1. Burkholderia
spp.
tolerance
to
desicca-on
and
their
environmental
niches
Madeline
Lummis1,
Kenzie
Shippy1,
Bri<any
Bayer1,
Carina
M.
Hall1,
Joseph
D.
Busch1,
Jennifer
Ginther1,
Vanessa
Theobald2,
Mark
Mayo2,
Bart
J.
Currie2,
Paul
Keim1,
David
M.
Wagner1
1Center
for
Microbial
Gene-cs
and
Genomics,
Northern
Arizona
University,
1298
S
Knoles
Drive,
Flagstaff,
AZ
86011,
USA
2Menzies
School
of
Health
Research,
Casuarina,
NT,
Australia
Burkholderia
pseudomallei
is
a
gram-­‐nega-ve
soil
bacterium
that
is
the
causa-ve
agent
of
melioidosis.
Closely
related
species
of
Burkholderia
(B.
pseudomallei
near-­‐neighbors)
also
dwell
in
the
soil
but
generally
are
non-­‐pathogenic
to
healthy
individuals.
Currently,
there
is
li<le
understanding
of
the
types
of
environmental
stresses
these
soil
bacteria
endure
and
what
their
tolerance
level
is
for
these
stressors.
Our
goal
is
to
expand
our
knowledge
on
this
unique
group
of
soil
dwelling
bacteria.
Chemical
desicca-on
was
produced
by
using
polyethylene
glycol
(PEG)
to
dehydrate
the
cell
at
various
concentra-ons.
We
measured
the
density
of
cells
(amount
of
bacterial
growth)
over
a
30
hour
period
then
compared
their
growth
curves.
We
started
by
inves-ga-ng
desicca-on
effects
on
three
geographically
diverse
strains
of
B.
vietnamiensis
from
Arizona,
Florida,
and
Louisiana.
Due
to
the
lack
of
rainfall
Arizona
experiences,
we
expected
to
find
that
the
Arizona
strain
would
be
able
to
withstand
higher
desicca-on
pressures
unlike
the
strains
from
higher
moisture
environments.
However,
we
found
the
Arizona
strain
was
the
most
suscep-ble
to
desicca-on.
Next,
to
determine
if
tolerance
to
desicca-on
is
species
specific
we
tested
various
near
neighbor
species
from
the
same
geographic
loca-on
in
Northern
Australia.
We
found
B.
ubonensis
had
the
highest
tolerance
to
desicca-on
while
B.
humptydooensis
had
the
lowest
desicca-on
tolerance.
To
confirm
this
species
specific
trait
we
then
tested
mul-ple
strains
from
the
two
species
of
interest.
These
experiments
provided
us
with
insight
to
how
various
Burkholderia
species
are
affected
by
desicca-on
stress
in
their
environment.
Abstract
Introduc-on
•
Burkholderia
spp.
are
soil
dwelling
bacteria
found
worldwide
•
B.
pseudomallei
is
the
most
virulent
species
of
the
Burkholderia
genus
causing
the
disease
melioidosis
in
humans
•
B.
pseudomallei
is
endemic
in
Southeastern
Thailand
and
Northern
Australia
•
Northern
Australia
has
a
high
diversity
of
Burkholderia,
which
is
why
it
was
the
primary
focus
to
compare
species
(Fig.
1)
Methods
Results
Discussion
Over
a
30
hour
period
the
cell
density
was
measured
by
op-cal
density
at
a
wavelength
of
600nm
(OD600).
Burkholderia
spp.
were
grown
in
Luria-­‐Bertani
broth
with
0%,
10%,
and
20%
PEG
(polyethylene
glycol)
in
duplicates.
•
All
Burkholderia
spp.
were
affected
by
PEG
seen
either
in
the
log
phase
(Fig.
5A
and
Fig.
4)
or
in
the
sta-onary
phase
(Fig.
5B)
•
Some
samples
were
more
affected
by
desicca-on
than
others
(Fig.
3)
but
there
does
not
appear
to
be
a
species
specific
associa-on
(Fig.
4
and
5)
•
Possibly
the
samples
least
affected
by
PEG
are
able
to
survive
in
the
upper
(drier)
sec-on
of
the
soil,
whereas
those
more
affected
are
found
in
deeper
(more
moist)
soil,
resul-ng
in
the
occupa-on
of
various
niches
in
the
same
environment
•
B.
vietnamiensis
from
Arizona
had
a
much
greater
doubling
-me
than
all
other
samples,
while
having
the
smallest
difference
between
the
untreated
and
20%
treatment
maximum
OD600
•
Possibly
a
greater
doubling
-me
results
in
a
longer
log
phase
•
Future
research
could
include
increasing
the
sample
size
to
determine
if
there
is
more
varia-on
within
species
Sources
Ginther
J,
Mayo
M,
Warrington
D,
et.
al
(2013)
Iden-fica-on
of
Burkholderia
pseduomallei
near-­‐neighbors
in
the
Northern
Territory
of
Australia
Hamid
S,
Bae
W,
Kim
S,
Amin
M
(2014)
Enhancing
co-­‐metabolic
degrada-on
of
trichloroethylene
with
toluene
using
Burkholderia
vietnamiensis
G4
encapsulated
in
polyethylene
glycol
polymer
Figure
1:
Distribu-on
of
Burkholderia
spp.
found
in
Northern
Australia.
y
=
0.214x
-­‐
0.6107
R²
=
0.9866
y
=
0.0982x
-­‐
0.481
R²
=
0.99171
y
=
0.0246x
-­‐
0.1226
R²
=
0.97606
0
1
2
3
0
5
10
15
20
25
30
OD600
Time
(hour)
B.
humptydooensis
log
phase
y
=
0.2071x
-­‐
0.4389
R²
=
0.99365
y
=
0.1327x
-­‐
0.5099
R²
=
0.95748
y
=
0.0844x
-­‐
0.4083
R²
=
0.98235
0
1
2
3
0
5
10
15
20
25
30
OD600
Time
(hour)
B.
vietnamiensis
log
phase
Figure
3:
Growth
curves
of
B.
vietnamiensis
and
B.
humptydooensis
(red
box
represents
data
points
used
for
the
log
phase).
Figure
2:
Map
of
B.
vietnamiensis
sampling
loca-ons
within
the
Southern
United
States.
•
Our
ques-ons:
1. Do
various
species
from
the
same
geographical
loca-on
respond
differently
to
desicca-on?
2. Does
geographical
distribu-on
play
a
role
in
tolerance
to
desicca-on?
Polyethylene
glycol
(PEG)
is
a
molecular
desiccator
that
imposes
a
stress
on
bacterial
cells.
0.0
0.1
1.0
10.0
0
5
10
15
20
25
30
OD600
Time
(hour)
B.
vietnamiensis
growth
curve
0.0
0.1
1.0
10.0
0
5
10
15
20
25
30
OD600
Time
(hour)
B.
humptydooensis
growth
curve
Data
were
collected
to
calculate
the
effects
PEG
had
on
growth.
Figure
4:
Average
doubling
-mes
of
replicates
for
0%
and
20%
PEG
treatments.
(doubling
-me=
-me/number
of
genera-ons)
(A)
(B)
•
Other
members
of
the
Burkholderia
genus
can
both
cause
disease
in
immune
comprised
individuals
and
be
used
for
bioremedia-on
(Hamid
et.
al
2014)
•
All
B.
vietnamiensis
samples
(n=5)
were
collected
in
the
United
States
(Fig.
2)
while
all
other
samples
(n=7)
were
collected
in
Northern
Australia
(Fig.
1)
0%
20%
40%
60%
80%
100%
Percent
affected
by
PEG(%)
Sample
PEG-­‐Reduced
Final
PopulaTon
Density
Figure
5:
The
average
percent
decrease
of
the
(A)
slope
during
the
log
phase
from
the
0%
to
the
20%
PEG
treatments
(B)
maximum
OD600
from
the
0%
to
the
20%
PEG
treatments.
0%
20%
40%
60%
80%
100%
Percent
affected
by
PEG
(%)
Sample
Reduced
Efficiency
of
PopulaTon
Growth
least
Response
to
PEG
greatest
Legend:
0%
PEG
10%
PEG
20%
PEG
0
50
100
150
200
250
Doubling
Time
(minutes)
Sample
Doubling
Time
Untreated
Treated
Typical
Bacterial
Growth
Curve