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Viable but Not Culturable Forms of Legionella pneumophila Generated After Heat Shock Treatment Are Infectious for Macrophage-Like and Alveolar Epithelial Cells After Resuscitation on Acanthamoeba polyphaga.
[legionellosis]
Legionella
pneumophila
,
the
causative
agent
of
legionellosis
is
transmitted
to
human
through
aerosols
from
environmental
sources
and
invades
lung
's
macrophages
.
It
also
can
invade
and
replicate
within
various
protozoan
species
in
environmental
reservoirs
.
Following
exposures
to
various
stresses
,
L
.
pneumophila
enters
a
non-replicative
viable
but
non-culturable
(
VBNC
)
state
.
Here
,
we
evaluated
whether
VBNC
forms
of
three
L
.
pneumophila
serogroup
1
strains
(
Philadelphia
GFP
008
,
clinical
044
and
environmental
RNN
)
infect
differentiated
macrophage-like
cell
lines
(
U
937
and
HL-
60
)
,
A
549
alveolar
cells
and
Acanthamoeba
polyphaga
.
VBNC
forms
obtained
following
shocks
at
temperatures
ranging
from
50
to
70
Â
°
C
for
5
to
60
Â
min
were
quantified
using
a
flow
cytometric
assay
(
FCA
)
.
Their
loss
of
culturability
was
checked
on
BCYE
agar
medium
.
VBNC
forms
were
systematically
detected
upon
a
70
Â
°
C
heat
shock
for
30
Â
min
.
When
testing
their
potential
to
resuscitate
upon
amoebal
infection
,
VBNC
forms
obtained
after
30
Â
min
at
70
Â
°
C
were
re
-cultivated
except
for
the
clinical
strain
.
No
resuscitation
or
cell
lysis
was
evidenced
when
using
U
937
,
HL-
60
,
or
A
549
cells
despite
the
use
of
various
contact
times
and
culture
media
.
None
of
the
strains
tested
could
infect
A
.
polyphaga
,
macrophage-like
or
alveolar
epithelial
cells
after
a
60
-
min
treatment
at
70
Â
°
C
.
However
,
heat-treated
VBNC
forms
were
able
to
infect
macrophage-like
or
alveolar
epithelial
cells
following
their
resuscitation
on
A
.
polyphaga
.
These
results
suggest
that
heat-generated
VBNC
forms
of
L
.
pneumophila
(
i
)
are
not
infectious
for
macrophage-like
or
alveolar
epithelial
cells
in
vitro
although
resuscitation
is
still
possible
using
amoeba
,
and
(
ii
)
may
become
infectious
for
human
cell
lines
following
a
previous
interaction
with
A
.
polyphaga
.
Diseases
Validation
Diseases presenting
"alveolar epithelial cells after a 60"
symptom
legionellosis
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