C). Error bars show ± SEM (n=14; 2 biofilm/planktonic
samples read at 7 different spatial points). *Relative Florescent Units.

9. Anti-biofilm compound D
50 100 150 200 250 300 350 400 450 500 550
-70
-60
-50
-40
-30
-20
-10
0
Time (seconds)
%changeRFU*
(emissionat500nm)
Figure 4. Kill rates of P. aeruginosa when exposed to antibiofilm compound D.
Black line = biofilm control (TSB + 30% poloxamer + Sterile H2O). Blue line = biofilm test (TSB +
30% poloxamer + compound D). Green line = planktonic control (TSB + Sterile H2O). Red line =
planktonic test (TSB + compound D). Error bars show ± SEM (n=14; 2 biofilm/planktonic samples
read at 7 different spatial points). *Relative Florescent Units.

10. Summary
• The use of Poloxamer grown biofilm cells could be a useful tool for screening potential
antibiofilm compounds.
• Using Poloxamer grown biofilms enabled the examination of 4 novel compounds to be
assessed for potential activity against P. aeruginosa biofilms.
• Comparisons of biofilm and planktonic curves demonstrated that planktonic P. aeruginosa
had more rapid reduction in fluorescence compared to biofilm cells after exposure to
antimicrobials.
• This method highlights the known increased tolerance to antimicrobials observed in biofilm
developed cells.
• This method can readily be expanded to cover a wide range of pathogenic organisms from
wounds including multi-drug resistant organisms, anaerobes, yeasts and mixed culture
consortia.
Acknowledgements
• The authors wish to acknowledge the support received by a Knowledge Transfer Partnership (KTP)
program to facilitate this research.
References
• Clutterbuck AL, Cochrane CA, Dolman J & Percival SL (2007) Evaluating antibiotics for use in medicine using
a poloxamer biofilm model. Ann. Clin. Microbiol. Antimic. 6:2
• Gilbert P, Jones MV, Allison DG, Heys S, Maira T & Wood P (1998) The use of poloxamer hydrogels for the
assessment of biofilm susceptibility towards biocide treatments. J. Appl. Microbiol. 85(6) 985-90.
• Yamada H, Koike N, Ehara T & Matsumoto T (2011) Measuring antimicrobial susceptibility of Pseudomonas
aeruginosa using Poloxamer 407 gel. J. Infect. Chemother. 17(2):195-9.