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Applied and Environmental Microbiology, November 1998, p. 4307-4312, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Infectious Enteroviruses, Enterovirus
Genomes, Somatic Coliphages, and Bacteroides fragilis
Phages in Treated Wastewater
C.
Gantzer,1
A.
Maul,2
J. M.
Audic,3 and
L.
Schwartzbrod1,*
Faculté de Pharmacie, Laboratoire de
Virologie, 54000 Nancy,1
I.U.T. de Metz,
57000 Metz,2 and
Lyonnaise des Eaux,
CIRSEE, 78230 Le Pecq,3 France
Received 13 January 1998/Accepted 13 July 1998
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ABSTRACT |
In this study, three types of treated wastewater were tested for
infectious enteroviruses, the enterovirus genome, somatic coliphages,
and Bacteroides fragilis phages. The aim of this work was
to determine whether the presence of the two types of bacteriophages or
of the enterovirus genome was a good indicator of infectious enterovirus contamination. The enterovirus genome was detected by
reverse transcription-polymerase chain reaction. Infectious enteroviruses were quantified by cell culturing (BGM cells), and the
bacteriophages were quantified by plaque formation on the host
bacterium (Escherichia coli or B. fragilis) in
agar medium. Forty-eight samples of treated wastewater were
analyzed. Sixteen samples had been subjected to a secondary
treatment for 8 to 12 h (A), 16 had been subjected to a secondary
treatment for 30 h (B1), and 16 had been subjected to both
secondary and tertiary treatments (B2). The mean concentrations of
somatic coliphages were 4.9 × 104 PFU · liter
1 for treatment line A, 9.8 × 103
PFU · liter1 for B1, and 1.4 × 103 PFU · liter1 for B2, with all the
samples testing positive (100%). The mean concentrations of B. fragilis phages were 1.7 × 103 PFU · liter1 for A (100% positive samples), 17 to 24 PFU
· liter1 for B1 (44% positive samples), and 0.8 to 13 PFU · liter1 for B2 (6% positive samples). The
mean concentrations of infectious enteroviruses were 4 most
probable number of cytopathogenic units (MPNCU) · liter1 for A (31% positive samples) and <1 MPNCU
· liter1 for B1 and B2 (0% positive samples). The
percentages of samples testing positive for the enterovirus genome were
100% for A, 56% for B1, and 19% for B2. The percentages of samples
testing positive for the enterovirus genome were significantly higher
than those for infectious enteroviruses. This finding may have been due
to the presence of noninfectious enteroviruses or to the presence of infectious enteroviruses that do not multiply in BGM cell cultures. However, under our experimental conditions, nondetection of the genome
implies the absence of infectious viruses. There was a significant
correlation between the concentration of somatic coliphages or
B. fragilis phages and the presence of infectious
enteroviruses or the presence of the enterovirus genome. However, the
somatic coliphage concentration did not lead to fluctuations in the
infectious enterovirus concentration, whereas the B. fragilis phage concentration did.
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INTRODUCTION |
Water in the environment may be
contaminated by more than 140 serotypes of viruses via wastewater.
Hepatitis A virus, caliciviruses, adenoviruses, rotavirus, and
enteroviruses have the greatest effect on public health. A large number
of epidemics due to the presence of these viruses in the environment
have been reported (2, 3, 24). It is thus necessary to
monitor the levels of these viruses in the aqueous environment,
particularly in wastewater discharged into surface water.
The microbiological quality of water is currently evaluated by use of
indicators of fecal contamination (fecal coliforms, Escherichia
coli, and fecal streptococci). Such fecal contamination may lead
to the presence in the water environment of organisms that cause
diseases in humans. Some studies have shown that these indicators do
not provide adequate information about viruses, particularly in terms
of their fate in the environment and their resistance to treatment
(6, 9, 17). Thus, studies have been directed toward
identifying more specific indicators of viral contamination. Two
approaches are possible. One involves the detection of enteroviruses
(poliovirus, coxsackievirus groups A and B, and echovirus) by cell
culture or molecular biology techniques. The other involves looking for
particular types of bacteriophages commonly found in human feces.
The isolation of enteroviruses by cell culturing is the reference
method because it is the only way in which the infectious nature of the
virus isolated can be determined. However, it is time-consuming (1 to 2 weeks) and difficult to perform, and not all viral serotypes can be
detected. Molecular biology techniques, such as reverse
transcription-polymerase chain reaction (RT-PCR), can be used for the
sensitive, specific, and rapid (24 to 48 h) detection of
the enterovirus genome (13). Certain areas of the 5'
noncoding region of the enterovirus genome are highly conserved among all serotypes. Primers binding to these areas can be used to
amplify sequences common to most enteroviruses. Thus, the detection of
the enterovirus genome by RT-PCR is a valuable alternative to cell
culturing for evaluating the virological status of the water
environment. However, the detection of the viral genome by itself does
not provide any information about the infectivity of the viruses detected.
Three types of bacteriophages have also been proposed as
specific indicators of viral contamination: the somatic
coliphages (18), the F-specific RNA phages
(8), and the Bacteroides fragilis phages
(12). The size, structure, and survival rate in the
environment of these bacteriophages are similar to those of
enteroviruses. The B. fragilis HSP40 phages seem to
be specific indicators of human fecal contamination, whereas the
other two types of phages may be indicators of both human and nonhuman
animal fecal contamination (8, 12).
The aim of this work was to determine whether B. fragilis phages or somatic coliphages and the enterovirus genome
are reliable indicators of contamination of wastewater by
infectious enteroviruses. We therefore tested for infectious
enteroviruses, the enterovirus genome, somatic coliphages,
and B. fragilis phages in various treated-wastewater samples.
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MATERIALS AND METHODS |
Sampling.
Forty-eight samples of treated wastewater were
taken from two wastewater treatment plants. The first treatment line
(A) included a pretreatment step (sand and oil removal), primary
settling, activated sludge treatment, and secondary settling. The
treatment time was between 8 and 12 h. The sampling site was
situated at the plant outlet before the point at which treated
wastewaters were discharged into the environment. Sixteen samples were
taken. The second treatment line included two phases. The first phase included a pretreatment step (sand and oil removal), primary settling, biological treatment (alternating aeration and anoxia), and secondary settling. The second phase involved tertiary phosphate removal and
enhanced settling. The treatment time was about 30 h. Two sampling
sites were used: one at the outlet of the secondary treatment (B1) and
the other at the outlet of the tertiary treatment (B2). Sixteen samples
were taken at B1, and 16 samples were taken at B2. The 48 samples were
taken between 18 March and 7 May 1997.
Enterovirus and enterovirus genome concentration.
The
enteroviruses were concentrated by adsorption-elution on glass wool
(23). Twenty liters of water was filtered at a rate of 20 liters · h1 through 50 g of sodocalcic glass
wool (type 725; Rantigny, Saint-Gobain, France) compacted (0.4 g
· cm3) in a stainless steel cartridge (Sartorius SM
16249). The viruses adsorbed to the glass wool were eluted with 300 ml
of 0.05 M glycine buffer-3% beef extract-0.0005% phenol red at pH
9.5. The eluate was rapidly neutralized to pH 7.2 to produce the concentrate.
B. fragilis phage concentration.
B.
fragilis phages were concentrated on an inorganic membrane
(Whatman 6809-5022) as described by Lucena et al. (15). The membrane was saturated with protein by filtration of 3 ml of a solution
containing 3% beef extract. Water (100 ml) was filtered through the
membrane to facilitate phage adsorption. The membrane was removed and
ground in 5 ml of 0.05 M glycine buffer (pH 9.5) to elute the
bacteriophages. The eluate was rapidly neutralized to pH 7.2 to produce
the concentrate.
Sample decontamination.
Fungi and bacteria were removed from
the water and concentrate samples by filtration through a
0.22-µm-pore-size Millex GV membrane (Millipore) saturated with fetal
calf serum. The decontaminated samples were the filtrates.
Infectious enterovirus quantification.
Analysis of the
samples was carried out directly and after concentration. Water or
concentrate (25 µL) was added to a well containing 175 µl of a
suspension of 7.5 × 104 BGM (monkey kidney cell line)
cells ml1 in Eagle's minimal essential medium (Eurobio)
containing 2% fetal calf serum. Four hundred wells were used for each
sample (five plates each containing 80 wells), so that 10 ml of each
sample was tested. Sixteen wells per plate contained only the 175 µl of cells to serve as controls.
The plates were incubated for 6 days at 37°C in a 5%
CO2-95% air atmosphere. The plates were then examinated
under a light microscope, and wells in which there was a cytopathogenic
effect (CPE) on the cells were identified. Two subcultures were made for each well with the same protocol. The number of wells with a CPE
were counted, and the most probable number of cytopathogenic units
(MPNCU) was calculated (16). The results are expressed as
MPNCU · liter1.
Viral genome detection.
Analysis of the samples was carried
out directly and after concentration.
(i) Extraction.
Viral genome RNA was extracted as described
by Chomczynski and Sacchi (4). Sample aliquots (100 µl)
were treated with 500 µl of extraction solution (4 M guanidine
thiocyanate, 25 mM sodium citrate [pH 7], 0.5%
N-lauroylsarcosine, 0.1 M 
-mercaptoethanol). Fifty
microliters of 2 M sodium acetate (pH 5.2) was added, followed by 600 µl of phenol-chloroform-isoamyl alcohol (25:24:1). The RNA in the
aqueous phase after centrifugation (10,000 × g for 20 min at 4°C) was precipitated with 1 volume of absolute ethanol in the
presence of 20 µg of glycogen ml1 for 1 h at
20°C. A pellet obtained by centrifugation (10,000 × g for 10 min at 4°C) was suspended in 300 µl of
extraction solution-600 µl of absolute ethanol. A second
precipitation (1 h at 20°C) was carried out, followed by
centrifugation. The pellet was washed with 1 ml of 70% ethanol and
dried, and then the resulting RNA precipitate was dissolved in 20 µl
of diethyl pyrocarbonate-treated deionized water.
(ii) Primers.
The primers corresponded to the 5' noncoding
region (14) and were selected because they corresponded to
sequences present in many enterovirus serotypes: primer 2 (nucleotides
164 to 184), primer 3 (nucleotides 584 to 603), and primer f2
(nucleotides 516 to 530).
(iii) cDNA synthesis.
cDNA was synthesized from the
extracted RNA by use of oligo (dT)15 (Promega). We used a
reaction volume of 20 µl containing 4 µl of 5× reverse
transcription buffer (250 mM Tris-HCl [pH 8.4], 50 mM
MgCl2, 350 mM KCl, 15 mM dithiothreitol, 2.5 mM
spermidine), 40 U of RNase inhibitor (Promega), 0.25 mM each
deoxynucleoside triphosphate (Perkin-Elmer Cetus), 1 mM
oligo(dT)15, 10 U of avian myeloblastosis virus reverse
transcriptase (Promega), 6 µl of diethyl pyrocarbonate-treated
H2O, and 5 µl of RNA extract. Reverse transcription was
performed at 42°C for 30 min. RNA-DNA hybrids were denatured, and
reverse transcriptase was inactivated by heating to 95°C for 5 min.
The resulting cDNA was then amplified by PCR.
(iv) PCR amplification.
PCR was carried out with 5 µl of
cDNA mixed with 95 µl of PCR mix, containing 10 µl of 10× PCR
buffer (500 mM KCl, 100 mM Tris-HCl [pH 8.3], 15 mM
MgCl2, 0.001% [wt/vol] gelatin), 0.25 mM each
deoxynucleoside triphosphate, 0.1 mM each primer (primers 2 and 3), and
2.5 U of Taq polymerase (Perkin-Elmer Cetus). Thirty cycles
of amplification were performed as follows: 30 s at 96°C for
denaturation, 45 s at 50°C for hybridization, and 60 s at 72°C for elongation. A fragment of 439 bp was amplified.
(v) Seminested PCR.
One microliter of the first PCR
amplification product was mixed with 99 µl of PCR mix as described
above, except that primers 2 and f2 were used. The temperature cycles
were as described above. A 366-bp fragment was amplified. The amplified
fragment was separated by electrophoresis on a 2% agarose gel
containing 0.5 µg of ethidium bromide ml1 and examined
under UV light.
Phage quantification.
B. fragilis HSP40 grown on
Bacteroides phage recovery medium was used in the
quantification of B. fragilis phages. Phages contained
in 1 ml of wastewater or concentrated wastewater were quantified by the
double-agar-layer method (21). E. coli C strain ATCC 13706 grown in modified Scholten's broth was used in the quantification of somatic coliphages. Phages contained in 1 ml of
wastewater were quantified by the single-agar-layer method (10). The results for both phages are expressed in PFU
· liter1.
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RESULTS |
Sixteen samples from treatment line A, 16 samples from treatment
line B1, and 16 samples from treatment line B2 were tested for somatic
coliphages, infectious enteroviruses, the enterovirus genome, and
B. fragilis phages.
Preliminary study.
A preliminary study was carried out to
determine whether the two types of bacteriophages and the enteroviruses
were present in sufficient amounts to be detected directly in the
samples or whether concentration of the samples was necessary. The 16 samples from each treatment line (A, B1, and B2) were analyzed directly without concentration (Table 1): somatic
coliphages were found in all 48 samples, B. fragilis
phages were detected in 6 of the 48 samples, the enterovirus genome was
detected in 9 of the 48 samples, and no infectious enteroviruses were
isolated. The small number of samples testing positive by direct
analysis showed that preliminary concentration was necessary for
B. fragilis phage and enterovirus detection. The high
density of somatic coliphages present in all samples made direct
detection possible for these phages.
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TABLE 1.
Number of samples containing somatic coliphages,
B. fragilis phages, infectious enteroviruses, and the
enterovirus genome, as detected by direct water analysis
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The levels of somatic coliphages were determined directly from water
samples, those of the infectious enteroviruses and the
enterovirus
genome were determined after concentration from 20
liters of water, and
those of
B. fragilis phages were determined
after
concentration from 100 ml of
water.
Water from line A.
The results obtained for the four types of
samples are shown in Fig. 1. Somatic
coliphages were detected in the 16 samples analyzed at concentrations
of 1.1 × 104 to 9.7 × 104 PFU
· liter1. The mean somatic coliphage concentration for
the first 10 samples (10 April to 19 April) was 1.9 × 104 PFU · liter1, and the mean
concentration for the last six samples (2 May to 7 May) was 7.9 × 104 PFU · liter1. There was a
significant difference (P < 0.01; Mann-Whitney test) between these two series of samples, which differed in somatic coliphage content by a factor of 4. B. fragilis phages
were found in the 16 samples analyzed at concentrations of 1.3 × 101 to 5.1 × 103
PFU · liter1. There was an increase in phage
contamination between the first 10 samples (10 April to 19 April) and
the last 6 samples (2 May to 7 May). The mean concentrations of
B. fragilis phages were 3.5 × 101
PFU · liter1 between 10 April and 19 April and
3.4 × 103 PFU · liter1 between 2 May and 7 May. There was a significant difference (P < 0.01; Mann-Whitney test) between these two series of
samples, which showed a 97-fold increase in B. fragilis phage content.
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FIG. 1.
Qualitative detection of the enterovirus genome and
concentrations of infectious enteroviruses, B. fragilis
phages, and somatic coliphages in treatment line A water. L, liter.
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Infectious enteroviruses were isolated from 5 of the 16 samples
analyzed at concentrations of 1.5 to 22.5 MPNCU · liter
1. The mean concentration was between 3.5 and 4.5 MPNCU · liter
1. The lower value of this interval
was the average concentration
calculated assuming that the samples
testing negative had a virus
concentration of zero, and the upper value
was that calculated
assuming that the samples testing negative
had a virus concentration
equal to the detection threshold
(1 MPNCU · liter
1). Dissociation of the two sample
series described above showed
that only 1 of 10 samples was positive
between 10 April and 19
April. It contained 1.5 MPNCU · liter
1 (mean concentration, 0.15 to 1 MPNCU · liter
1). Four of six samples were positive between 2 May
and 7 May (mean
concentration, 9.5 to 10 MPNCU · liter
1). Thus, the concentration of infectious
enteroviruses increased
by factors of 10 to
67.
The viral genome was present in all of the samples
analyzed.
Water from line B1.
The results for B1 are shown in Fig.
2. Somatic coliphages were present in all
16 samples analyzed at concentrations of 6.5 × 103 to
1.7 × 104 PFU · liter1, with a
mean concentration of 9.8 × 103
PFU · liter1. B. fragilis
phages were present in 7 of the 16 samples analyzed at concentrations
of 1.7 × 101 to 6.7 × 101 PFU
· liter1, with a mean concentration between 1.7 × 101 and 2.4 × 101 PFU · liter1. No infectious enteroviruses were detected,
although the enterovirus genome was detected in 9 of the 16 samples
analyzed. There was no significant difference between the number of
samples testing positive for B. fragilis phages and the
number testing positive for the enterovirus genome (P > 0.1). However, the number of samples testing positive for somatic
coliphages was significantly higher than that testing positive for the
enterovirus genome or for B. fragilis phages
(P = 0.01).
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FIG. 2.
Qualitative detection of the enterovirus genome and
concentrations of infectious enteroviruses, B. fragilis
phages, and somatic coliphages in treatment line B1 water. L, liter.
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Water from line B2.
The results for B2 are shown in Fig.
3. Coliphages were present in the 16 samples analyzed at concentrations of 2.5 × 102 to
5.5 × 103 PFU · liter1, the mean
concentration being 1.4 × 103 PFU · liter1. B. fragilis phages were present
in only one sample, at a concentration of 13 PFU · liter1, for a mean concentration between 0.8 and 13 PFU · liter1. No infectious enteroviruses were
detected, although the enterovirus genome was detected in 3 of the 16 samples analyzed. As so few samples tested positive for B. fragilis phages and the enterovirus genome, statistical comparison
was not possible. However, the number of samples testing positive for
somatic coliphages was significantly higher than that testing positive
for the enterovirus genome or for B. fragilis phages
(P < 0.01).
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FIG. 3.
Qualitative detection of the enterovirus genome and
concentrations of infectious enteroviruses, B. fragilis
phages, and somatic coliphages in treatment line B2 water. L, liter.
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Figure
4 shows some results obtained for
the detection of the enterovirus genome in three unconcentrated
wastewater samples
from each treatment line (A, B1, and B2) and the
same three wastewater
samples after concentration. The three samples
(3, 4, and 5 April)
taken from treatment line B2 were negative for the
enterovirus
genome before concentration on glass wool (A1, A2, and A3
in Fig.
4), but one sample became positive after concentration (B10,
B11,
and B12 in Fig.
4). For treatment line B1, one of three samples
(3, 4, and 5 April) was positive before concentration (A4, A5,
and A6
in Fig.
4), and two of three samples were positive after
concentration
(B13, B14, and B15 in Fig.
4). Finally, for treatment
line A, two of
three samples (15, 16, and 17 April) were positive
before
concentration (A7, A8, and A9 in Fig.
4), and all three
samples
were positive after concentration (B16, B17, and B18 in
Fig.
4).
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FIG. 4.
Detection of the enterovirus genome in unconcentrated
(A) and 67-fold-concentrated (B) treated wastewaters. The seminested
RT-PCR products were analyzed by gel electrophoresis. Lanes: C+,
positive control (deionized water with 10 MPNCU of poliovirus 1 ml1); C, negative control (deionized water); 1, 2, and
3, unconcentrated treatment line B2 wastewater (3, 4, and 5 April); 4, 5, and 6, unconcentrated treatment line B1 wastewater (3, 4, and 5 April); 7, 8, and 9, unconcentrated treatment line A wastewater (15, 16, and 17 April); 10 to 18, samples from lanes 1 to 9 concentrated
(67-fold) by adsorption-elution on glass wool; M, markers.
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Correlation between phage concentration and the presence of
enteroviruses.
We used the results obtained for the three
categories of treated wastewater to evaluate the possible relationships
between bacteriophage concentration and the presence of enteroviruses (infectious or genome).
There was a significant correlation (
t test) between the
concentration of somatic coliphages and the presence of infectious
enteroviruses (
P = 0.0014) and between the somatic
coliphage concentration
and the presence of the enterovirus genome
(
P < 0.0001) (Table
2).
Infectious enteroviruses were isolated from 22% of the samples
in
which the somatic coliphage concentration was greater than
10
4 PFU · liter
1, whereas the
enterovirus genome was detected in 87% of these
samples. No
enteroviruses were isolated when the somatic coliphage
concentration
was between 10
2 and 10
4 PFU · liter
1, and the enterovirus genome was detected in only
29 to 33% of
these samples.
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TABLE 2.
Relationship between the concentration of somatic
coliphages and the presence of enteroviruses (infectious
or genome)
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There was a significant correlation (
t test) between the
concentration of
B. fragilis phages and the presence of
infectious
enteroviruses (
P = 0.0004) and between the
B. fragilis phage concentration
and the presence
of the enterovirus genome (
P = 0.0001) (Table
3). Infectious enteroviruses were
isolated from 71% of the samples
in which the
B. fragilis phage concentration was greater than
10
2
PFU · liter
1, and the enterovirus genome was
detected in 100% of these samples.
No enteroviruses were isolated
at phage concentrations of 10
1 to 10
2 PFU
· liter
1, although the enterovirus genome was
detected in 75% of these
samples. At phage concentrations below
the detection threshold,
no infectious enteroviruses were isolated, but
the enterovirus
genome was detected in 29% of the samples.
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TABLE 3.
Relationship between the concentration of B. fragilis phages and the presence of enteroviruses (infectious
or genome)
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DISCUSSION |
This study evaluated the value of a concentration step for the
detection of bacteriophages, enteroviruses, and the enterovirus genome
in treated wastewater. It also investigated whether somatic coliphages, B. fragilis phages, and the enterovirus
genome could be used as indicators of enterovirus contamination. The
concentration step was necessary for all of the samples tested except
for the somatic coliphages. Enough somatic coliphages were present in treated wastewater for direct analysis.
The number of samples testing positive after concentration was
significantly higher than that before concentration for the infectious enteroviruses (5 versus 0), the enterovirus genome (28 versus 9), and B. fragilis phages (24 versus 6). The
requirement for a concentration step is consistent with some studies of
infectious enteroviruses (20) and the enterovirus genome
(5). However, it conflicts with the genome detection results
of other studies (1, 13) showing the inhibition of
RT-PCR after concentration of surface water on electropositive
filters. Simultaneous concentration of both the viral genome and
substances inhibiting RT-PCR may occur during the virus concentration
step. The concentration of inhibitors may depend on the support medium
used; there may be less coconcentration of inhibitors with glass wool
than with electropositive filters.
Based on the number of positive samples, the concentration of somatic
coliphages (P < 0.01) was significantly higher
than that of B. fragilis phages, which were present at
a frequency similar (P > 0.1) to that of the
enterovirus genome. The enterovirus genome was present in significantly
more samples (P < 0.01) than infectious enteroviruses.
This classification is consistent with that of Tartera et al.
(22), who showed that the somatic coliphage concentration
is, on average, 100 times higher than the B. fragilis phage concentration, which is 10 times higher than
the concentration of enteroviruses.
The number of samples testing positive for the enterovirus genome was
significantly higher than the number of samples containing infectious enteroviruses (28 versus 5). Free viral RNA is rapidly broken down in wastewater (13), so the difference in the
rates of detection of these two factors may have been due to the
presence of noninfectious viral capsids containing the genome or to the presence of infectious enteroviruses that do not have a CPE on BGM cell
cultures, such as particular coxsackie virus group A serotypes. Thus,
the presence of the enterovirus genome may be regarded only as an
indicator of more or less recent viral contamination. It is not
possible to determine the relationship between the presence of the
enterovirus genome and infectious enterovirus concentration unless
experiments are carried out under conditions optimal for the isolation
of all types of enteroviruses, which would mean the inoculation of a
large number of cell culture systems. However, the genome may be a
useful indicator because it is rapidly detected by this sensitive
method and, in this study, no infectious enteroviruses were detected
when the enterovirus genome was not detected.
As the level of treatment increased among treatment line A (biological
treatment, 8 to 12 h), treatment line B1 (biological treatment
with alternating phases of aeration and anoxia, approximately 30 h), and treatment line B2 (secondary and tertiary treatments), the
concentrations of infectious enteroviruses, somatic coliphages, and
B. fragilis phages decreased, as did the number of
samples testing positive for the enterovirus genome. There was a
significant correlation between infectious enterovirus concentration
and that of somatic coliphages and between infectious enterovirus
concentration and B. fragilis phage concentration for
all treatments. This correlation made it possible to define
bacteriophage concentration thresholds over which enteroviruses were
detected in the samples. These thresholds were 104 PFU
· liter1 for somatic coliphages and 102
PFU · liter1 for B. fragilis
phages. Above these concentrations, infectious enteroviruses were
detected in 22% (coliphages) and 71% (B. fragilis phages) of the samples, whereas below these concentrations, no infectious enteroviruses were isolated. Similar correlations were found
between these phages and the enterovirus genome. At somatic coliphage
concentrations between 102 and 103 PFU · liter1 and B. fragilis phage
concentrations of <10 PFU · liter1, the
enterovirus genome was detected in almost 30% of the samples.
The study of various markers is particularly useful in cases of failure
of a treatment line. There was a destabilization of the bacterial flora
involved in biological treatment on treatment line A between 2 May and
7 May. It was accompanied by a decrease in the yield from the secondary
settling step. This treatment failure resulted in increases in the
levels of suspended solids from 12.5 mg · liter1
to 22 mg · liter1, in the 5-day biochemical oxygen
demand from 19.5 mg · liter1 to 24 mg · liter1, and in the chemical oxygen demand from 67 mg
· liter1 to 83 mg · liter1,
whereas these factors were constant at the plant inlet over the entire
sampling period. During this period, the concentration of enteroviruses
increased by factors of 10 to 67. The results obtained during this
treatment failure showed that the detection of the enterovirus genome
cannot be used to evaluate changes in the concentration of infectious
enteroviruses. This result could only be achieved by adapting existing
genome quantification protocols to wastewater.
B. fragilis phages were good indicators during
treatment failure. The phage concentration increase (by a factor
of 97) was higher than that for enteroviruses (by factors of 10 to
67). This strong correlation between enterovirus and B. fragilis phage concentrations has been observed in other
environments, such as marine sediments (11). Changes in
somatic coliphage concentration were not related to changes in
enterovirus concentration because somatic coliphage concentration
increased by a factor of only 4. This result may have occurred because
the maximum percentage of adsorbed suspended solids in secondary
effluents is about 24% for somatic coliphages, whereas it may be as
high as 100% for enteroviruses (7). The significant
increase in the levels of suspended solids at the treatment line outlet
therefore may have caused a larger increase in the enterovirus
concentration than in the coliphage concentration. Somatic coliphages
may simply indicate fecal contamination in the broad sense of the term,
as suggested by Nieuwstad et al. (19), who observed a strong
correlation between the concentrations of these phages and fecal
coliform bacteria.
Thus, in the three different types of wastewater tested, B. fragilis phages were good indicators of enterovirus contamination. Despite a significant correlation between somatic coliphage
concentration and the presence of infectious enteroviruses, this
factor was a poor indicator of fluctuations in enterovirus
concentration. The enterovirus genome could be used to evaluate viral
contamination of treated wastewater if nucleic acid quantification is
carried out and a threshold for the presence of infectious
enteroviruses is determined.
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ACKNOWLEDGMENTS |
We thank Lyonnaise des Eaux (CIRSEE) for financial support. This
study was carried out within the framework of the Franco-Israeli cooperation (AFIRST) water management program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Faculté de
Pharmacie, Laboratoire de Virologie, 5 rue Albert Lebrun, 54000 Nancy, France. Phone: 33.3.83.17.88.25. Fax: 33.3.83.17.88.79. E-mail: lschwart{at}pharma.u-nancy.fr.
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Abstract
Introduction
