This document presents research finding that filamentous bacteriophage produced by Pseudomonas aeruginosa biofilms organize the biofilm matrix into a liquid crystalline structure. This liquid crystalline structure increases the biofilm's tolerance to aminoglycoside antibiotics by binding and sequestering the antibiotics within the matrix. Specifically, mixtures of Pf4 phage and host/bacterial polymers were found to spontaneously assemble into liquid crystals. P. aeruginosa biofilms producing high levels of Pf4 phage also exhibited a liquid crystalline biofilm matrix structure. This liquid crystalline structure enhanced the biofilm's resistance to killing by tobramycin and increased the binding of fluorescent-labeled tobramycin within the matrix.

1. Mucin + DNA
0 107 108 109 1010
0.10
0.15
0.20
PFU/ml
!sin(δ)!
p<0.05
tobramycin
WT Pf++
0
1
2
killing,log10
p<0.02
WT Pf++
0
1
2
3
5×1004
7×1004
9×1004
Pf4/104CFUs
p<0.01
WT Pf++ WT Pf++
0.0
0.2
0.4
0.6
!sin(δ)!
washed
p<0.01
A
WT
Pf++
WT
Pf++
C
0.75
0.38
0.0
|sin(δ)|
References
1.
Costerton
JW
et
al.
(1999),
Science.
2.
Whiteley
M
et
al.
(2001),
Nature.
3.
Webb
JS,
Lau
M
&
Kjelleberg
S.
(2004),
J
Bacteriol
.
4.
Dogic
Z
&
Fraden
S.
(2006),
Curr
Opin
Colloid
In.
5.
Glazer
AM
et
al.
(1996),
P
Roy
Soc
Lond
a
Mat.
Filamentous
bacteriophage
organize
biofilms
into
liquid
crystals,
increasing
anTbioTc
tolerance
IntroducTon
Lia
A.
Michaels1,
Patrick
R.
Secor1,
William
C.
Parks2,
and
Pradeep
K.
Singh1
1
Departments
of
Medicine
and
Microbiology,
University
of
Washington,
SeaYle,
WA;
2
Department
of
Pulmonary
and
CriTcal
Care
Medicine,
Cedar
Sinai
Medical
Center,
Los
Angeles,
CA
Biofilms
are
aggregates
of
bacteria
within
a
polymer-­‐rich
extracellular
matrix.
The
formaTon
of
biofilms
protects
bacteria
from
environmental
stresses.
For
example,
bacteria
within
biofilms
become
tolerant
to
desiccaTon,
immune
defenses,
and
anTbioTc
treatment,
contribuTng
to
persistent
infecTons
(1).
Therefore,
understanding
the
structure
and
funcTon
of
the
biofilm
matrix
is
important
in
understanding
the
pathogenesis
of
chronic
bacterial
infecTons
such
as
diabeTc
ulcers,
burn
wounds,
and
airway
infecTons
associated
with
cysTc
fibrosis
.
Fig
3.
The
liquid
crystalline
matrix
enhances
an=bio=c
tolerance
by
binding
aminoglycosides.
A.
Killing
of
biofilms
by
tobramycin
(10
µg/ml)
relaTve
to
untreated
controls.
B.
Tobramycin
or
ciprofloxacin
were
added
to
DNA
(2.5
mg/ml),
Pf4
(1010
PFUs/ml),
and
DNA
+
Pf4
to
invesTgate
binding.
C.
Binding
of
tobramycin
to
DNA,
Pf4,
and
DNA
+
Pf4
was
visualized
by
adding
fluorescently
conjugated
tobramycin
(Cy5-­‐
tobramycin,
40
µg/ml).
Scale
bars,
20
µm.
Conclusions
• Mixtures
of
Pf4
and
host
&
microbial
polymers
spontaneously
assemble
into
liquid
crystals.
•
P.
aeruginosa
biofilms
producing
Pf4
have
a
liquid
crystalline
matrix.
• Liquid
crystalline
structure
of
the
biofilm
matrix
increases
tolerance
to
aminoglycoside
anTbioTcs.
• Liquid
crystalline
structures
enhance
the
binding
of
aminoglycosides.
New
therapeuTc
strategies
might
be
developed
targeTng
phage
producTon
or
the
liquid
crystalline
matrix.
Given
that
several
species
of
Gram-­‐negaTve
bacteria
harbor
filamentous
phage,
our
observaTons
might
be
applied
to
other
pathogens
such
as
E.
coli
or
Vibrio
cholerae.
A
Results
(cont.)
Acknowledgments
This
work
was
funded
by
a
CysTc
Fibrosis
FoundaTon
Postdoctoral
Fellowship
to
PRS
and
a
BasseY
scholarship
supported
LAM.
Fig
2.
The
P.
aeruginosa
biofilm
matrix
is
organized
by
Pf4
into
a
liquid
crystal.
A.
Pf4
producTon
by
wild
type
(WT)
and
Pf
over-­‐
producing
(Pf++)
biofilms
were
enumerated
and
normalized
to
bacterial
CFUs.
B.
Birefringence
(normalized
for
thickness)
was
quanTfied
in
WT
and
Pf++
biofilms
pre
and
post
washing
to
remove
the
extracellular
matrix.
C.
QualitaTve
image
of
birefringence
in
WT
and
Pf++
biofilms.
Results
ObservaTons
&
Hypothesis
• Pseudomonas
aeruginosa
is
an
opportunisTc
bacterial
pathogen.
• As
P.
aeruginosa
biofilms
develop,
many
laboratories
have
observed
the
producTon
of
filamentous
Pf
phage
(up
to
1011
PFU/ml)
under
a
variety
of
growth
condiTons
(2,3).
• Clinical
isolates
and
laboratory
strains
of
P.
aeruginosa
harbor
Pf
phage.
For
example,
P.
aeruginosa
strain
PAO1
harbors
Pf4.
• We
observe
~108
Pf
phage/ml
in
sputum
from
cysTc
fibrosis
paTents.
• Like
other
filamentous
phage,
Pf
phage
are
long,
filamentous,
and
negaTvely
charged.
• When
suspended
in
polymer
soluTons,
filamentous
phage
spontaneously
assemble
liquid
crystals
due
to
their
physical
properTes
(4).
• Liquid
crystals
are
a
state
of
maYer
between
that
of
a
liquid
and
a
solid.
Hypothesis
1:
Pf
phage
spontaneously
assemble
liquid
crystals
in
the
presence
of
host
and
bacterial
polymers.
Methods:
A
custom
built
microscope
(5)
was
used
to
measure
an
opTcal
property
of
liquid
crystals
called
birefringence,
which
is
the
splijng
of
passing
light
into
two
beams.
This
device
measures
birefringence
as|sin
(δ)|,
a
measure
of
the
phase
difference
between
the
two
beams.
Birefringence
is
a
direct
measurement
of
the
molecular
alignment
of
a
sample.
Physiologically
relevant
polymers
were
mixed
with
Pf
phage
and
birefringence
was
quanTtated.
Hypothesis
2:
The
matrix
of
phage
producing
biofilms
show
liquid
crystalline
organizaTon.
Methods:
Birefringence
was
quanTtated
as|sin
(δ)|in
biofilms
producing
either
basal
amounts
of
Pf
phage
or
abundant
amounts
of
Pf
phage.
Results:
Pf
phage
assemble
the
biofilm
matrix
into
a
birefringent,
liquid
crystalline
structure.
Hypothesis
3:
The
liquid
crystalline
matrix
enhances
anTbioTc
tolerance
by
binding
aminoglycoside
anTbioTcs.
Methods:
Biofilms
were
treated
with
the
indicated
anTbioTcs.
To
test
the
effects
of
liquid
crystal
assembly
on
anTbioTc
tolerance,
bacteria
were
added
to
the
indicated
phage
+
polymer
soluTons
followed
by
anTbioTc
treatment.
Fluorescently
conjugated
tobramycin
was
added
to
liquid
crystalline
mixtures
of
Pf
phage
and
DNA
to
directly
visualize
binding.
A
B
Pf4
DNA
0.5
0.25
0.0
|sin(δ)|
DNA
+PF4
Fig
1.
Pf4
assemble
disease
relevant
polymers
into
birefringent
liquid
crystals.
A.
Purified
Pf4
(1011
PFU/ml)
and
DNA
(10
mg/ml)
alone
are
not
birefringent,
but
1:1
mixtures
of
Pf4
and
DNA
assemble
birefringent
liquid
crystals.
Scale
bar,
10
µm.
B.
Birefringence
was
quanTfied
in
disease
relevant
concentraTons
of
mucin
(8%
solids)
mixed
with
DNA
(4
mg/ml)
supplemented
with
Pf4.
E. coli
Control DNA Pf4 DNA+Pf4
0.00
0.01
1
2
3
antibiotic,µg/ml
tob
cipro
liquid crystal
B
DNA
Pf4
DNA+Pf4
C
Results:
Host
and
microbial
polymers
interact
with
Pf
phage
at
disease
relevant
concentraTons
to
spontaneously
assemble
birefringent
liquid
crystals,
similar
to
other
filamentous
phage
in
the
presence
of
syntheTc
polymers
(4).
Liquid
crystals
are
inherently
viscoelasTc
in
nature
and
viscoelasTc
materials
generally
display
reduced
rates
of
diffusion.
Altering
the
viscoelasTc
and
diffusion
properTes
of
the
biofilm
matrix
would
likely
impact
several
disease
phenotypes
such
as
anTbioTc
tolerance.
Primary
hypothesis:
The
matrix
of
phage
producing
biofilms
show
liquid
crystalline
organiza=on,
enhancing
an=bio=c
tolerance.
B
250
µm
250
µm
Results:
Biofilms
with
a
liquid
crystalline
matrix
are
tolerant
to
aminoglycosides.
Liquid
crystals
formed
from
Pf
phage
and
DNA
efficiently
bind
aminoglycosides.