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Appl Environ Microbiol, March 1998, p. 955-959, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Comparative Recoveries of Naegleria
fowleri Amoebae from Seeded River Water by Filtration and
Centrifugation
P.
Pernin,1,*
M.
Pélandakis,1
Y.
Rouby,2
A.
Faure,3 and
F.
Siclet4
Laboratoire de Biologie
Cellulaire1 and
Laboratoire
d'Informatique Appliquée aux Systèmes
Finalisés,3 Faculté de Pharmacie,
69373 Lyon Cedex 08, Laboratoire de Biologie Médicale,
Hôpital des Armées Desgenettes, 69275 Lyon
Cedex,2 and
Direction des Etudes et
Recherches, Département Environnement Aquatique,
Electricité de France, 78400 Chatou
Cedex,4 France
Received 2 September 1997/Accepted 2 January 1998
 |
ABSTRACT |
Detection of pathogenic Naegleria fowleri in
environmental water samples, which is necessary for the prevention of
primary amoebic meningoencephalitis, generally requires concentrating the samples. Two concentration techniques, filtration and
centrifugation, were used to study the recovery of N. fowleri, in vegetative or cystic form, that had been mixed with
the two other thermotolerant Naegleria species, N. lovaniensis and N. australiensis. Counting of amoebae
was performed by the most probable number method on 10 water replicates
of 100 ml and 10 ml each. With both concentration methods, recovery was
better for cysts than for trophozoites (53% ± 21% versus 5% ± 5%
by filtration and 57% ± 25% versus 22% ± 5% by centrifugation).
The recovery of Naegleria trophozoites by filtration was
very low, and centrifugation was significantly better than filtration
in recovery of Naegleria trophozoites (22% ± 5% versus
5% ± 5%; P < 0.001). For cysts, however,
filtration appeared as efficient as centrifugation, with equivalent
values for recovery (53% ± 21% versus 57% ± 25%;
P > 0.7). Although the recovery of cysts of N. fowleri obtained by filtration (51% ± 24%) appeared higher
than that by centrifugation (36% ± 23%), the difference was not
significant (P > 0.1). Both concentration methods
have highly variable recovery rates, making accurate quantification of
low concentrations (<100/liter) of N. fowleri in the
environment difficult.
| |
INTRODUCTION |
Naegleria is a ubiquitous
free-living amoeba found in diverse freshwater environmental sites.
Within this genus, the pathogenic species Naegleria fowleri
is the causative agent of primary amoebic meningoencephalitis, a rare
but rapidly fatal central nervous system disease occurring after
exposure to contaminated water.
Isolation of pathogenic free-living amoebae and evaluation of their
abundance in water samples are difficult and require concentrating the
samples because the amoeba content in the natural environment is
generally too low to allow a direct evaluation method. The two
procedures commonly used for Naegleria concentration are
filtration and centrifugation. Numerous workers (3, 5-7,
12) use membrane filtration, while others (4, 8, 9,
14) prefer centrifugation or use one of the two methods,
depending on the sample volumes (13). Until now, the
accuracy, precision, and analytical sensitivity of the two
concentration methods have not been studied in a comparative fashion
for free-living amoebae. However, the validity of count results depends
on the accuracy of the recovery procedure used for the isolation of
amoebae. In the present study, we have attempted to quantify the yield
of filtration and we have compared the recovery efficiencies of
filtration and centrifugation by using both techniques on the same
seeded river water samples.
| |
MATERIALS AND METHODS |
The experiments have been conducted with different mixtures of
both vegetative and cystic forms of the pathogenic N. fowleri associated with another thermotolerant
Naegleria species, either N. lovaniensis or
N. australiensis.
River water.
All the experiments were carried out with river
water. Amoebae in the river water had been removed by two filtrations
(with a 3-µm-pore-size cellulose acetate filter). The concentration of suspended matter in the water samples was adjusted to 20 mg/liter by
addition of autoclaved suspended solids. The river water specimens thus
obtained (4 to 5 liters) were then seeded with an amoebic suspension in
vegetative or cystic form. This decontamination procedure was very
effective, since in the course of our study, we never observed or
isolated any amoeba other than the Naegleria species used
for each experiment.
Amoebae.
Amoeba strains (N. fowleri Na 1104c,
N. lovaniensis Ar9Ml, and N. australiensis
4.5.c.3) were grown for 2 to 5 days at 37°C on nonnutrient agar
plates (NNA) spread with Escherichia coli. Naegleria
trophozoites or cysts were harvested in 2 to 3 ml of amoeba saline
solution (10) by gentle scraping of either an advancing
front of amoebae or an encysted area. The amoebic concentration of this
initial suspension was determined by four counts on a Thoma
hemacytometer. After an appropriate dilution, this suspension was added
to the sample of river water to obtain a final mixed concentration
ranging from 50 to 100 amoebae/liter. The concentration of viable
amoebae was controlled for each species from a dilution of the initial
suspension by directly spreading on 10 petri plates a volume of 0.2 to
0.5 ml in order to theoretically have 5 to 10 lytic areas per plate.
The cumulative number of lytic areas observed on the 10 plates was
recorded to calculate the number of viable amoebae actually present in
the sample before processing.
Filtration.
To determine the recovery efficiency of
filtration, the river water specimens seeded with 50 to 100 amoebae/liter were submitted to continuous magnetic stirring and
replicate samples (10 100-ml and 10 10-ml samples) were pressure
filtered (with about 3 to 5 kPa) through 1.2-µm-pore-size cellulose
acetate filters (Millipore). Filters cut in half were inverted on NNA
plates overlaid with E. coli. Plates incubated at 42°C
were microscopically examined daily for amoeba outgrowth over a period
of 8 to 9 days.
Experiments were performed with the following mixtures of vegetative or
cystic forms in different ratios to detect possible interspecific
competition: (i) N. fowleri (Na 1104)-N.
lovaniensis (Ar9Ml) (ratio from 1:1 to 4:1) and (ii) N. fowleri (Na 1104)-N. australiensis (4.5.c.3) (ratio of
1:1).
Every plaque emerging along the two membrane halves was picked for
isolation and subcultured in order to identify the amoebic clone by
isoenzyme typing as previously described (11).
Comparison of filtration and centrifugation.
The comparative
study of filtration and centrifugation was carried out by processing 10 replicates of 10 ml and 100 ml each from the same seeded river water
specimen by the two methods. The river water specimen was seeded with
vegetative or cystic forms (100 amoebae/liter) of the following species
mixtures: (i) N. fowleri (Na 1104)-N. lovaniensis
(Ar9Ml) (ratio of 1:1 or 3:1 for trophozoites and 3:1 for cysts) and
(ii) N. fowleri (Na 1104)-N. australiensis
(4.5.c.3) (ratio of 1:1 for both trophozoites and cysts). Filtration
was performed as described above for the 10- and 100-ml volumes.
Centrifugation was carried out at 1,000 × g for 15 min
for the 10-ml volumes and at 3,000 × g for 10 min for
the 100-ml volumes in a FIRLABO SV11 centrifuge with an SV11E11 swinging rotor. After centrifugation, all but 750 µl of the
supernatant was discarded by vacuum aspiration. The tube contents,
after vortex stirring, were spread on an NNA plate overlaid with
E. coli. Each tube was rinsed carefully with sterile water
(750 µl), and the rinsing material was also spread on the same plate.
All the plates for both procedures were incubated at 42°C. Careful
daily macroscopical and microscopical examinations of plates
allowed us
to observe amoebic growth by noting the development
of lytic areas over
the bacterial coating, and each amoebic plaque
was subcultured for
species identification by isoenzyme typing.
The number of positive
plates for total
Naegleria and for each
Naegleria
species was recorded to compare the two concentration
methods.
Expression of results and statistical analysis.
Both for
filtration and comparison of filtration-centrifugation, the
concentration of total Naegleria and of each
Naegleria species was expressed as the most probable number
(MPN) per liter after recording the number of positive plates for the
10 10-ml replicates and the 10 100-ml replicates (2). The
recovery efficiencies were calculated by dividing the number (MPN per
liter) of amoebae recovered by each method by the number of viable
amoebae, as determined by control plating before processing.
For comparison of filtration and centrifugation, a
2
test was applied to the paired MPN/liter results obtained with the two
methods concurrently applied to the same samples.
| |
RESULTS |
Filtration recovery.
The filtration method was better for the
recovery of cysts than for vegetative forms, as the yield of
filtration, always higher for cysts, demonstrated. For the competing
mixed species N. fowleri plus N. lovaniensis and
N. fowleri plus N. australiensis, the filtration
mean recovery, all species taken as a whole, was only 10% ± 10.3%
for vegetative forms but was 54.5% ± 15.6% for the cysts (including
the results of comparative experiments on filtration-centrifugation) (Tables 1 and
2). The yield for each species considered
separately showed the same tendency, particularly for N. fowleri (10.2% for trophozoites versus 55.3% for cysts).
Recovery efficiencies were highly variable for trophozoites and cysts,
as evidenced by the standard deviations.
Filtration generally allowed recovery of the association of the two
species in 7 of the 12 tests for trophozoites (Table
1)
and in 10 of
the 11 tests for the cysts (Table
2) (Fig.
1 and
2).
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FIG. 1.
Recovery of competing Naegleria trophozoites
from seeded river water by filtration and centrifugation for mixtures
of N. fowleri-N. lovaniensis (a) and N. fowleri-N.
australiensis (b). Values plotted for each experiment are the
number (number of viable amoebae per liter according to control
plating) of the two species in the inoculum (I) and the MPN of each
species obtained by filtration (F) and centrifugation (C). The
theoretical ratio of the two species (in parentheses) and the
theoretical total amoebic concentration are shown at the bottom of the
figure for each experiment.
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FIG. 2.
Recovery of competing Naegleria cysts from
seeded river water by filtration and centrifugation for mixtures of
N. fowleri-N. lovaniensis (a) and N. fowleri-N.
australiensis (b). Values plotted for each experiment are given as
described in the legend to Fig. 1.
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|
The study of
N. fowleri-N. lovaniensis mixtures of
trophozoites where theoretical species ratios varied significantly
(from
1:1 to 4:1) showed that the recovery of
N. fowleri
tended to be
higher when it made up a larger proportion of the mixture
(Table
1). However, when the ratio of the two species was close to 1:1,
the presence of competing
N. lovaniensis species decreased
the
recovery of
N. fowleri trophozoites, which were
overgrown especially
when the
N. lovaniensis concentration
reached a threshold value
of 30 to 40 amoebae/liter in the mixture
(Fig.
1a), as was the
case in some experiments with a theoretical ratio
of 1:1 or 3:1.
For
N. fowleri-N. australiensis mixtures, trophozoites of
N. fowleri have been recovered with their usual low yield,
but in
these experiments the observed ratio, theoretically 1:1,
fluctuated
less (Table
1 and Fig.
1b).
In the mixtures of cysts (Table
2 and Fig.
2), independently of the
species ratio (1:1 to 4:1), filtration appeared more
favorable to
recovery of
N. fowleri cysts (55.3%) than to that
of cysts
of the two other species,
N. lovaniensis and
N. australiensis (23.8 and 21.3%, respectively).
Comparative recoveries of filtration and centrifugation.
Generally, for the Naegleria trophozoites, without regard to
the species, the centrifugation recovery rate was significantly higher
than the filtration recovery rate (22% ± 5% versus 5% ± 5%;
2 on paired MPN values = 29.65; P 
0.001) (Table 3 and Fig. 1). However,
filtration was as efficient as centrifugation for isolation of cysts:
the mean recoveries were approximately equivalent with nonsignificant
differences (53% versus 57%; 2 = 2.54;
P > 0.7), and the mean recovery by centrifugation was superior to that by filtration in only one of five experiments with
cysts.
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TABLE 3.
Comparison of recovery efficiencies of filtration and
centrifugation for competing Naegleria species in vegetative
and cystic forms
|
|
The results of the recovery of each
Naegleria species by the
two methods were as follows. (i) Typically for
N. fowleri in
the vegetative form, centrifugation was always significantly superior
to filtration, with mean recoveries of 25% ± 14% and 2% ± 2%,
respectively (
2 = 16.64;
P < 0.01). For
N. fowleri cysts, although the results
appeared better for
filtration than for centrifugation (51% ±
24% versus 36% ± 23%),
the differences were not statistically
significant (
2 = 7.91;
P > 0.1). (ii) For
N. lovaniensis, in
the vegetative
and cystic forms, centrifugation always produced better
results
than filtration (72% ± 57% versus 41% ± 50% for cysts and
40%
± 13% versus 14% ± 5% for trophozoites), but these
differences
between the two methods were significant only for the
trophozoites
(
2 = 10.06;
P < 0.02) and
not for the cysts (
2 = 3.37;
P > 0.1).
(iii) For
N. australiensis in cystic form,
centrifugation
was superior to filtration in two of the three
tests (
2 = 14.05;
P < 0.01). No conclusion can be drawn from
the experiments
with
N. australiensis trophozoites because
they were not recovered.
In addition, the results of the comparative
study carried out
on
N. fowleri-N. lovaniensis or
N. fowleri-N. australiensis mixtures
showed that, whatever the
concentration method, the recovery rates
obtained with the cysts were
better than with vegetative forms
(Table
3). This is in agreement with
the results obtained previously
for filtration.
| |
DISCUSSION |
Among the numerous environmental surveys, most aimed at detecting
the presence of pathogenic N. fowleri in correlation with the elevation of water temperature. Only a few investigators have tentatively considered exact numeration of amoebae in the water (1, 4, 9, 13). Indeed, the quantitative determination of
Naegleria in water by filtration necessarily requires an
indirect counting method because of the presence of the filter membrane on the agar surface, which hides the development of lytic plaques subsequently coalescing under the two filter halves. Therefore, the
response expected for the presence of amoebae in a given volume of
water treated by filtration (volume 
10 ml) can be only of an
all-or-none type: i.e., the presence or absence of at least one amoeba
in the volume considered. The precision and accuracy of the best method
of determining MPN will be reached only by increasing the serial
repetition of different sample volumes. Therefore, the determination of
amoebic concentration by the MPN method is tedious and time-consuming,
which may explain the scarcity of such studies. Probably for the same
reasons, no systematic comparative study between filtration and
centrifugation for amoeba recovery has ever been attempted. From a
limited number of environmental samples treated simultaneously by the
two methods, De Jonckheere (4) demonstrated the isolation of
a larger number of strains of Naegleria by centrifugation
than by filtration but did not estimate the amoebic concentration.
All the results presented here demonstrate a greater efficiency in
recovery of cysts than of vegetative forms regardless of the
concentration method used. The filtration isolation procedure largely
used by most investigators showed a particularly low recovery yield for
vegetative forms of Naegleria, since most of the time it was
below 10%, whereas it reached 54.5% for the cysts. These results show
that the evaluation of the number of amoebae by filtration leads to an
underestimation of the real situation that should be taken into
consideration for the interpretation of environmental surveys. Although
the filtrates have not been checked for the presence of amoebae, it is
very unlikely that this low efficiency was due to a lack of retention
of amoebae on the filter membrane. Indeed, because of their size and
despite their great plasticity, the Naegleria trophozoites
(10 to 35 µm), as well as the cysts (7 to 17 µm), cannot pass
through a 1.2-µm-pore-size filter membrane. This fact is clearly
confirmed by the total absence of exogenous amoebae during all our
experiments (even those normally smaller than Naegleria,
like some Hartmannellidae), resulting from decontamination of the river water by 3-µm-pore-size filtration. By comparison, centrifugation allowed higher recovery rates than filtration for vegetative forms but did not appear to improve cyst recovery (Table 3).
These results can be explained as follows. The cytoplasmic membrane of
the vegetative form, which is directly exposed to the external
conditions, is probably very sensitive to the various adverse
conditions met during successive operations of isolation. For example,
during filtration, trophozoites may be suddenly lysed by a too rapid
and pronounced vacuum. Immediately after filtration, the trophozoites
are then covered by the inverted filter membrane and can remain trapped
by the layer of suspended matter, which can be very compact when there
is a high load of this suspended matter in the water. On the other
hand, centrifugation is probably better tolerated by vegetative forms.
Indeed, after the pellet resulting from centrifugation is plated,
vegetative forms dispersed on the agar surface are in better
oxygenation conditions to resume outgrowth and multiplication than when
they are flattened by the filter membrane. For cysts, the approximately
equivalent mean recovery efficiencies with the two methods can be
explained by the fact that they are protected by their cyst wall and
resist adverse conditions better than trophozoites whatever the
concentration procedure used. Furthermore, centrifugation could allow a
direct plaque count of lytic areas on individual plates instead of an estimate of the MPN as we have attempted in our comparative scheme. Consequently, according to our results, since the Naegleria
amoebae can assume three different stages during their life cycle
(vegetative, cystic, and even flagellate forms), the concentration
method should ideally be chosen with the knowledge of the precise form
of the pathogen in the environmental sample. If the water contains a high proportion of cysts, the two concentration methods seem
equivalent, and conversely, if trophozoites predominate, centrifugation
will obviously be better than filtration. Unfortunately, we have no means to foresee the precise Naegleria status at the time of
sampling in natural waters.
The second major conclusion that may be drawn from our data is that
regardless of the concentration method, there is important imprecision
and inaccuracy in the evaluation of Naegleria concentrations in water samples. This imprecision probably results in part from the
low Naegleria concentration, which was arbitrarily fixed at 50 to 100 Naegleria per liter in our experiments. However,
amoebic contaminations higher than this threshold value do not require concentration any longer: their MPN can be estimated by direct spreading of small volumes (0.1 and 1 ml) on the agar surface. Furthermore, the specific isolation of N. fowleri by
filtration, especially for vegetative forms, becomes more uncertain in
the presence of direct competitors, such as any other thermotolerant free-living amoebae, and particularly other thermotolerant
Naegleria species (N. lovaniensis and N. australiensis). The importance of this competitive phenomenon is
evidenced by the fact that according to our own experience of
environmental survey (data not published), the small volumes (0.01 liter) used for MPN determination are often more positive for N. fowleri strains than larger volumes (1 liter and 0.1 liter).
Similar observations have already been made by De Jonckheere
(4) and Tyndall et al. (13). Therefore, for all
these reasons, in practice, it will be better to include in the MPN
procedure, whenever possible, the use of small volumes (10 ml) while
increasing the number of replicates. Therefore, this process has two
main advantages: first, a limitation of the phenomena of interspecific
competition, and second, the elimination of the interfering effects of
concentration methods (for 1- and 0.1-ml volumes).
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from Electricité de
France (Direction des Etudes et Recherches).
We acknowledge the skillful technical assistance of M. C. Testard
and thank J. F. De Jonckheere for helpful comments on the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Biologie Cellulaire, Faculté de Pharmacie, 8 Avenue Rockefeller,
69373 Lyon Cedex 08, France. Phone: 33 04 78 77 71 08. Fax: 33 04 78 77 71 58. E-mail: pernin{at}rockefeller1.univ-lyon1.fr.
| |
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Appl Environ Microbiol, March 1998, p. 955-959, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
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(2001). Assessing the Risk of Primary Amoebic Meningoencephalitis from Swimming in the Presence of Environmental Naegleria fowleri. Appl. Environ. Microbiol.
67: 2927-2931
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Abstract
Introduction
