Acanthamoeba spp.

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.

FEMS Microbiol Ecol 70 (2009) 20–29c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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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

FEMS Microbiol Ecol 70 (2009) 20–29 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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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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.


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.



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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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