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Appl Environ Microbiol, January 1998, p. 279-286, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Production of Respirable Vesicles Containing Live
Legionella pneumophila Cells by Two
Acanthamoeba spp.
Sharon G.
Berk,1,*
Rebecca S.
Ting,1
Glenn W.
Turner,2,
and
Rebecca
J.
Ashburn1
Center for the Management, Utilization and
Protection of Water Resources, Tennessee Technological University,
Cookeville, Tennessee 38505,1 and
Biology Department, Middle Tennessee State University,
Murfreesboro, Tennessee 371322
Received 23 June 1997/Accepted 31 October 1997
 |
ABSTRACT |
Two Acanthamoeba species, fed at three temperatures,
expelled vesicles containing living Legionella pneumophila
cells. Vesicles ranged from 2.1 to 6.4 µm in diameter and
theoretically could contain several hundred bacteria. Viable L. pneumophila cells were observed within vesicles which had been
exposed to two cooling tower biocides for 24 h. Clusters of
bacteria in vesicles were not dispersed by freeze-thawing and
sonication. Such vesicles may be agents for the transmission of
legionellosis associated with cooling towers, and the risk may be
underestimated by plate count methods.
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INTRODUCTION |
In 1980, Rowbotham (10)
demonstrated that Legionella spp. could reproduce within
environmental isolates of amoebae; since then, there have been numerous
laboratory studies of interactions between amoebae and
Legionella spp., mainly in coculture experiments. Most
studies concur that amoebae must play a major role in the epidemiology
of legionellosis through their ability to amplify numbers of
legionellae. Rowbotham (10, 11) has previously described the
cycle of amplification of bacteria in amoeba cultures. At some stage
after the ingestion of bacteria, amoebae produce vesicles that contain
high numbers of legionellae. At 35°C, amoebae usually lyse, releasing
numerous free bacteria or a few vesicles. Rowbotham (11)
hypothesized that the inhalation of vesicles of tightly packed
legionellae is dangerous when the particles are of respirable size (1 to 5 µm in diameter) and contain motile bacteria. He stated that such
a particle, protected by a membrane, may reach the alveoli of the lungs
and contain an infectious dose of legionellae.
The present work reports the production of vesicles from two species of
amoebae fed a cooling tower isolate of Legionella pneumophila at temperatures found in cooling towers. Some
temperatures allowed amoebae to remain viable and thereby able to serve
as a continuous source of such vesicles filled with legionellae. Vesicles were also exposed to cooling tower biocides to determine whether bacteria could remain viable within vesicles after such treatment.
| |
MATERIALS AND METHODS |
Organisms and growth conditions.
Acanthamoeba
polyphaga (ATCC 30461) and A. castellanii (ATCC 30010)
were obtained from the American Type Culture Collection and were
maintained axenically in plate count broth (PCB) (Difco, Detroit,
Mich.) at 28°C. L. pneumophila (ATCC 33216) was obtained from the American Type Culture Collection and was originally isolated from a cooling tower. It was maintained on Legionella
selective agar (Becton-Dickinson, Cockeysville, Md.) in a 5%
CO2 atmosphere at 35°C. For feeding studies, bacteria
(48-h old) were washed from agar plates with a Tris-buffered salts
solution (TBSS) (2 mM KCl, 1 mM CaCl2, 0.5 mM
MgCl2, 1 mM Tris, [pH 6.8 to 7.2]). Escherichia
coli (ATCC 25922) was used for a control as described below.
Feeding experiments.
Axenic amoeba trophozoites were washed
by replacing the medium from their tissue culture flasks with TBSS.
They were fed either live or heat-treated L. pneumophila
cells in 4 ml of TBSS or in PCB. Heat-treated bacteria were exposed for
45 min at 85 to 90°C and then plated on buffered charcoal-yeast
extract (BCYE) agar to determine whether they were killed. Previous
treatment at 70°C was not effective in killing all bacteria. A
multiplicity of infection (MOI) of 10,000 produced many vesicles;
however, vesicles were also produced at MOIs of approximately 30 to
300. The numbers of vesicles per amoeba were determined for A. polyphaga at MOIs of 100, 400, 700, 1,000, and 10,000; since
A. castellanii produced fewer vesicles per amoeba, MOIs of
1,000 and 10,000 were tested.
Amoebae were allowed to feed on L. pneumophila cells for
24 h in PCB or TBSS before the medium was examined for the
presence of vesicles expelled from amoebae. Vesicle production was
tested at 25, 30, and 35°C. Experiments in which the numbers of
vesicles per amoeba were calculated were conducted only at 25 and
30°C, since amoebae lysed at 35°C when they were fed live L. pneumophila cells. Cellular debris from lysed amoebae (at 35°C)
made the enumeration of small vesicles difficult, and the actual
numbers of amoebae involved in vesicle production could not be
determined. Samples of vesicles were examined by differential
interference contrast microscopy for measurements of vesicle diameters
and to confirm the presence of bacteria within vesicles. One hundred
randomly selected vesicles from each species were measured. Other
vesicle samples from amoebae fed live and killed L. pneumophila cells were treated with the respiratory indicator
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) (Eastman Kodak, Rochester, N.Y.) at a final
concentration of 0.05% to test the viability of bacteria within free
vesicles. During active respiration, INT becomes reduced to an
insoluble INT-formazan precipitate. The treatment of heat-killed bacteria served as a control for the performance of the INT technique, i.e., to show that formazan crystals would not form in the vesicles of
amoebae that were fed dead bacteria. In addition, since free bacteria
outside of vesicles had INT-formazan products after exposure of the
suspension to biocides (see below), L. pneumophila
suspensions were exposed in a separate test to up to 80 ppm of MBC 115 (MBC represents microbiocide) and to up to 200 ppm of MBC 215 for
5 h, after which they were plated on BCYE agar to show they were still viable and that the positive INT reaction was a valid indicator of viability.
Biocide treatment.
To test whether bacteria in free vesicles
could survive exposure to cooling tower biocides, washed amoeba
suspensions containing free vesicles were exposed separately to two
biocides in wells of 96-well microplates after amoebae had fed on live
Legionella cells for 4 h. The two cooling tower
biocides were an isothiazolone derivative, MBC 215 (a mixture of
5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin
[Nash-Chem, Nashville, Tenn.]), and a quaternary ammonium compound,
MBC 115 {poly[oxyethylene (dimethyliminio)ethylene (dimethyliminio)ethylene dichloride] (Nash-Chem)}. Both biocides are
registered with the U.S. Environmental Protection Agency under their
MBC numbers. They were prepared by dilution with TBSS. Vesicles in TBSS
were exposed to biocides for 4 h (a period of exposure in which
biocide concentrations may remain effective in an actual cooling tower)
and 24 h, after which they were treated with INT. Sterile PCB was
added with INT as the substrate for respiration. Final concentrations
of 15 ppm (vol/vol) of quaternary ammonium compound and 100 ppm of
isothiazolone were used for exposure. These concentrations were
selected on the basis of previous studies of amoeba tolerances to
cooling tower biocides (15) and because they fell within the
manufacturer's recommended dosage for cooling tower maintenance.
Samples of vesicles were examined at high magnifications with a Nikon
Microphot microscope with differential interference contrast to confirm
that vesicles contained bacteria.
Freeze-thawing.
To demonstrate that the presence of vesicles
with high numbers of legionellae may result in underestimates of the
total numbers of legionellae, based on CFU, vesicles were subjected to
three cycles of freeze-thawing (
70 and +35°C) and sonication in an ultrasonic water bath to disperse bacteria. After such treatment, vesicles were examined microscopically to determine whether bacteria were released from vesicles.
Bacterial mixtures.
To determine whether amoebae select
against L. pneumophila when they are presented with a choice
of E. coli or L. pneumophila, a mixture of equal
numbers of both bacteria were fed simultaneously to amoebae. After
24 h, free vesicles were enumerated and fluorescent antibodies to
L. pneumophila were used to detect L. pneumophila cells within such vesicles. The numbers of vesicles produced by amoebae
which fed on the mixture at 25 and 30°C were compared with the
numbers produced when amoebae fed on each bacterial species separately.
An MOI of 10,000 was used in these experiments. Vesicle diameters were
determined from A. polyphaga and A. castellanii organisms fed E. coli cells alone and the mixture of
L. pneumophila and E. coli cells.
Electron microscopy.
Vesicles from L. pneumophila-fed amoebae were fixed for electron microscopic
examination in a solution of 3% glutaraldehyde and 0.5% (wt/vol)
OsO4 in 0.05 M PIPES
[piperazine-N,N'-bis(2-ethanesulfonic acid]
buffer at pH 7.1. The fixative was combined in a ratio of 2 parts
fixative to 1 part vesicles suspended in TBSS. Fixation proceeded for
2 h at 4°C. After fixation, specimens were dehydrated in an
ethanol series and embedded in Epon-araldite resin. Propylene oxide was
used as a transition solvent. Microscopy was performed with a Zeiss 109 electron microscope at 50 kV.
Antibody treatment of vesicles.
As an additional treatment
for the possible application of flow cytometry to detect such
Legionella-packed vesicles, fluorescent antibodies to
L. pneumophila (Genetic Systems Corp., Redmond, Wash.) were
added to samples of vesicles to determine whether vesicles fluoresced.
Controls consisted of vesicles from E. coli-fed amoebae
treated in the same manner.
Data analysis.
Statistical analysis software was used to run
an analysis-of-variance test, followed by Tukey's multiple comparisons
to determine significant differences between treatments in MOI and
bacterial-mixture studies. Significant differences are reported at the
P < 0.05 level.
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RESULTS |
Feeding experiments.
The results show that A. polyphaga and A. castellanii amoebae fed L. pneumophila cells produced vesicles ranging from 2.1 to 6.4 µm
in diameter, with an average (±1 standard error) of 3.3 ± 0.08 to 3.6 ± 0.06, depending on the species (Fig.
1). Over 90% of vesicles from both
species fell within the size range considered to be respirable. A large
number of vesicles were released from amoebae after amoebae were fed
live L. pneumophila cells (Fig. 2A), and these contained live L. pneumophila cells, based on INT reduction (Fig. 2C and D). Greater
than 90% of vesicles had several cells with red formazan crystals
inside, whereas vesicles from amoebae which fed on heat-treated
bacteria rarely had any crystals inside vesicles (they were probably
from bacteria that had not been killed by heat treatment). Legionellae
exposed to biocides and plated onto BCYE agar formed colonies,
indicating that a positive INT reaction after biocide exposure was a
valid indicator of viability. No INT-formazan products were observed
outside of cells, indicating that abiotic reduction of INT did not
occur to create artifacts. Amoebae which fed on heat-treated L. pneumophila cells produced vesicles a day later than did those
which fed on living bacteria. Amoebae produced vesicles with live
L. pneumophila cells within 24 h at all three
temperatures tested in PCB and TBSS. At 35°C in PCB and TBSS, amoebae
did not appear to be healthy and many lysed, as reported by other
investigators (1, 4, 6). Amoebae fed at 25 and 30°C
produced normal viable cysts that were capable of excysting. The
percentage of amoebae infected, i.e., having bacteria within a food
vacuole, ranged from 73 to 98% for A. polyphaga, depending
on the MOI and temperature, and from 96 to 100% for A. castellanii. The number of amoebae infected may have been slightly
underestimated, since it was occasionally difficult to ascertain that
there were no bacteria in any vesicle within an amoeba. A. polyphaga produced up to 25 vesicles per amoeba in 24 h under
certain conditions (Fig. 3); however, the
number of vesicles per amoeba was lower for A. castellanii,
ranging from 0.08 to 0.11, depending on the temperature and MOI. In
general, a higher MOI resulted in a higher number of vesicles produced.
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FIG. 1.
Frequency distribution of vesicle sizes from A. polyphaga and A. castellanii. One hundred vesicles from
each species were measured. Over 90% of vesicles from each species
fell within the size range (1 to 5 µm) considered to be respirable.
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FIG. 2.
Vesicles from amoebae. (A) Low magnification of
numerous small vesicles among amoebae (three larger objects)
photographed by differential interference contrast microscopy. (B)
Higher magnification of three vesicles containing rods, observed by
differential interference contrast microscopy. These vesicles were
exposed for 3.5 h to MBC 215. (C) Vesicle containing formazan
crystals. It was produced by A. polyphaga at 35°C. The
light was altered to enhance the visualization of formazan. (D)
Unusually large vesicle containing bacteria with formazan crystals.
This vesicle was exposed for 4 h at 25°C to MBC 115 and then for
6 h to INT. Bar, 50 (A) and 20 (B through D) µm.
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FIG. 3.
Numbers of vesicles produced per amoeba at various MOIs
with respect to time and temperature. Data are averages from duplicate
flasks. Error bars were omitted for visual clarity.
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Biocide treatment.
The appearance of vesicles exposed to
biocides was the same as that of controls, and these vesicles
maintained their form with clusters of bacteria (Fig. 2B). The
respiration of bacteria within vesicles was intense at 35°C, based on
red-formazan precipitates associated with bacteria (Fig. 2C). After
exposure to biocides, bacteria in vesicles contained formazan products
(Fig. 2D).
Freeze-thawing.
The structural integrity of vesicles, based on
differential interference microscopy, was apparent even after 3 months
in culture flasks. The freeze-thawing treatment did not disperse
bacteria clustered in vesicles, whereas amoeba trophozoites were
completely destroyed. A count of intact vesicles before and after three
cycles of freeze-thawing showed no significant change in the numbers of
intact vesicles.
Electron microscopy.
Electron microscopy revealed that
bacteria in vesicles appeared to be wrapped in membranes with myelin
forms (Fig. 4 and
5). These structures were present even
within amoebae (Fig. 6).
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FIG. 5.
Transmission electron micrograph of a vesicle expelled
from A. castellanii and exposed to MBC 215 for 5 h.
Bar = 1 µm.
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Bacterial mixtures.
The number of vesicles produced when
amoebae fed on a mixture of L. pneumophila and E. coli was significantly higher than when they fed on either one
alone (Fig. 7). Fluorescent antibody treatment of vesicles from amoebae fed the bacterial mixture indicated that L. pneumophila cells were present in vesicles. Vesicles
from E. coli-fed A. polyphaga amoebae
averaged (±1 standard error) 2.9 ± 0.13 µm in diameter, and
those from E. coli-fed A. castellanii amoebae
averaged 2.14 ± 0.15 µm (Fig. 8).
Vesicles from amoebae fed the mixture of bacteria appeared the same
microscopically as did vesicles from amoebae fed L. pneumophila alone and averaged 3.65 ± 0.2 µm in diameter
for A. polyphaga and 3.5 ± 0.2 µm in diameter for
A. castellanii (Fig. 9).
Vesicles from amoebae fed E. coli alone did not fluoresce
with antibodies to L. pneumophila. Therefore, vesicles from
amoebae fed the bacterial mixture contained L. pneumophila
cells, indicating that amoebae did not select against legionellae when
they were presented with a choice between L. pneumophila and
E. coli. Vesicles from Legionella-fed amoebae contained brightly fluorescing rods after treatment with fluorescent antibodies to L. pneumophila, and vesicle membranes
also fluoresced faintly. It was not possible to enumerate bacteria
within vesicles, since they were packed in clusters.
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FIG. 7.
Numbers of vesicles produced per amoeba within 24 h
after feeding on E. coli, L. pneumophila
(Leg.), or a mixture of both species at 25 and 30°C with
an MOI of 10,000.
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FIG. 8.
Size frequency distribution of vesicles from A. polyphaga amoebae fed E. coli alone or a mixture of
E. coli and L. pneumophila (Lp and
Ec).
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FIG. 9.
Size frequency distribution of vesicles from A. castellanii amoebae fed E. coli alone or a mixture of
E. coli and L. pneumophila (Lp and
Ec).
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DISCUSSION |
Schuster (12) reported that an Acanthamoeba
sp. expels food vacuoles prior to encystment rather than retaining them
within the cyst. In the present study, most vesicles appeared in the medium just prior to encystment of the population. The addition of
cooling tower biocides may induce encystment (16) and
thereby facilitate the release of vesicles from amoebae. The fact that vesicles were produced much earlier when amoebae were fed live L. pneumophila cells may reflect the theory that live infectious legionellae resist digestion (5). Using the same A. polyphaga cultures as those employed in the present study, Newsome
et al. (8) examined the acid phosphatase activities in
amoeba fed live L. pneumophila cells, heat-killed
L. pneumophila cells, live E. coli cells,
and heat-killed E. coli cells. They found that amoebae fed
live L. pneumophila cells had small acid
phosphatase-positive vacuoles, presumably lysosomes, evenly distributed
in the cytoplasm, whereas those fed heat-killed L. pneumophila cells or live or heat-killed E. coli cells
had large acid phosphatase-positive vacuoles, presumably acidified food
vacuoles. Others have previously reported the same phenomenon
(2). Heat-treated legionellae may not contain the chemical
signals for avoiding digestion; therefore, the vesicles produced a day
later may have contained digested cell material. In addition, any
control amoebae fed live E. coli cells produced smaller and
clearer vesicles than did those containing L. pneumophila
cells. Live E. coli cells may have been digested, although
we did not test for this. The reason for amoebae to produce significantly more vesicles when they were fed the mixture of two
bacterial species is not understood.
Calculating from the volume of a sphere and the estimated volume of a
single bacterium by using the formula of Bratbak (3) [V = (
/4)w2(L w/3), where w is the width and L is the
length of the bacterium, we estimated that each vesicle could
theoretically contain between 20 and 200 bacteria. Rowbotham
(11) made similar calculations and reported that for a
5-µm-diameter vesicle, there may be a range of 365 to 1,483 bacteria
for a vesicle that is 90% full, depending on the size of the
bacterium. Such numbers may constitute an infectious dose (9,
11). Furthermore, Cirillo et al. (4) reported that
L. pneumophila is more invasive, even for macrophages, after
growth with amoebae; therefore, the infectious dose may be smaller for
bacteria that have passed through amoebae.
Most investigations of the numbers of legionellae in the environment
and in laboratory coculture experiments base such numbers on CFU on
agar. Since 1 CFU may result from one vesicle, the risk for
legionellosis may be underestimated severalfold. The fact that bacteria
within vesicles were viable after biocide exposure and freeze-thawing
underscores the significance of such vesicles in assessing the numbers
of legionellae in environments where amoebae may be present and where
many legionellae may be in vesicles. Amoebae from cooling towers have
also previously been found to be resistant to cooling tower biocides
(13-15).
These results demonstrate that respirable vesicles containing living
legionellae can be produced by at least two species of amoebae at
temperatures which allow the amoebae to carry out normal biological
processes, such as encystment, excystment, and feeding. In a study of
40 cooling towers, Yamamoto et al. (17) found cooling tower
temperatures to range from 8.3 to 35.2°C, depending on the season.
Amoebae may therefore serve as continuous sources of such potentially
infectious particles. In the present study, vesicles remained free in
the medium, whereas amoeba trophozoites and cysts generally adhered
tenaciously to the physical substrate. This indicates that such
vesicles may be agents for the transmission of legionellosis from
cooling towers, where aerosols may carry small respirable vesicles
rather than amoebae or cysts. The existence of vesicles may help to
explain some infectious-dose paradoxes (9), such as how
certain cooling towers with relatively low numbers of legionellae can
be sources of legionellosis and how aerosols can carry an infective
dose miles from a cooling tower without bacteria dying from
desiccation. There is a need to determine whether such vesicles can be
found in environments which may be sources of Legionnaires' disease.
The fact that vesicles in the present study reacted with antibodies to
L. pneumophila or with fluorescently tagged gene probes
to L. pneumophila (7) indicates that flow
cytometric analyses for such vesicles could be a tool in risk
assessment.
Although the MOI used in the present study may be high, only bacteria
which reach amoebae settled on the bottoms of flasks are encountered by
amoebae. It is unknown how such numbers of bacteria relate to the
numbers of legionellae attached to substrates and in biofilms, where
amoebae most likely feed in cooling towers. In addition, the ratio of
legionellae to amoebae on the surfaces of flasks may change as amoebae
change their pattern of distribution on these surfaces. Ongoing studies
in our laboratory aim to determine factors which lead to the production
and release of vesicles containing legionellae. Such factors include
the use of actual cooling tower water, the ratio of legionellae to
other non-Legionella bacteria as food sources, and biofilms
versus suspended bacteria. Also under investigation is the biocide
resistance of vesicle-bound legionellae compared with that of
legionellae free in medium.
| |
ACKNOWLEDGMENTS |
This work was supported in part by grant 1 R15 AI 31171-O1A1 BM
to S.G.B. from the National Institute of Allergy and Infectious Diseases and by the Center for the Management, Utilization and Protection of Water Resources at Tennessee Technological University. The research described in this article was funded wholly or in part by
the U.S. Environmental Protection Agency NCERQA environmental biology
program through grant R 825352-01-0 to S.G.B.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for the
Management, Utilization and Protection of Water Resources, P.O. Box 5033, Tennessee Technological University, Cookeville, TN 38505. Phone:
(931) 372-3451. Fax: (931) 372-6346. E-mail: SBERK{at}TNTECH.EDU.
Present address: Institute of Biological Chemistry, Washington,
State University, Pullman, WA 99164-6340.
| |
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Appl Environ Microbiol, January 1998, p. 279-286, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
