United States
Environmental Protection
Agency
Water Engineering
Research Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S2-87/010 Apr. 1987
Project Summary
Evaluation of Bacteroides as
Indicator Bacteria in Drinking
Water
James T. Staley, Thomas T. Moench, N. Robert Ward, and
Donald L. Johnstons
Research was undertaken to examine
the feasibility of using the Bacteroides
fragilis group of intestinal bacteria as
indicators of fecal contamination in
drinking water. This group surpasses
Escherichia coli in fulfilling criteria for
an ideal microbial indicator of fecal con-
tamination.
Two immunological approaches—
fluorescent antibody (FA) and enzyme-
linked immunosorbent assay (ELISA)—
were used to detect and quantify
intestinal Bacteroides spp. in the labo-
ratory and in simulated contaminated
drinking water supplies that were
seeded with fecal material from raw
sewage.
FA procedures did detect intestinal
Bacteroides spp. in sewage-contami-
nated waters, but full quantitative re-
covery was not attained. Although an
extensive effort was made to develop
an ELISA test, considerable difficulty
was encountered in the nonspecific ad-
sorption of goat anti-rabbit peroxidase
to filters. Thus this approach is not ad-
visable until this problem can be over-
come.
Viable counting of intestinal Bac-
teroides spp. from simulated raw-
sewage-contaminated drinking waters
indicates that there is a need for better
selective and differential med" for this
purpose.
This Project Summary was devel-
oped by EPA's Water Engineering
Research Laboratory, Cincinnati. OH.
to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering
information at back).
Introduction
The ability to determine the quality of
drinking water is essential to protecting
the public health. In particular, it is nec-
essary to know whether drinking water
contains fecal material from humans or
animals, since its presence indicates the
possibility of intestinal pathogens in the
water. Because pathogens would be ex-
pected to occur in low numbers relative
to the total bacteria from feces, tests
have been designed to detect Es-
cherichia coli, a normally nonpatho-
genic bacterium that is indigenous to
the intestinal tract of humans and other
animals. Unfortunately, no simple and
rapid test exists to determine whether
E. coli is present in a water sample. In-
stead, lengthy cultivation tests (requir-
ing at least 24 to 48 hr of incubation)
must be used to determine the presence
of total and fecal coliform species,
which are only presumptive indicators
of the presence of E. coli.
The ideal microbial indicator of fecal
contamination should have the follow-
ing features: (a) it should occur in large
numbers in the intestinal tract of
humans, (b) it should grow only in the
intestinal tract and not in the habitat,
and (c) it should be sufficiently distinc-
tive so that it can be identified quickly
with simple tests that are inexpensive.
This research project examined the
feasibility of using the Bacteroides frag-
ilis group of intestinal bacteria as indica-
tors of fecal contamination because this
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group surpasses E. coli in fulfilling the
three criteria outlined above. First,
members of the enteric Bacteroides
group (B. fragilis, B. vulgatus, B. ovatus,
B. thetaiotaomicron, and B. distasonis)
occur in much higher numbers (100 to
1000-fold greater concentrations) in the
normal human intestinal tract than
E. coli. Second, because the B. fragilis
group comprises obligate rather that
facultative anaerobes, they cannot grow
in aerobic receiving waters. Finally, un-
like E. coli, there are specific immuno-
logical tests for this group that could
potentially be used to quantitate them
rapidly in water samples.
This project examined the use of two
immunological approaches—fluores-
cent antibody (FA) and enzyme-linked
immunosorbent assay (ELISA) proce-
dures—to detect and quantify intestinal
Bacteroides spp. in the laboratory and
in simulated contaminated drinking
water supplies that were seeded with
fecal material from raw sewage.
Materials and Methods
Pure cultures of Bacteroides spp., pri-
marily B. fragilis and B. vulgatus, were
grown anaerobically m either Gas Pak
containers* (BBL Laboratories) or in an
anaerobic glove bag (Coy Laboratories,
Ann Arbor, Michigan). Several media
were used for pure culture work and
also for viable enumeration of environ-
mental Bacteroides spp., including
brain heart infusion (BHI), which is a
nonselective medium, as well as a vari-
ety of selective and differential media,
including the medium of Wilkins and
Chalgren (WC), Bacteroides bile esculin
(BBE), and kanamycin bile (KB). Faculta-
tive anaerobes were grown aerobically
on R2A medium.
Antisera against Bacteroides spp.
were tested. These included a polyva-
lent commercial preparation, Fluorotec-
F (General Diagnostics, Morris Plains,
New Jersey), as well as specific rabbit
preparations against B. fragilis and 6.
vulgatus. The Fluorotec-F serum was
used for direct fluorescent microscopic
counting (dFA), whereas antisera for
6. fragilis and B. vulgatus were labeled
with goat anti-rabbit serum conjugated
to fluorescein (Miles-Yeda Ltd., Israel)
for indirect fluorescent microscopic
counting (iFA). These latter two sera
were used for the ELISA testing in which
'Mention of trade names or commercial products
does not constitute endorsement or recommenda-
tion for use.
goat anti-rabbit peroxidase (Miles-
Yeda) was used as the enzyme.
FA microscopic counting was per-
formed on 0.2-u.m pore size Nuclepore
filters that were prestained with irgalen
black to reduce background fluores-
cence. After filtration of the sample, 1.0
ml of a 2% hydrolyzed gelatin solution
was added before exposure of the finer
to Fluorotec-F (dFA) or anti-Bacteroides
serum (iFA). Following appropriate in-
cubation, washing, and mounting pro-
cedures, dFA counts were made using
an epifluorescence microscope. For iFA
counting, an additional incubation with
fluorescent goat anti-rabbit serum was
included.
ELISA tests were performed using a
variety of filters and pore sizes, includ-
ing polysulfone (0.45 u,m), polypropy-
lene (10 |xm), polycarbonate (0.2 n,m),
polyvinylidine-fluoride (0.45 u,m), cellu-
lose acetate (0.45 u.m), cellulose nitrate
(0.45 u.m), and teflon (0.45 urn). To pre-
vent the nonspecific binding of goat
anti-rabbit peroxidase (GARP) to filters,
a variety of proteins (including bovine
serum albumin, gelatin, and goat nor-
mal serum) were tested under a variety
of conditions before exposure to GARP.
To simulate the contamination of a
drinking water supply, some experi-
ments were performed, including the
addition of one part of raw untreated
wastewater to nine parts of lake water
samples. In some experiments, pure
cultures of B. vulgatus and/or B. fragilis
were seeded in addition. Samples were
withdrawn at periodic intervals and
enumerated by iFA and viable plating
procedures. Oxygen concentrations
were measured, .and all flasks were
found to be aerobic (near oxygen satu-
ration) shortly after initiation of the ex-
periment and 24 hr later.
Experimental Results
Fluorescence Microscope
Counting of Intestinal
Bacteroides spp.
Pure culture studies of 6. vulgatus
showed that the iFA counts remained
high throughout the ^rowth curve, par-
alleling culture turbidity even after vi-
ability declined appreciably (Table 1).
This result indicates that the iFA proce-
dure closely follows the optical density
and total microscopic count in cultures
of this numerically important intestinal
Bacteroides spp.
Recovery experiments were per-
formed in which B. fragilis was added to
buffered water, tap water, or primary
wastewater and recovered by viable
plating and dFA counting. Although
high recoveries (36% to 96% of the vi-
able count) were obtained from buf-
fered water suspensions, lower recover-
ies were obtained from tap water (8% to
32%) and primary wastewater (5% to
11%). The low recoveries from waste-
waters were partly due to the large
amount of detritus that interfered with
the fluorescence microscope counting.
In another experiment, a pure culture
of 6. vulgatus was inoculated into filter-
sterilized lake water and incubated aer-
obically at 17°C. Cell counts by iFA indi-
cated there was no decline in numbers
following 3 days of incubation.
These experiments demonstrate that
fluorescent antibody counts of Bac-
teroides spp. can be made with pure
cultures in media and when inoculated
into natural environmental samples of
lake water, tap water, and wastewater.
ELISA Test for Bacteroides spp.
The ELISA methodology was thought
to be a more effective means of assess-
ing intestinal Bacteroides spp. in natural
samples than fluorescent antibody pro-
cedures for several reasons. First, the
microscopic procedure is tedious and
requires considerable time to perform.
Also, it was thought that the problem of
detrital interference might be partly cir-
cumvented by the use of the ELISA pro-
cedure.
Because drinking water samples that
would be tested for Bacteroides would
have to be concentrated before exami-
nation, efforts of this study were di-
rected toward the development of an
ELISA procedure that would use a mem-
brane filtration step. However, prob-
lems were encountered in preventing
the goat anti-rabbit peroxidase from
nonspecifically binding to filters. De-
spite the use of various types of pore
sizes of filters and several pretreatment
procedures, this problem could not be
resolved.
Fluorescent Antibody
Enumeration of B. fragilis and
B. vulgatus in Simulated
Contaminated Drinking Waters
Because of the difficulties encoun-
tered in the development of an ELISA
test, efforts were directed toward exam-
ining fluorescent antibody and viable
counting procedures for recovering
Bacteroides spp. from drinking waters.
Since there was no assured source of
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drinking water that was contaminated
by Bacteroides spp., a simulated one
was used. Raw wastewater was added
to lake water and incubated aerobically.
In some experiments, a pure culture of
B. vulgatus (the most common human
intestinal species) or a combination of
6. vulgatus and B. fragilis was seeded
into the contaminated water. Bac-
teroides spp. were enumerated on sev-
eral selective and differential media and
also by use of iFA techniques.
The data in Figure 1 show the results
of one of the experiments using artifi-
cially contaminated water. In this exper-
iment, pure cultures of Bacteroides
were not seeded, so the counts that are
shown are representative of what
would be found in a natural situation.
First note that the iFA counts were the
highest of all, remaining in excess of
105/mL at zero time on days 1 and 2, and
declining only slightly below that level
on day 3. This result indicates that the
FA technique can detect Bacteroides
spp. (a pooled antiserum of B. vulgatus
and B. fragilis was used) in a simulated
contamination of drinking water.
Viable counts of presumptive Bac-
teroides spp. on WC medium were
higher than total coliforms at zero time,
whereas other media gave results com-
parable with those found for total and
fecal coliform bacteria (Figure 1). How-
ever, when cultures of the presumptive
Bacteroides were isolated and charac-
terized, it was discovered that most
could not be Bacteroides because they
were facultative anaerobes rather than
obligate anaerobes. Thus the actual lev-
els of viable Bacteroides spp. in this ex-
periment (as well as another that was
performed) indicated that this group
died off much more quickly than would
have been predicted from pure culture
studies.
Thus no adequate selective and dif-
ferential procedure presently exists for
viable counting of Bacteroides spp. for
water samples contaminated with raw
sewage.
Conclusions
The results of this study indicate that
FA procedures can detect intestinal Bac-
teroides spp. in waters that have been
contaminated with raw sewage. How-
ever, full quantitative recovery was not
attained by this procedure for two rea-
sons: a) problems occurred with detrital
interference, which obscured fluoresc-
ing cells and gave rise to background
fluorescence, and b) several serotypes
Table 1. Comparison of Various Enumeration Procedures during Growth ofB. vulgatus
Number of Cells/mL (x W7)
Hours After
Inoculation
7.5
11.5
25
36
218
Optical
Density
(660 nm)
0.08
0.21
0.46
0.46
0.49
Direct Micro-
scopic Count
5.6
29
63
N.D.*
58
(Fa Count
6.1
27
42
64
45
Viable Count
3.7
22
35
54
0.07
*Not determined.
Ol
o
D
Figure 1.
Time (days)
Comparison of various techniques for enumeration of bacteria from a simulated
contaminated drinking water source at daily intervals following contamination
FA is the indirect fluorescent antibody count using pooled B vulgatus and B
fragilis sera. Total coliform and fecal coliform bacteria were enumerated by
standard methods, and three media (BBE. WC, and KB agar) were used for plating
of viable Bacteroides spp. (see text for details).
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existed among the intestinal Bac-
teroides spp., and a better polyvalent
antiserum (comparable to or better than
the Fluorotec-F, which is no longer com-
mercially available) would be needed to
obtain better recovery. Furthermore,
microscopic counting procedures are
tedious and laborious and therefore not
as desirable as other procedures such
as ELISA testing.
Although an extensive effort was
made to develop an ELISA test, consid-
erable difficulty was encountered in the
nonspecific adsorption of goat anti-
rabbit peroxidase to filters. Until this
problem can be overcome, it is inadvis-
able to consider using this approach.
Viable counting of intestinal Bac-
teroides spp. from simulated raw-
sewage-contaminated drinking waters
indicates that there is a need for better
selective and differential media for this
purpose. However, even if a better
medium were to be developed, its use-
fulness is questionable for two reasons:
a) it would be no more rapid than cur-
rent methods already developed for the
enumeration of coliform bacteria, and
b) Bacteroides spp. appear to die off
quickly in raw sewage (i.e., much more
quickly than Escherichia coli). One pos-
sible advantage of such a test would be
to assess how recently a water supply
had been contaminated by raw sewage.
Thus if a high ratio of Bacteroides spp.
to fecal coliforms was found, it would
suggest recent contamination, whereas
a low ratio would suggest earlier con-
tamination.
The full report was submitted in fulfill-
ment of Cooperative No. CR-810477-01-
3 by the University of Washington
under the sponsorship of the U.S. Envi-
ronmental Protection Agency.
James T. Staley, Thomas T. Moench, and N. Robert Ward are with University
of Washington, Seattle, WA 98195; and Donald L Johnstone is with
Washington State University, Pullman, WA 99164.
Donald Reasoner is the EPA Project Officer (see belowj.
The complete report, entitled "Evaluation of Bacteroides as Indicator Bacteria
in Drinking Water," (Order No. PB 87-145 892/AS; Cost: $11.95, subject
to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Water Engineering Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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