Animal Waste Pathogens Laboratory, U.S. Department of
Agriculture-Agricultural Research Service, Beltsville, Maryland
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INTRODUCTION |
Enterohemorrhagic Escherichia
coli (EHEC) has emerged as a serious food-borne and waterborne
pathogen. There are an estimated 73,000 cases of E. coli
O157 infections per year in the United States, of which approximately
62,000 are food-borne and 11,000 are waterborne (14).
E. coli O157 was first reported in the United States in
1982, when it was associated with a multistate outbreak of hemorrhagic
colitis (20). EHEC outbreaks (including O157 and O111
strains) have since been reported in Europe and Australia
(2). In 1986, E. coli O157 was recovered from
healthy dairy cows, suggesting that dairy and beef herds could serve as a reservoir (13). Subsequent studies have confirmed that
E. coli O157 and other EHEC strains are commonly found in
beef and dairy cattle (6, 19, 21, 24) as well as animals
associated with farm environments: birds, flies, rodents, and companion
animals (8). Recent studies suggest that deer may also be
a source of E. coli O157 (11, 18).
Although the predominant mode of transmission to humans is contaminated
meat or meat products, infection via contaminated water has also been
documented. For example, Ackman et al. (1) reported an
outbreak of E. coli O157 (six confirmed and six probable cases) among swimmers in a freshwater lake in New York State. The lake
was closed by the county department of health 8 days after the
presumptive exposure, and extensive water testing was conducted.
However, no water samples exceeded New York standards (70 fecal
coliform CFU ml
1), and E. coli O157 was not
detected in lake water samples, presumably due to die-off and/or dilution.
This case illustrates the difficulty associated with the detection and
enumeration of E. coli O157 cells in surface waters. Cultural methods are laborious and expensive. For example, the current
USDA-Food Safety and Inspection Service method entails four enrichment
and culturing steps for preliminary identification and 10 biochemical
tests for confirmation of E. coli O157 (5). These require a minimum of 3 days to perform. The use of sorbitol MacConkey agar has been proposed as a presumptive indicator of E. coli O157 because most O157 strains are sorbitol negative while generic E. coli strains typically are sorbitol positive
(10). However, many genera of enteric bacteria contain
sorbitol-negative species or strains. Consequently, multiple
sorbitol-negative colonies must be screened via genetic, biochemical,
or serological methods to accurately quantify E. coli O157
populations. In addition, standard enrichment methods for detection and
enumeration of fecal E. coli cells via most-probable-number
techniques may fail to detect E. coli O157. Ferenc et al.
(7) reported that many O157 strains did not grow at
44.5°C in EC broth (ECB) when their initial populations densities
were <100 CFU ml1. Kusunoki et al. (12)
evaluated the growth of E. coli O157 at 37 and 42°C in six
different media. E. coli O157 grew more rapidly at 37°C in
all media, with the exception of Trypto-soya broth. Therefore,
incubation at 37°C is advisable; however, because many bacteria have
growth optima at or near 37°C, E. coli O157 may be
out-competed by other bacterial strains when present at lower
population densities.
Recently, a variety of immunological methods have been developed for
the detection and enumeration of E. coli O157 cells. The
common denominator among all methods is the use of monoclonal or
polyclonal anti-E. coli O157 antibodies to selectively
capture, or capture and label (sandwich assay), E. coli O157
cells. For example, several investigators have developed protocols for
the enumeration of viable E. coli O157 cells using
immunomagnetic (IM) bead separation (IMS) techniques in conjunction
with plating (3, 6). Several sandwich assays have also
been described that use a variety of methods for capture, labeling, or
detection. Pyle et al. (17) described a method utilizing
IMS and immunofluorescent antibody (IFA) techniques for capture and
labeling, respectively, followed by enumeration via solid-phase laser
cytometry. Kusunoki et al. (12) described a similar method
utilizing immunolatex beads in conjunction with IFA, followed by flow
cytometry. Park and Durst (15) reported a sandwich assay
in which E. coli O157 cells were immobilized by
anti-E. coli O157 antibodies bound to nitrocellulose and
detected using immunoliposomes containing a marker dye. DeMarco et al.
(4) developed a sandwich assay using anti-E.
coli O157 antibodies bound to silica fibers for capture, IFA for
labeling, and a fiber-optic sensor for quantitation. Finally, Yu and
Bruno (23) described a method utilizing IMS in conjunction with electrochemiluminescence (ECL) for labeling and detection. Although technically identical to the method reported here, they optimized their protocol only for the qualitative detection of E. coli O157 and Salmonella enterica serovar Typhimurium
in various foods and water samples. All of the above methods have been
demonstrated to be suitable for the detection of E. coli
O157 in extraction buffers or enrichment media derived from fecal,
food, or water samples. However, none have been shown to be applicable
to the detection of E. coli O157 in raw or concentrated
surface water samples containing variable levels of sediments, organic
particulates, and unidentified microflora.
We report here on the development and optimization of an IM-ECL
protocol for the quantitative detection of E. coli O157 in raw and concentrated surface water samples. The dynamic range of the
assay in raw surface water samples is ca. 101 to
105 E. coli O157 cells ml1. In
conjunction with vortex filtration concentration (100-fold), the
detection limit is ca. 25 E. coli O157 cells 100 ml1. When concentration and enrichment are combined, the
potential detection limit is 1 to 2 viable cells liter1.
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MATERIALS AND METHODS |
Bacteria, growth conditions, and reagents.
EC broth (ECB)
and membrane fecal coliform (mFC) media were purchased from Difco
Laboratories (Detroit, Mich.). Minimal lactose broth (MLB) consisted of
the basal salts medium of Hylemon and Phibbs (9) (50 mM
potassium phosphate, 15 mM ammonium, and trace nutrients) modified by
the addition of 8.5 g of NaCl, 1.5 g of Bacto Bile Salts
(Difco), and either 3.6 g of lactose (10 mM) or 0.36 g of
lactose (1 mM) liter1. Phosphate-buffered saline (PBS-2)
consisted of 150 mM potassium phosphate buffer (pH 7.2), 150 mM NaCl,
and 0.1% azide, while PBS-1 consisted of potassium phosphate plus 150 mM NaCl at pH 7.8. Diluent consisted of PBS-2 amended with 4% (wt/vol)
bovine serum albumin (BSA) and 1% (vol/vol) thesit (polyoxyethylene 9 lauryl ether).
E. coli O157 strains Odwalla and B6914 were obtained from
Pina Fratamico (USDA-Agricultural Research Service, Eastern Regional Research Laboratory, Wyndmoor, Pa.). Strain ATCC 35150 was purchased from the American Type Culture Collection (Manassas, Va.). A
sorbitol-negative non-O157 E. coli strain, strain 794, was
isolated from a lactating dairy cow at the Beltsville Agricultural
Research Center. Bacteria were cultured in Lennox broth for 18 h
at 37°C with shaking. Bacteria were harvested from the cultures by
centrifugation at 6,000 × g for 15 min at 4°C and
washed by suspension in cold 50 mM potassium phosphate buffer (pH 7)
containing 0.85% sodium chloride (PBS). Sodium azide (0.1%) was added
to the suspended cells as a preservative, and they were stored at
4°C. Cell numbers were determined in a hemocytometer using
phase-contrast microscopy (magnification, ×400; average of six
determinations). Concentrations of bacterial stock solutions were
2.0 × 109, 2.4 × 109, and 3.2 × 109 cells ml1 for B6914, Odwalla, and 35150 strains, respectively. To assess the effect of growth media on ECL
signal, E. coli O157 (Odwalla) cells were grown in 1 mM MLB
with or without bile salts to ca. 2 × 108 cells
ml1; precise numbers were determined using a
hemocytometer (six determinations). Cultures were serially diluted with
PBS-2, and the 102 to 104 dilutions were
analyzed by IM-ECL (data are means of three cultures).
Glycerol, Triton X-100, polyethylene glycol 1000 (PEG 1000), PEG 8000, BSA, and polyvinylpolypyrrolidone (PVPP) were purchased from Sigma (St.
Louis, Mo.) while hexa meta-phosphate and EDTA were purchased from
Fisher (Pittsburgh, Pa.).
Surface water collection and concentration.
Ten-liter water
samples were collected on 27 October 1999 from Little Cove Creek and
Licking Creek, and on 7 July 2000 from Little Cove Creek, located in
the Conococheague-Opequon watershed (U.S. Geological Survey no.
02070004) in south-central Pennsylvania. The creeks drain a watershed
containing a combination of forest and pasture (dairy). The creeks flow
into the Potomac River near Hancock, Md.
Ten liters of water was concentrated 100-fold using a Benchmark GX
vortex filtration unit, manufactured by Osmotics (Minnetonka, Minn.).
Raw water was pumped through the unit using a peristaltic pump (flow
rate of 150 ml min1) followed by an additional liter of
distilled water to rinse the tubing. An MX 500 (50-nm-pore-size)
200-cm2 cylindrical filter cartridge spinning at 2,000 rpm
was used. The unit was drained (ca. 40 to 50 ml), a liter of distilled
water was pumped through the unit to wash the filter, and the unit was drained again (ca. 40 to 50 ml). The concentrated water and wash water
were combined, and the total volume was adjusted to 100 ml with
distilled water. The filtration unit was cleaned with 1 liter of 0.1 N
NaOH between runs. Concentrated samples were preserved either by adding
10× PBS-2 (10%, vol/vol) or by adding the equivalent amounts of
phosphate, salt, and azide dry reagents to give a 1× PBS-2
concentration. Based on preliminary data which indicated that removal
of particulates enhanced cell capture, prior to analysis, 13-ml
aliquots (in 15-ml tubes) were centrifuged at 100 × g
for 15 min to sediment soil particulates and the top 10 ml was
transferred to a clean tube.
Turbidity (in nephelometric turbidity units) was determined before and
after concentration using a model 965-10A Turbidimeter manufactured by
Orbeco Analytical Systems, Inc. (Farmingdale, N.Y.). Selected
concentrated water samples were filtered using cellulose acetate
syringe filters with a pore size of 0.2 µm (Gelman Sciences, Ann
Arbor, Mich.).
Water spiking and enrichment.
Water spiking experiments were
conducted with E. coli O157 Odwalla cells grown to
stationary phase in 1 mM MLB at 37°C. Cultures were diluted ca.
10,000-fold in PBS, and 1 ml was added to 10 liters of Little Cove
Creek water samples (collected 7 July 2000). Just prior to
concentration, total coliforms counts were determined by filtering 100 ml of water sample and transferring the filters to mFC media at 37°C.
After spiking and concentration, E. coli O157 CFU were
determined on MacConkey or Lennox agar with an Autoplate 4000 spiral
plater (Spiral Biotech, Bethesda, Md.). CFU were counted manually or
with a Protocol plate reader (Synoptics, Cambridge, United Kingdom).
Enrichment experiments were conducted by mixing 4 ml of raw or
concentrated water (three replicates) with 4 ml of 2× ECB or 2× MLB
and incubating the mixtures statically at 37°C until turbid. When
turbid, 1 ml of sample was transferred to a microcentrifuge tube and
centrifuged for 10 min at high speed (ca. 13,000 × g), the supernatant was discarded, and the pellet was suspended in 0.5 ml
of PBS-2. Samples were diluted 102-, 104-, and
106-fold with PBS-2, and 0.5 ml was analyzed via the IM-ECL
protocol as subsequently described.
IM bead preparation.
Commercial E. coli O157
beads (Dynabeads) and 2.8-µm-diameter streptavidin beads (Dynabeads
M-280) were manufactured by Dynal A.S. (Oslo, Norway) and purchased
from IGEN International, Inc. (Gaithersburg, Md.). A monoclonal
antibody to O157 LPS (1 mg) was obtained as a liquid suspension from
BioDesign International (Kennebunk, Maine). The antibody was passed
through a gel filtration column that was equilibrated with 150 mM
PBS-1. To the monoclonal antibody solution in PBS-1 was added 75 µg
of Biotin-LC-sulfoNHS ester (IGEN International, Inc.) from a stock
solution of 2 mg ml1 in water. After 1 h at ambient
temperature the reaction was halted by the addition of 40 µmol of
glycine (from a 2 M stock in water) followed by 10 min of incubation at
room temperature to inactivate unreacted material. The biotinylated
antibody was purified by passage through a gel filtration column
equilibrated with 150 mM PBS-2. Biotinylated antibody was stored at
4°C.
An affinity-purified polyclonal antibody to E. coli O157
from a goat was obtained from Kirkegaard and Perry Laboratories
(Gaithersburg, Md.) as a freeze-dried preparation (1 mg). The antibody
was dissolved in 0.3 M sodium phosphate buffer (pH 7.4) to a
concentration of 10 mg ml1 according to the
manufacturer's instructions and then diluted to 1 mg ml1
with PBS-1. To 1 mg of the antibody was added 56 µg of ORIGEN TAG-NHS
ester [ruthenium (ii) tris-bipyridyl (referred to hereafter in this
work as TAG) IGEN International, Inc.] from a stock solution of 1.5 mg
ml1 in dimethyl sulfoxide. The conditions for the
reaction, termination of the reaction with glycine, purification of the
antibody, and storage were identical to those described above for the
biotinylation of antibodies. TAG-labeled antibodies were purified and
stored in PBS-2 at 4°C. Protein (antibody) concentrations were
determined using the bicinchoninic acid protein assay of Pierce
Chemical Co. (Rockford, Ill.) with BSA as standard. Working antibody
solutions were prepared by diluting with diluent.
IM-ECL protocol.
The sandwich assay procedure is illustrated
in Fig. 1. Preliminary experiments with
PBS-2 and raw water samples were conducted with IM beads prepared
immediately prior to use. Fifty microliters of strepavidin beads (0.4 mg ml1) was incubated for 30 min with 50 µl of
biotinylated monoclonal antibody (1 µg ml1). To capture
cells 0.5 ml of E. coli O157 cells was added to prepared IM
beads and shaken for 2 h. To label cells 50 µl of the TAG
antibody (1 µg ml1) was added, and the mixture shaken
for an additional 2 h. (The final total volume was 650 µl. All
water samples were run in triplicate. PBS-2 blanks and/or unspiked
water samples (background) were run at the beginning and end of all
standard curves.
For experiments with concentrated surface water samples, monoclonal IM
beads were prepared in bulk and used as needed. Five milliliters of
strepavidin beads (0.4 mg ml1) was incubated for 1 h
with 5 ml of biotinylated monoclonal antibody (1 to 2 µg
ml1). Beads were harvested using an MPC-1 magnetic
particle collector (Dynal) and suspended in 1 ml of PBS-2, providing a
10-fold concentration. They were stored at 4°C until use. For cell
capture, 20 µl of IM beads and 100 µl of diluent were added to 0.5 ml of water sample and shaken for 2 or 3 h. For sequential bead
capture (SBC), 20 µl of IM beads and 100 µl of diluent were added
to 1 to 3 ml of concentrated water sample and incubated for 3 h,
the beads were recovered for 15 min using an MPC-S magnetic particle
collector (Dynal), the supernatant was transferred to new
microcentrifuge tubes, an additional 20 µl of IM beads, was added,
and the tubes incubated for 3 h. Preliminary experiments indicated
that a 15-min bead capture was adequate for quantitative recovery of
beads from concentrated water samples (data not shown). To label cells,
50 µl of the TAG antibody (1 to 2 µg ml1) was added
and the mixture was shaken for an additional 2 h. The final total
volume was 650 µl. All water samples were run in triplicate or quadruplicate.
ECL instrumentation.
Samples were analyzed using the ORIGEN
device manufactured by IGEN International, Inc. Briefly, 550 µl of
samples (85% of total volume) was pumped through a flow cell where the
bead-cell-TAG complexes were magnetically captured on a platinum
electrode, the sample was washed to remove contaminants and unused
reagents, and a voltage was applied to create an electron transfer
reaction in the presence of tripropylamine, resulting in the emission
of multiple photons from the Ru-chelate component of TAG. The
adjustable instrument parameters were an assay gain of 100, instrument
background subtraction, and signal averaging. Approximate analysis time
per tube was 75 s. Net ECL units are values for spiked water
samples minus values for blank water samples.
Statistics.
Detection limits for the sequential bead capture
protocol were determined using Student's t test
(23) comparing mean ECL signals from the first and second
bead captures as follows: t = d/s
,
where d is the difference between means,
S is
(2s2/n)1/2,
s2 is the sample variance, and n is
the replicate number of tubes. Values of Student's t for 6 df are P = 0.05, 2.447; P = 0.01, 3.707; and
P = 0.001, 5.959.
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RESULTS |
Protocol optimization with PBS and raw water.
Three antibody
sandwich formats were evaluated for quantitative detection of E. coli O157: commercial IM beads-polyclonal TAG, polyclonal IM
beads-polyclonal TAG, and monoclonal IM beads-polyclonal TAG. Standard
curves with commercial, polyclonal, and monoclonal IM beads all gave a
dynamic range of ca. 101 to 105 cells
ml1 (data not shown). However, there were substantial
differences in regression slopes and background ECL values. Average
slopes ± standard deviations SDs) for commercial, polyclonal, and
monoclonal IM beads were 0.83 ± 0.05 (n = 4),
0.68 ± 0.05 (n = 4), and 1.0 ± 0.03 (n = 4), respectively, while mean background values
were 740 ± 130 (n = 24), 820 ± 230 (n = 24), and 164 ± 22 (n = 36) ECL units, respectively.
Monoclonal IM beads were tested with E. coli O157 strains
Odwalla, B6914, and ATCC 35150. All strains gave comparable results (Fig. 2, upper panel). Monoclonal IM beads selectively captured E. coli O157 cells in the presence of ca. 106,
107, or 108 cells ml1 of a
sorbitol-negative non-O157 strain of E. coli (Fig.
2, lower panel). The dynamic range with
monoclonal IM beads was from ca. 101 to 105
cells ml1 (Fig. 3),
although values for 105 cells ml1 were
somewhat variable, indicating that the maximum limit of linear
detection was between 104 and 105 cells
ml1. ECL signals at cell concentrations of
>105 ml1 decreased, presumably because cell
concentrations exceeded IM bead or antibody-TAG concentrations.
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FIG. 2.
The upper panel shows standard curves with
101 to 105 E. coli O157 strains
Odwalla, B6914, and 35150 cells ml1, with monoclonal IM
beads in PBS-2. The lower panel shows standard curves with
101 to 105 cells of E. coli O157
(Odwalla) per ml, using monoclonal IM beads in PBS-2 in the presence of
106, 107, or 108 non-O157 E. coli. Linear regression slopes were computed using 101
to 104 samples.
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FIG. 3.
Dynamic range of IM-ECL assay with 101 to
108 E. coli O157 (Odwalla)) cells
ml1 in PBS-2, with monoclonal IM beads; the linear
regression slope was computed using 101 to 104
samples.
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Results obtained with spiked Little Cove Creek and Licking Creek water
samples and monoclonal IM beads were similar to those obtained with
PBS-2 (Fig. 4). Background ECL values for
raw creek water samples were slightly higher than for PBS-2 blanks
(cited above; Table 1). By comparison,
background ECL values with commercial or polyclonal IM beads were
substantially higher (Table 1). Filtering of water samples through a
0.2-µm-pore-size cellulose acetate filter had little effect on
background values.
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FIG. 4.
Standard curves with 101 to 105
E. coli O157 (Odwalla) cells ml1 in Little
Cove Creek and Licking Creek water samples, with monoclonal IM beads;
linear regression slopes were computed using 101 to
104 samples.
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TABLE 1.
Background ECL signals from unfiltered and filtered
(0.2-µm pore size) Little Cove Creek and Licking Creek water samples
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Protocol optimization with concentrated water.
Preliminary
studies with concentrated water samples indicated that background ECL
signals were substantially higher than with PBS-2 or raw water,
apparently due to nonspecific binding by unidentified materials in
water samples. Experiments with concentrated water samples serially
diluted with PBS-2 demonstrated that there was apparent saturation of
binding sites (Table 2). Eight detergents or polymers were evaluated for their ability to minimize background nonspecific binding: glycerol, Triton X-100, PEG 1000, PEG 8000, BSA,
PVPP, hexa meta-phosphate, and EDTA. Only PVPP at 0.1% reduced background values to near those of PBS-2 (data not shown). However, the
reaction of 0.1% PVPP with cell cleaning solution routinely clogged
the ORIGEN inlet tubing. Experiments with lower concentrations indicated that the maximum concentration which would not plug the
tubing was 0.005%. PVPP reduced background ECL signals ca. 50% in 2-h
incubations and ca. 30% in 3-h incubations (data not shown). Limited
studies were conducted to assess the nature of the nonspecific binding.
When concentrated water samples were incubated with antibody-conjugated
beads or nonconjugated beads (2 h), background ECL values (with 0.005%
PVPP) were higher for nonconjugated beads (646 ± 98) than for
antibody-conjugated beads (349 ± 42; n = 6).
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TABLE 2.
Background ECL signals from concentrated Little Cove
Creek and Licking Creek water samples diluted with PBS-2
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Standard curves were run with Little Cove Creek and Licking Creek
concentrated (100-fold) water; turbidity was 400 to 500 NTU. ECL
signals were somewhat diminished relative to PBS-2 or raw water
standard curves (Fig. 5). The combination
of higher backgrounds and diminished ECL signal per cell resulted in
lower net ECL signals. Consequently, the sensitivity of the assay was reduced to ca. 102 E. coli O157 cells ml of
concentrated water1 (Fig. 5). However, neither turbidity
nor PVPP adversely effected the linearity or dynamic range.
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FIG. 5.
Standard curves for E. coli O157 (Odwalla)
cells in concentrated (100-fold) Little Cove Creek and Licking Creek
water samples with (closed symbols) or without (open symbols) 0.005%
PVPP; linear regression slopes were computed using 101 to
104 samples.
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The ability of consecutive cell capture incubations, termed SBC, to
establish baseline values for individual water samples was
investigated. These experiments were conducted with a new preparation
of polyclonal TAG-antibody which gave a higher and somewhat more
variable ECL signal. For all experiments, monoclonal IM beads were
prepared in advance as previously described. PVPP 0.1% was added to
stock bead preparations, providing a final PVPP concentration in tubes
of ca. 0.0033%.
Based on ECL signals, ca. 80% of E. coli O157 cells were
captured in the initial cell capture incubation (2 h), with the
remainder being captured in the successive incubation (data not shown). Extending the cell capture incubation to 3 h resulted in enhanced cell recoveries of >90% (data not shown). At cell concentrations of
103 cells ml1, ECL values for the
consecutive cell capture (second bead capture) were comparable to
background values; i.e, the differences in ECL signal between the first
and second bead captures (
ECL) were consistent with net ECL
responses from standard curves. At cell concentrations of
>104 cells ml1, however, ECL values for the
consecutive cell capture (second bead capture) were substantially
higher than background values due to the ECL signal from cells captured
during the consecutive cell capture. Since the typical background
levels observed for concentrated waters were 1,000 ECL units, a more
accurate estimate of cell concentrations of >104
ml1 was obtained by combining ECL signals from the first
and second bead captures. The SBC assay gave a linear dynamic range of
ca. 102 to 105 cells ml1 in
concentrated water (Fig. 6). ECL
values were correlated with the sample volume; increasing sample size
from 1 to 3 ml resulted in an ~2.5-fold enhancement in sensitivity
(Fig. 6).
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FIG. 6.
Standard curves for E. coli O157 (Odwalla)
cells in concentrated (100-fold) and centrifuged (100 × g) Little Cove Creek water samples, obtained after SBC with 1-, 2-, and 3-ml sample volumes; combined net ECL signals from the first
and second bead captures are shown for a cell concentration of
105.
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Protocol validation with concentrated water.
Triplicate
10-liter Little Cove Creek water samples were spiked with ca. 5,000 E. coli O157 cells and concentrated, and 1- and 3-ml
aliquots were analyzed by SBC (Fig. 7;
Table 3). Concentration via vortex
filtration did not appear to affect cell viability; total coliform
recoveries on agar plates (including E. coli O157) from
concentrate were consistent with initial populations of spiked plus
background coliforms (Table 3). In the absence of low-speed centrifugation (100 × g), there was no difference in
ECL signals between the first and second bead captures (data not
shown); i.e., E. coli O157 cells were not detected.
Low-speed centrifugation decreased NTU values ca. 10-fold; however,
based on plating before and after centrifugation, low-speed
centrifugation did not result in a detectable loss of viable bacteria
(data not shown). Increasing the sample volume from 1 to 3 ml increased
the ECL values more than twofold, consistent with previous
experiments (Fig. 7; Table 3). The ECL values from two of three 1-ml
samples were highly statistically significant (P < 0.01), while all three values from 3-ml samples were highly
statistically significant (P < 0.01) (Table 3).
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FIG. 7.
Mean ECL signals and SDs (error bars) (n = 4) for ca. 5,000 E. coli O157 (Odwalla) cells spiked
into 10 liters of Little Cove Creek water after concentration
(100-fold) and centrifugation (100 × g).
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Experiments were conducted to detect viable E. coli O157
cells via enrichment culture. With spiked raw water samples, a higher percentage of tubes were positive for E. coli O157 with MLB
than ECB (>10,000 ECL units) (Table 4).
With concentrated water samples, all tubes (both MLB and ECB) were
positive for E. coli O157 (Table 4). MLB was more selective
than ECB. All ECB tubes became turbid, including unspiked, spiked, and
concentrated water samples, while only those MLB tubes which were
positive for E. coli O157 became turbid.
The effect of bile salts on ECL response was investigated. Net ECL
signals (normalized to 102, 103, or
104 cells ml1) were highest for stock culture
cells (grown in L broth), while net ECL signals for MLB cells grown
with bile salts were ca. three-fold higher than those for cells grown
without bile salts (Fig. 8).
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FIG. 8.
Net ECL signal (normalized) for E. coli O157
(Odwalla) cells in stock culture or cultured in 1 mM MLB with or
without bile salts (mean of three cultures). ND, not determined.
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DISCUSSION |
Although commercial, polyclonal, and monoclonal anti-O157:H7 IM
beads gave comparable dynamic ranges of ca. 101 to
105 cells ml1 in PBS-2, the monoclonal IM
beads were chosen for further development and optimization based on
background ECL levels, selectivity, and linearity. Background ECL
values with commercial and polyclonal IM beads for raw creek water
samples ranged from ca. 2,000 to 8,000, effectively masking E. coli O157 cell concentrations of 100 cells ml1.
Similar ECL values were obtained with filtered and unfiltered surface
water samples, suggesting that high background ECL values were not due
to E. coli O157 but rather to nonspecific binding to beads
by unidentified organic materials. Background ECL values with
monoclonal IM beads were only slightly higher in surface water samples
than in PBS-2. This suggests that for low turbidity samples (NTU < 5) it may be feasible to use PBS-2 values as baseline values.
An anti-O157 LPS monoclonal antibody was chosen for immunomagnetic
capture to maximize selectivity. Other investigators have reported
cross-reactivity between commercial polyclonal E. coli O157
IM beads and non-O157 strains (16). Using monoclonal IM beads, small numbers of E. coli O157 cells were detected and
quantified in the presence of large numbers of a non-O157 strain,
strain 794. The 794 strain was isolated as a presumptive E. coli O157 because it was sorbitol negative. Based on information
from BioDesign, the monoclonal immunoglobulin G O157 antibody does not
react with related E. coli strains. However,
cross-reactivity with other bacteria is unknown and should be evaluated.
An anti-O157 polyclonal antibody was chosen as the TAG-antibody based
on the hypothesis that this would maximize TAG binding sites, hence ECL
signal. The combination of monoclonal IM beads and polyclonal
TAG-antibody consistently gave linear slopes of ca. 1.0 in PBS-2 and
raw water (from >101 to 105 cells
ml1), indicative of a constant ECL response per cell.
Consequently, analyzing a single cell concentration should be adequate
to establish a standard response curve.
Low ECL background levels in low-turbidity raw water samples (relative
to PBS-2 blanks) allowed for rapid screening. However, assay
sensitivity (ca. 25 cells ml1 or 2,500 cells 100 ml1) was inadequate to detect E. coli O157 at
levels which could present a public health threat. Preliminary
experiments with concentrated water samples (vortex filtration)
indicated that background levels were substantially elevated over PBS-2
blanks due to nonspecific binding. Experiments with nonconjugated IM
beads suggested that unidentified materials were binding directly to
the bead matrix or streptavidin sites, as opposed to the monoclonal
antibody. PVPP partially inhibited this nonspecific binding. PVPP was
added directly to stock IM bead preparations (0.1%), resulting in a final concentration of ca. 0.0033% in incubation tubes. At this concentration, background ECL signals were reduced ca. 50% during a
2-h incubation or ca. 30% during a 3-h incubation. Although helpful,
PVPP is only a partial remedy. A more ideal solution would be to
identify and permanently block the IM bead binding sites to prevent
nonspecific binding to the bead matrix or streptavidin sites.
Attempts to establish baseline values for individual water samples
using filtration were unsuccessful (data not shown). Substantial numbers of cells from the stock culture passed through the
0.2-µm-pore-size filter. Although these results are not typical for
viable E. coli cells, they do suggest that filtration could
provide incorrect baseline data if small cells or cell fragments are
present in water samples.
The SBC method was developed to accurately account for background ECL
signals. The SBC method is based on the premise that the ECL signal
from the initial cell capture incubation is due to cell capture plus
background binding while the second incubation is due predominately to
background binding. This assumes that background ECL signals are
consistent between the first and second bead captures. Our studies
indicated that the material(s) in Little Cove Creek and Licking Creek
concentrated water samples responsible for the background signal were
present in excess. As a consequence, background ECL signals from the
first and second bead captures were consistent. Further research is
required to determine if this is universally true.
Experiments were conducted to evaluate and optimize the SBC protocol. A
2-h incubation resulted in ca. 80% cell capture. Increasing the
incubation time to 3 h improved recoveries to >90%. At cell concentrations of >104 ml1 (ca. 20,000 ECL
units), the ECL signal from the second bead capture was higher than the
background signal, due apparently to incomplete capture. At these high
cell concentrations, the addition of ECL signals from the first and
second bead captures provided a more accurate estimate of E. coli O157 populations. Increasing the sample volume from 1 to 3 ml
increased sensitivity ca. 2.5-fold. The protocol consistently provided
linear data over the effective dynamic range (ca. 102 to
105), indicative of a constant ECL signal per cell.
Spiking experiments were conducted to validate the protocol and
estimate detection limits. Vortex filtration provided essentially complete recoveries of E. coli O157 cells spiked into water
samples. The SBC protocol successfully detected E. coli O157
(Odwalla) cells in the spiked water samples, and the ECL values
obtained were generally consistent with standard curves. Utilizing
regression data for the 3-ml volume standard curve shown in Fig. 6
resulted in an estimated cell concentration of 60 cells
ml1, after correcting for growth medium effects (see
below), which was comparable to the expected value of 50 cells
ml1. The maximum sensitivity of the assay is dependent on
the magnitude of the difference in ECL signals between first and second
bead captures (ECL), variability within replicates, and the number of replicates. We chose four replicates as a compromise between cost,
labor, and statistical power. Student's t test for paired comparisons was used to determine assay sensitivity. These data suggest
that with a 3-ml incubation the detection limit could be as low as 25 E. coli O157 cells 100 ml1 (P < 0.01), assuming comparable variability for other water samples.
Enrichment cultures with raw spiked samples suggest that both ECB and
MLB are potentially capable of enriching one viable cell. Consequently,
the combination of concentration, enrichment, and IM-ECL analysis can
potentially achieve a detection limit of 1 to 2 viable cells
liter1, depending on the volume of water concentrated
(
10 liters) and the volume of concentrated sample used for
enrichment. MLB appeared to be more selective than ECB, because it
selected only for organisms capable of growth with lactose as the sole
carbon source. In the absence of spiking, no MLB tubes were turbid.
Further research is necessary to determine the rates of false negatives
with both ECL and MLB. False negatives may arise with ECB if E. coli O157 cells are out-competed by organisms growing on casein,
while false negatives may arise with MLB if E. coli O157
cells are unable to adapt to the minimal medium. The use of monoclonal
IM beads should minimize false positives, although this also requires
further investigation.
Standard curves with stock culture Odwalla cells versus MLB-grown
cells, with and without bile salts, indicate that it may be impossible
to establish a definitive standard curve for E. coli O157.
The ECL signal per cell was highest with the stock culture. It is
plausible that the original hemocytometer counts for the stock culture
were underestimated. However, note that stock cells were of a
diminished size, as evidenced by the fact that a majority of cells
passed through a 0.2-µm-pore-size filter. Since the ECL photochemical
reaction only occurs within close proximity to the electrode surface,
cell geometry (i.e., total cell surface area within the reaction zone)
directly affects the magnitude of the ECL signal. The differences
between MLB with and without bile salts are not attributable to
incorrect cell numbers, because cells were cultured simultaneously on
similar media and gave essentially identical CFU and hemocytometer
counts. These data suggest that E. coli O157 (Odwalla) cells
modulate their outer cell membrane composition in response to bile
salts. Consequently, the IM-ECL signal is directly dependent upon the growth culture media, both in vivo and in vitro.
The IM-ECL assay appears suitable for routine analysis and screening of
water samples. Several similar immunological methods have been
described with varying sensitivities, analysis times, and levels of
sophistication. It is difficult to compare the IM-ECL protocol to these
methods, however, because of limited information regarding their
applicability or optimization for raw or concentrated surface water
samples. Since the IM-ECL assay detects nonviable, viable but
nonculturable, and culturable cells, it may be useful in locating and
"tracking" E. coli O157 plumes or identifying point
sources after waterborne infections have occurred. In conjunction with
enrichment procedures, the protocol can also be used to detect viable
cells. The generic protocol is potentially applicable to a wide range
of microorganisms (bacteria, protozoa, and viruses). The application of
the assay to other specific pathogens, however, will be dependent on
the availability of appropriate antibodies with the selectivity to
allow capture and labeling of the target population.
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Abstract
Introduction
