R E S E A R C H A R T I C L E
Viabilityof Listeriamonocytogenes in co-culturewithAcanthamoeba spp.Alisha Akya1, Andrew Pointon2 & Connor Thomas1
1University of Adelaide, School of Molecular and Biomedical Science, Adelaide, SA, Australia; and 2Food Safety Group, South Australian Research and
Development Institute, Glenside, SA, Australia
Correspondence: Alisha Akya, Microbiology
Group, School of Medicine, Kermanshah
University of Medical Sciences, Shirudi
Boulevard, PO Box 67148, 69914
Kermanshah, Iran. Tel. (mobile):
09183853123; fax: 198 831 427 6477;
e-mail: [email protected]
Received 28 February 2009; revised 7 May
2009; accepted 18 June 2009.
Final version published online 23 July 2009.
DOI:10.1111/j.1574-6941.2009.00736.x
Editor: Alfons Stams
Keywords
Listeria monocytogenes; Acanthamoeba;
interaction.
Abstract
Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and
can grow and survive under a wide range of environmental conditions. It
contaminates foods via raw materials or food-processing environments. However,
the current knowledge of its ecology and, in particular, the mode of environmental
survival and transmission of this intracellular pathogen remains limited. Research
has shown that several intracellular pathogens are able to survive or replicate within
free-living amoebae. To examine the viability of L. monocytogenes in interaction
with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated
amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanth-
amoeba lenticulata. The survival of bacteria and amoebae was determined using
culture techniques and microscopy. Under the experimental conditions used, all
amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did
not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew
saprophytically on materials released from amoebae, which may play an important
role in the survival of bacteria under extreme environmental conditions.
Introduction
Listeria monocytogenes is an important human pathogen
that produces severe diseases in pregnant women, neonates
and immunocompromised individuals including the
elderly and people with cancer (Farber & Peterkin, 1991;
Vazquez-Boland et al., 2001). Given the increase in the
proportion of aged populations over recent decades and
the longer survival of cancer patients, listeriosis remains a
major food-associated public health concern in developed
countries (Pearson & Marth, 1990). In fact, a number of
reports have indicated that the prevalence of listeriosis in
developed countries has increased during recent years
(Garcia-Alvarez et al., 2006; Koch & Stark, 2006). One of
the main reasons for this trend is the fact that L. mono-
cytogenes can grow in refrigerated foods, even in the
presence of commonly used inhibitory agents used as
preservatives.
However, the current knowledge of the ecology of this
important bacterium and, in particular, the mode of trans-
mission and survival in the environment, remains very
limited. Filling this information gap may assist the food
industry to minimize the potential for contamination of food
materials by this opportunistic pathogen. Recent studies of
the microbial ecology of other intracellular pathogens has
highlighted the potential role of protozoan hosts to act as
reservoirs of these bacteria and, indeed, act as vectors for
dissemination to mammalian hosts (Winiecka-Krusnell &
Linder, 1999, 2001). In particular, Acanthamoeba spp. have
been studied as an alternative host for these pathogens
(Greub & Raoult, 2004). Because Acanthamoeba spp. are
widespread and naturally feed on free-living bacteria, it is
reasonable to assume that this group of protozoa could
interact with Listeria spp. in the environment. Ly & Muller
(1990), in a co-cultivation assay, argued that L. monocytogenes
survived within amoeba cells. Most recently, Zhou et al.
(2007) showed that L. monocytogenes in a co-culture with
Acanthamoeba castellanii survived after predation by amoeba
trophozoites. However, they did not unequivocally show the
replication of bacteria within amoeba cells. Further, Huws
et al. (2008) found no evidence of survival or multiplication
of L. monocytogenes within Acanthamoeba polyphaga cells.
Conversely, they found that Listeria cells were eliminated by
A. polyphaga trophozoites.
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It is possible that these observations are peculiar to
certain Acanthamoeba species or strains. There are published
studies that indicate that not all Acanthamoeba isolates can
harbour intracellular bacteria (Lamothe et al., 2004; Tezcan-
Merdol et al., 2004). Therefore, to determine whether the
ability of A. polyphaga to kill L. monocytogenes is shared by
other Acanthamoeba species, the survival of bacteria in long-
term co-cultivation with the freshly isolated amoebae was
assessed. Further, the role of the major virulence factor of
L. monocytogenes, LLO, in their interaction with amoeba
trophozoites was investigated.
Materials and methods
Bacterial strains and culture conditions
Listeria monocytogenes strains (Table 1) were routinely
cultivated in brain heart infusion (BHI) broth or on BHI
agar plates with incubation at 37 1C overnight with aeration.
When required, DRDC8 was cultured in amoeba-condition
medium (ACM) (Marolda et al., 1999). Briefly, amoebae
were cultured in proteose peptone–yeast extract–glucose
(PYG) (Smirnov & Brown, 2004) supplemented with anti-
biotics (gentamicin, 10 mg mL�1; streptomycin and penicil-
lin, 200mg mL�1) to grow as a confluent monolayer. To
remove any antibiotics and nutrients, the monolayer was
washed three times by adding 10 mL of modified Neff ’s
amoeba saline (AS) (Smirnov & Brown, 2004) to the flask
and decanting the washout. Finally, 5 mL AS was added to
the flask and incubated overnight at room temperature. The
flask was vigorously shaken to resuspend the amoebae. The
cell suspension was subsequently filter sterilized using a
0.45-mm filter. The flow throw was used as ACM.
Escherichia coli DH5a was from laboratory stocks (Table 1).
Escherichia coli was cultivated in Luria media with incubation
at 37 1C. The E. coli cells used to feed amoebae were cultured,
harvested by centrifugation and washed in AS to remove
residual nutrients and resuspended in AS.
Amoebae and culture conditions
All Acanthamoeba spp. were isolated from water and soil for
this study (Table 1). Acanthamoeba polyphaga AC012 was
isolated from fresh water and kindly provided by Brett
Robinson (South Australian Water Quality Centre).
Acanthamoeba castellanii and Acanthamoeba lenticulata were
isolated from Torrens River water and river mud and soil (in
Adelaide) by the techniques described by Smirnov & Brown
(2004). Briefly, 10–20 mL of water samples were filtered
using Whatman number 1 paper (Lorenzo-Morales et al.,
2005). Filters containing trapped amoebae were placed on
the surface of a non-nutrient agar (NNA) plate spread with
live E. coli cells. The E. coli was spread on NNA plates in an X
figuration pattern. Consequently, amoeba trophozoites fed
on bacteria and grew outward into the marginal zones of
plates. To isolate amoebae from soil, 2–5 g of soil or mud was
placed in a small cut area (small cut in agar made using a
sterile scalpel) at the centre of NNA plates spread with live
E. coli. These small wells kept the samples in place and
prevented spreading of soil materials to contaminate whole-
plate surfaces. To clone the recovered amoebae, a small piece
of agar from the marginal area of plates (containing amoeba
cysts) was transferred onto a fresh plate spread with heat-
killed E. coli (marginal cloning method) (Smirnov & Brown,
2004). The growth of amoeba trophozoites was examined
daily with the aid of an inverted microscope.
To make them axenic, they were subcultured several times
by transferring few cysts to a fresh culture in the presence
of antibiotics (gentamicin, 10 mg mL�1; streptomycin and
penicillin, 200 mg mL�1). They were also cultured in PYG
free of antibiotic, followed by examining cultures to rule out
any bacterial growth. All amoebae were typed using PCR
amplification of subgenic 18S rRNA gene and direct PCR
amplicon sequencing (400–500 bp) (Schroeder et al., 2001).
Sequence analysis showed that these strains were highly
similar to A. polyphaga, A. lenticulata and A. castellanii. The
sequence data have been submitted to GenBank and their
accession numbers are presented in Table 1.
Amoebae were routinely cultured in PYG supplemented
with antibiotics as mentioned above in 25-cm2 flasks
(Falcon 3018; Becton Dickinson) at room temperature
(c. 22 1C). Confluent monolayers of amoebae, which
attached to the bottom of the flasks, were washed three
times with AS followed by addition of antibiotic-free PYG to
the flask and incubation overnight. Finally, the monolayers
Table 1. Bacteria and Acanthamoeba strains used in this study
Strains Sources
References/
GenBank accession
numbers
Listeria
DRDC8 Dairy NSW Dairy Corporation,
Sydney, Australia
LLO17 DRDC8: prfA <Tn917 C. Thomas, University of
Adelaide, Adelaide, SA,
Australia
KE1003 Clinical isolate King Edward Hospital,
Perth, WA, Australia
KE504 Clinical isolate Australia
E. coli DH5a Laboratory collection University of Adelaide,
Adelaide, SA, Australia
Acanthamoeba
Lenticulata
AS2
Torrens River water and soil
(river mud)
EF176004
Castellanii
AC328
EF176006
Polyphaga
AC012
South Australian Water
Quality Centre
DQ490964
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21Acanthmoeba eliminate L. monocytogenes cells
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were washed three times in AS buffer and harvested by
tapping the flasks. Amoeba cell counts were determined by
Trypan blue staining and using a hemocytometer.
Co-culture assays
Amoebae and bacteria were co-cultured in 24-well tissue
culture trays as described (Marolda et al., 1999), with some
modification. Briefly, 100mL of a suspension of AC012
(containing 104–105 amoebae) was mixed with 800mL of AS
in each well of a 24-well tray followed by incubation at room
temperature for 1 h to form a monolayer. The amoebae were
then inoculated with 100mL of bacterial suspension (contain-
ing 106–107 bacterial cells) to achieve a multiplicity of
infection (MOI) of 50–100 bacteria per amoeba cell. The
inoculated trays were centrifuged at 180 g for 5 min to
sediment bacteria onto amoebae followed by incubation at
room temperature (c. 22 1C) for 2 h. Monolayers of amoebae
in each well were then washed with 1 mL of AS followed by
the addition of 1 mL of AS containing gentamicin
(50mg mL�1) to kill extra-amoebic bacteria. The trays were
then incubated at room temperature for 2 h followed by
washing three times in AS. Finally, 1 mL of AS buffer was
added to each well and the trays were incubated for 1, 2, 3 and
4 days at 22 1C. To prevent starvation stress of amoebae, each
day, heat-killed E. coli (107 bacteria) was added to co-cultures.
The culture supernatant plus 1 mL of AS used to wash
each well was combined in sterile tubes. Aliquots (100 mL) of
three different dilutions were used for CFU counting and the
mean results of three wells used as extra-amoebic bacteria.
To count intra-amoebic bacteria, amoebae monolayers
attached to the bottom of wells were lysed by addition of
1 mL AS containing Triton X-100 (0.3% v/v). Three aliquots
(100 L) with different dilutions of the lysate in each well
were used for CFU counting and the mean of three wells
used as extra-amoebic bacteria. Counts of amoebae were
carried out using a hemocytometer after resuspension of
amoebae in AS buffer. At least three separate counts were
carried out for each well and the mean count of three wells
was used as the number of amoebae.
To determine the outcome of prolonged interaction,
A. polyphaga, A. lenticulata and A. castellanii were
co-cultured with L. monocytogenes strains. Co-cultivation
was performed in 75-cm2 tissue-culture flasks for 20 days.
Briefly, bacteria were washed three times and diluted in AS,
followed by addition of an aliquot of bacterial suspension to
obtain 106–107 of bacterial cells in 25 mL AS in each flask.
Amoebae were also washed three times, resuspended in AS
and counted using a hemocytometer. An aliquot of amoebae
(103 cells) was added to each flask to obtain an MOI of 103
bacteria per amoeba cell. Bacteria were also cultured as
controls in AS and ACM as described previously. The flasks
were incubated at 30 1C for 20 days and samples were
obtained at determined time points for bacterial CFU
counting. The number of amoebae was also counted in
flasks using inverted microscopy (Kahane et al., 2001).
Briefly, 10–20 microscopic fields were randomly selected
and counted using objective lens power 10 (LPF) and their
average was used to determine the number of amoebae in
each flask. Three flasks were used for each test. This method
of counting was calibrated using the hemocytometer and
microscopy method to determine the concentration of
different amoeba suspensions.
Microscopy
To localize intra-amoebic bacteria, amoebae were co-cultured
with bacteria in 24-well tissue-culture trays as described
previously, except that the amoebae were overlaid on sterile
13-mm-diameter round glass coverslips. At different time
points (1–28 h) following incubation, the coverslips were
gently rinsed in AS, fixed in formalin solution (10% v/v) for
30 min and then washed three times in phosphate-buffered
saline (PBS). The amoebae were then treated with Triton
X-100 (0.3% v/v) for 1 min, washed in PBS and incubated
with diluted (1/50 in PBS) polyclonal rabbit anti-Listeria
antibody (DifcoTM Listeria poly O Antisera Type 1, 4) for
30 min. Unbound antibody was removed by washing three
times in PBS. Coverslips were then treated with FITC-labelled
antibody (anti-rabbit immunoglobulin F(ab)2 fraction affi-
nity isolated fluorescein-conjugated antibody, Silenius),
washed, dried and mounted with Mowiol 4-88 containing
an antibleaching agent (p-phenylene diamine) face down on
slides. The coverslips prepared were examined with a fluores-
cence microscope.
Transmission electron microscopy (TEM)
Amoeba monolayers were prepared and washed, as described
previously, in 25-cm2 flasks. Monolayers of A. lenticulata were
infected with L. monocytogenes cells at an MOI of 50 bacteria
per amoeba cell and incubated at 22 1C for 1 h. Extra-
amoebic bacteria were removed, followed by incubation at
22 1C for 0, 1, 2 and 4 h. The monolayers were then washed
and harvested in AS. Amoebae were pelleted by centrifuga-
tion (180 g for 7 min). Uninfected amoebae were also
prepared in an identical manner as a control. The amoeba
pellets were processed for TEM as essentially described
previously (Gao et al., 1997; Smirnov & Brown, 2004).
Ultrathin sections of resin-embedded amoebae cells were
stained with uranyl acetate and Reynolds lead citrate and
examined in a Phillips EM300 electron microscope.
Statistical analysis
Data were statistically analysed using SPSS software program
to perform the following tests: ANOVA, paired t-test and
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Wilcoxon were used to determine significant changes in the
number of amoebae and bacteria and their trends. Tukey’s
post hoc test was performed to determine the significance
between controls and test data at each time point.
Results
Co-culture of L. monocytogenes withmonolayers of amoeba trophozoites
Counts of total and intra-amoebic bacteria declined during
co-culture with A. lenticulata (Fig. 1a). However, the reduc-
tion in counts of intra-amoebic bacteria was more signifi-
cant, and counts of bacteria changed from c. 105 to
c. 103 CFU mL�1 over 96 h of co-culture. By contrast, esti-
mates of the total numbers of bacteria (intra-amoebic plus
extra-attached bacteria) reduced by only 10-fold. The differ-
ence between these two reduced patterns was statistically
significant (Po 0.05). Counts of amoeba cells fluctuated
only slightly over the duration of the experiment (Fig. 1b)
(P4 0.05). As expected, a proportion of amoeba tropho-
zoites gradually encysted, and by the end of the experiment,
about 45% of all amoeba cells were present as cysts. Similar
results were also obtained for co-culture of bacteria with
A. polyphaga (data not shown).
When A. castellanii was co-cultured with L. monocyto-
genes, the overall progression of changes in counts of intra-
amoebic bacteria and amoeba cells was basically similar to
that reported for A. lenticulata (Fig. 1c). However, counts of
total bacteria increased by 10-fold over the period of
co-culture. This result indicated that extra-amoebic bacteria
grew quite well on nutrients released from amoeba cells or
grew on heat-killed E. coli cells routinely added to the
co-culture as a food source for amoebae to prevent early
encystation. Further, counts of intra-amoebic bacteria de-
creased from c. 104 CFU mL�1 by only 10–15-fold compared
with the 100-fold decrease obtained with A. lenticulata
(Fig. 1c). This lower rate of reduction of intra-amoebic
bacteria compared with that obtained for A. lenticulata
corresponded with the slower rate of growth observed when
this amoeba was co-cultured with L. monocytogenes on the
surface of NNA plates (data not shown). Counts of amoeba
Fig. 1. Monolayers of Acanthamoeba lenticulata (a and b) and Acanthamoeba castellanii (c and d) were infected with Listeria monocytogenes DRDC8
(MOI = 50–100). Results shown are the mean of three replicates. Error bars represent the SD about the mean counts obtained. (a) Counts of total (m)
and intra-amoebic bacteria (’). (b) Counts of amoebae (�) and percent cysts of amoebae (�). (c) Counts of total (m) and intra-amoebic bacteria (’).
(d) Counts of amoebae (�) and percent cysts of amoebae (�). The difference between intra-amoebic and total number of bacteria was statistically
significant for both amoebae (Po 0.05).
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cells declined by c. 40% over the entire period of co-culture
(Fig. 1d). Only about 7% of all amoeba cells were present as
cysts at the end of co-culture.
Prolonged co-cultures of bacteria withAcanthamoeba spp.
Counts of bacteria, amoeba and cysts were assessed dur-
ing co-culture of L. monocytogenes with A. polyphaga cells
(Fig. 2a and b) for 20 days. Over the first 3 days of
co-cultures, counts of bacteria decreased only slightly from
c. 2� 106 CFU mL�1. After 4–6 days of co-culture, counts of
both DRDC8 and LLO17 decreased significantly (by 1000–
10 000-fold) to c. 2� 102 CFU mL�1 (Po 0.05). Thereafter,
the counts of bacteria decreased only marginally for the
remainder of the experiment (Fig. 2a) (P4 0.05). By con-
trast, counts of amoeba cells for both co-cultures gradually
increased from c. 2� 103 cells mL�1 and reached a maximum
of 4.6� 103 cells mL�1 after 6 days of co-culture (Fig. 2b).
This increase was statistically significant in comparison with
the control (amoebae in AS) (Po 0.05). After 8–10 days,
counts of amoeba for both co-cultures and the control
decreased to c. 2.2� 103 cells mL�1 and, thereafter, gradually
decreased to c. 1.1� 103 cells mL�1 (P4 0.05). A gradual
increase in the proportion of amoeba cells converting from
trophozoites to cysts also occurred for both co-cultures and
the control over the course of the experiment (P4 0.05).
When cells of both strains of bacteria were suspended in
AS buffer alone, counts of viable bacteria decreased from
c. 5� 106 to c. 102 CFU mL�1 over a period of 4 days. The
rate of decrease in viability was significantly greater than
that observed when these bacterial strains were co-cultured
with A. polyphaga AC012 (Po 0.05). Similarly, when
DRDC8 and LLO17 were suspended in ACM under identical
conditions, the counts of viable bacteria were similar to
those observed for the co-culture experiments.
In order to rule out the possible impact of strain-specific
factors on interaction of L. monocytogenes with A. polyphaga,
two different clinical isolates of L. monocytogenes (KE504
and KE1003) and one environmental turkey isolate (2T)
were co-cultured with A. polyphaga. Significantly, no major
differences in the counts of bacteria were observed during
co-culture for any strain of L. monocytogenes used
(P4 0.05) (Fig. 3).
The results obtained for co-cultivation with A. lenticulata
and A. castellanii were almost similar to those obtained for
A. polyphaga. However, A. lenticulata trophozoites did not
actively reduce the counts of viable bacteria as rapidly as
A. polyphaga (Fig. 2c and d). Furthermore, trophozoites
underwent early encystations during co-culture (Fig. 2d),
suggesting that trophozoites were under nutrient/physiolo-
gical stress. Acanthamoeba castellanii, like A. lenticulata, was
unable to reduce the number of bacteria effectively during
co-culture. The rate of decline in the number of bacteria in
co-cultures was a little lower than that observed in the
counts of bacteria suspended in ACM alone (Fig. 2e). This
speculation is supported by the fact that numbers of amoeba
did not increase during co-culture, but instead declined to
low levels over the first 10–12 days of co-culture at a rate
similar to that observed for amoeba suspended in AS
(control) (Fig. 2f).
Microscopy of Acanthamoeba cells infected withL. monocytogenes
Fluorescent micrographs of A. lenticulata cells as monolayers
after infection with L. monocytogenes DRDC8 cells are shown
in Fig. 4. Immediately after infection of the amoeba, few
immunolabelled bacteria were observed within the amoeba
cells (Fig. 4a). After 1 and 3 h of co-culture, only a small
proportion of amoeba contained immunolabelled bacteria
(Fig. 4b). With longer co-culture, no intact immunolabelled
intra-amoebic bacteria were observed (Fig. 4c) and the
majority of amoeba cells were present as cysts. During
extended co-culture, immunolabelled bacteria were only
found to be associated with the surface of amoeba cells.
Similar results were obtained for A. castellanii (Fig. 4d–f).
Trophozoites contained labelled intra-amoebic bacteria
1–2 h postinfection (Fig. 4d). However, no labelled bacterial
cells were observed within amoeba cells after 3–4 h of
co-culture (Fig. 4e). With extended co-culture, the majority
of amoeba cells were present as cysts (Fig. 4f).
TEM of A. lenticulata AS2 co-cultured with L. mono-
cytogenes DRDC8 revealed more details of their interaction
(Fig. 5). Bacterial cells were not found in any section of
uninfected control A. lenticulata cells (Fig. 5a). However,
in infected amoeba cells, bacterial cells were found located
within tight vacuoles in thin sections of amoeba after 2 h of
co-culture (Fig. 5b). Bacterial cells within vacuoles had
identifiable cell walls and were apparently intact. After
4–5 h of co-culture, almost all sections of bacteria located
within vacuoles displayed loss of cell-wall structures
(Fig. 5c and d). Sections of vacuoles that contained
bacteria were surrounded by mitochondria and lysosome-
like vesicles, some of which appeared to have merged with
adjacent phagosomal vacuoles. No evidence of long-term
bacterial survival or multiplication within phagosomal
vacuoles or within the cytoplasm of amoeba trophozoites
was obtained.
Discussion
This work was designed to study the interaction of
L. monocytogenes with Acanthamoeba spp. Although the three
types of Acanthamoeba used in co-cultures were able to
eliminate L. monocytogenes, the rates of killing were differ-
ent. Whether this is the case for other types of bacteria
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remains to be determined. Furthermore, prolonged
co-culture of each of the three Acanthamoeba strains used
did not result in growth of amoeba populations – in fact, the
trophozoites used for these experiments rapidly converted
to the cyst form. Given the different results obtained for
co-cultures, it is reasonable to conclude that different
Fig. 2. Counts of bacteria and amoebae during co-culture at 221C for 20 days. Listeria monocytogenes strains DRDC8 and the avirulent variant LLO17,
were co-cultured with Acanthamoeba polyphaga (a and b), Acanthamoeba lenticulata (c and d) and Acanthamoeba castellanii (e and f) (MOI= 1000).
Bacteria were also cultured in AS and ACM as controls. Counts shown are the mean of three replicates. Error bars represent the SD about the mean
counts of bacteria. (a) Counts of viable DRDC8 with A. polyphaga (’), DRDC8 in AS (}), DRDC8 in ACM (�), LLO17 with amoebae (^), LLO17 in AS
(�), LLO17 in ACM (&). (b) Mean counts of amoeba cells in co-culture with DRDC8 (�), with LLO17 (^), in AS (’). Percent cysts of amoebae in co-
culture with DRDC8 (�), LLO17 (}), in AS (&). (c) Counts of viable DRDC8 with A. lenticulata (’), DRDC8 in AS (}), DRDC8 in ACM (�), LLO17 with
amoebae (^), LLO17 in AS (�), LLO17 in ACM (&). (d) Mean counts of A. lenticulata cells in co-culture with DRDC8 (�), with LLO17 (^), in AS (’).
Percent cysts of amoebae in co-culture with DRDC8 (�), LLO17 (}), in AS (&). (e) Counts of viable DRDC8 with A. castellanii (’), DRDC8 in ACM (�),
LLO17 with A. castellanii (^), LLO17 in ACM (&). (f) Mean counts of A. castellanii cells in co-culture with DRDC8 (�), with LLO17 (^), in AS (’).
Percent cysts of amoebae in co-culture with DRDC8 (�), LLO17 (}), in AS (&).
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25Acanthmoeba eliminate L. monocytogenes cells
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Acanthamoeba spp. respond differently during co-culture
with L. monocytogenes – a result that is in agreement with
published observations (Marolda et al., 1999).
Prolonged co-culture of L. monocytogenes with amoeba
cells in flasks provided interesting information concerning
the outcome of interaction between bacteria and amoeba
cells. Given the significantly longer survival time of bacteria
in co-cultures with amoebae compared with cultures sus-
pended in AS buffer, L. monocytogenes cells apparently
benefit from materials released from amoeba cells during
encystation, nonmicrobial-induced lysis and growth. In fact
L. monocytogenes is able to grow and multiply in ACM as
reported for Burkholderia cepacia and Mycobacterium ovium
during co-culture with Acanthamoeba spp. (Steinert et al.,
1998; Marolda et al., 1999). Thus, there is potential for
L. monocytogenes to grow and survive in the extracellular
environment of grazing amoeba populations as long as
numbers of bacteria do not exceed critical levels needed for
efficient predation by amoeba. Consequently, equilibrium
Fig. 4. Localization of Listeria monocytogenes within Acanthamoeba lenticulata (a, b and c) and Acanthamoeba castellanii (d, e and f) trophozoites at
221C. (a) Time = 1 h. The amoeba cell shown contains a large number of immuno-labelled bacterial cells. Bacteria are attached to the cell surface of
amoebae and some have been phagocytosed by amoeba cells. (b) Time = 3 h. Note the lower density of bacterial cells within amoeba compared with
that shown in (a). (c) Time = 19 h. The majority of amoeba cells are free of bacteria. By 19 h incubation, a significant number of amoeba cells had
commenced encystation. (d) Time = 1 h high magnification view of stained bacteria attached to amoeba cells. Fewer bacteria were internalized by this
amoeba in comparison to other amoebae. (e) Time = 3 h. Most bacteria have been phagocytosed by amoeba cells. (f) Time = 19 h. Bacteria attached to
the surface of amoeba cells undergoing encystations. Bar marker = 20mm.
Fig. 3. Counts of four strains of Listeria monocytogenes in co-culture
with Acanthamoeba polyphaga at 221C. Listeria monocytogenes strains
DRDC8 (’), KE504 (m), KE1003 (.) and 2T (^) were co-cultured with
A. polyphaga trophozoites in flasks (MOI=1000) (P4 0.05). Counts
shown are the mean of three replicates. Error bars represent the SD
about the mean counts of bacteria.
FEMS Microbiol Ecol 70 (2009) 20–29c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
26 A. Akya et al.
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populations of L. monocytogenes and amoeba may exist in
the environment.
It is significant that the observed rate of decline in counts
of L. monocytogenes cells during co-culture was not signifi-
cantly different from that observed for identical populations
of L. monocytogenes suspended in ACM. Thus, although the
amoeba may predate and kill bacterial cells at a low level,
survival of extra-amoebic L. monocytogenes cells may be
more significant. Data shown indicated that L. monocyto-
genes cells may survive saprophytically by utilizing nutrients
released by amoeba cells during co-culture. Indeed, this is a
result that may have implications for the persistence of this
pathogen under adverse environmental conditions.
Co-culture experiments with A. lenticulata and A. castella-
nii showed that L. monocytogenes cells could not survive more
than a few hours within phagosomes of these amoebae. This
conclusion is supported by light and TEM examination of
infected amoebae at different stages of co-culture. In fact,
TEM of thin sections of amoeba cells containing L. mono-
cytogenes clearly showed that the bacterial cells were rapidly
degraded within phagosomes and apparently never had an
opportunity to grow and multiply either within the phago-
cytic vacuoles or in the host amoeba cytoplasm. Moreover,
fluorescence micrographs also indicated that bacteria were
eliminated over few hours postco-cultivation. It has been
shown that this hly mutant strain is avirulent for mamma-
lian cells and it is, therefore, not able to establish a
prolonged intracellular invasion (Francis & Thomas, 1996;
Glomski et al., 2003; Kayal & Charbit, 2006).
However, in co-culture with Acanthamoeba spp., there
was no difference in the survival and growth between
virulent and avirulent strains of L. monocytogenes, providing
more evidence to support extra-amoebic growth of bacteria.
These data are also compatible with the results reported in
the study by Zhou et al. (2007). Further, Zhou et al. (2007),
in their comprehensive study, argued that L. monocytogenes
in co-culture with A. castellanii survived after predation by
amoebae trophozoites. However, they did not unequivocally
show evidence to confirm the replication of bacteria within
amoeba cells. One explanation for this discrepancy could be
the different strains of amoebae and bacteria used or, more
likely, the saprophytic growth of extra-amoebic bacteria on
amoeba-released materials, which was clearly observed in
our study. Our results are consistent with the results
Fig. 5. TEM of Listeria monocytogenes within vacuoles of Acanthamoeba lenticulata AS2. (a) Control, uninfected amoeba cell at time 2 h. Note the
large number of vacuolar structures within amoeba cell. (b) A bacterial cell (B) located within a vacuole in an amoeba cell after 2 h incubation. Note the
intact bacterial cell wall (CW) and the vacuolar membrane (M). (c) An infected amoeba cell after 2 h incubation. The micrograph of the cell shows
several bacterial cells (B) located within vacuoles. (d) A bacterial cell (B) within a vacuole (time = 4 h). Note the distinct loss of bacterial cell wall integrity.
Also note the cluster of lysosome-like structures clustered around the vacuole shown and in various stages of fusion with the vacuolar membrane.
FEMS Microbiol Ecol 70 (2009) 20–29 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
27Acanthmoeba eliminate L. monocytogenes cells
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reported by Huws et al. (2008) from co-culture of
A. polyphgaga with L. monocytogenes. They showed that
amoebae eliminated bacteria and, apparently, bacteria were
not able to grow or survive within amoeba trophozoites.
The outcomes of interaction of L. monocytogenes and the
three Acanthamoeba spp. used are in contrast with results
reported by Ly & Muller (1990). They argued that
L. monocytogenes could survive or multiply within Acanth-
amoeba cells. However, they did not show evidence to
unequivocally support the growth of bacteria within amoeba
cells. The contradictory conclusions may reflect differences
in strains of amoeba and the approaches used, or it more
likely reflects the saprophytic growth of extra-amoebic
bacteria, which was observed in our study.
In conclusion, this study has described some important
features of the L. monocytogenes interaction with several
free-living Acanthamoeba spp. The results indicate that, at
least under the in vitro conditions used, amoebae effectively
eliminate bacteria, although with different rates, irrespective
of virulence genes. However, L. monocytogenes cells clearly
grow on materials released from amoeba cells during
encystations, lysis and growth. This may render bacteria
more capable of surviving under different environmental
conditions, which needs further investigation.
Acknowledgements
A.A. gratefully acknowledges the receipt of a scholarship
from Kermanshah University of Medical Sciences in Iran.
We also acknowledge Brett Robinson, South Australian
Water Quality Centre, for providing a strain of amoeba and
Dr Mansour Rezaee (PhD), the Medical Statistic Group of
Kermanshah University of Medical Sciences, for his com-
ments on the statistical analysis of data.
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