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Applied and Environmental Microbiology, May 1999, p. 1966-1972, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Sensitive Detection of Escherichia coli
O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase
Laser Cytometry
Barry H.
Pyle,*
Susan
C.
Broadaway, and
Gordon A.
McFeters
Microbiology Department, Montana State
University, Bozeman, Montana 59717
Received 1 May 1998/Accepted 17 February 1999
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ABSTRACT |
Rapid, direct methods are needed to assess active bacterial
populations in water and foods. Our objective was to determine the
efficiency of bacterial detection by immunomagnetic separation (IMS)
and the compatibility of IMS with cyanoditolyl tetrazolium chloride
(CTC) incubation to determine respiratory activity, using the pathogen
Escherichia coli O157:H7. Counterstaining with a specific
fluorescein-conjugated anti-O157 antibody (FAb) following CTC
incubation was used to allow confirmation and visualization of bacteria
by epifluorescence microscopy. Broth-grown E. coli O157:H7
was used to inoculate fresh ground beef (<17% fat), sterile 0.1%
peptone, or water. Inoculated meat was diluted and homogenized in a
stomacher and then incubated with paramagnetic beads coated with
anti-O157 specific antibody. After IMS, cells with magnetic beads
attached were stained with CTC and then an anti-O157
antibody-fluorescein isothiocyanate conjugate and filtered for
microscopic enumeration or solid-phase laser cytometry. Enumeration by
laser scanning permitted detection of ca. 10 CFU/g of ground beef or
<10 CFU/ml of liquid sample. With inoculated meat, the regression
results for log-transformed respiring FAb-positive counts of cells
recovered on beads versus sorbitol-negative plate counts in the
inoculum were as follows: intercept = 1.06, slope = 0.89, and
r2 = 0.95 (n = 13).
The corresponding results for inoculated peptone were as
follows: intercept = 0.67, slope = 0.88, and
r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb
method, compared with recovery by sorbitol MacConkey agar
plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times;
water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method
detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or
solid-phase laser scanning cytometry gave results that compared
favorably with plating following IMS.
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INTRODUCTION |
In environmental microbiology, the
need for rapid methods to detect specific bacteria and confirm their
viability or metabolic activity has been widely acknowledged. It is
well known that traditional culture methods for detecting
indicator and pathogenic bacteria in food and water may underestimate
numbers due to sublethal environmental injury, inability of
target bacteria to take up nutrient components in the medium, and other
physiological factors which reduce culturability (18, 20,
28). Existing cultural methods for examining water and foods
generally require enrichment followed by identification of the
contaminating bacteria. More sensitive, rapid microbial detection
methods are needed for many applications to complement or replace these
traditional culture procedures, which require at least 1 day before
definitive results are available. In the food industry, the U.S.
Department of Agriculture has recently adopted Hazard Analysis and
Critical Control Point procedures and enhanced inspection protocols to
ensure the safety of meat products. Recently introduced food inspection
regulations will require postharvest microbiological assays. In the
event of an outbreak, rapid methods can improve the probability of
identifying the source and, ultimately, controlling the spread of
infections. Other examples include the monitoring of spacecraft water
prior to launch (23) and the examination of military
environments for possible biological warfare agents. The use of more
rapid methods for confirmation, including immunomagnetic separation (IMS) and fluorescent-antibody (FAb) identification, has become more
common in the last several years (9, 10, 12, 15, 24, 25, 31,
35).
Since the 1992 outbreak of enterohemorrhagic Escherichia
coli (EHEC) infections from the consumption of hamburger in some fast-food restaurants in the northwestern United States (6), other outbreaks of EHEC infections have been attributed to a variety of
sources (11), including ground beef (2), alfalfa
sprouts (3), apple cider (7), and water
(13, 30). EHEC infections caused by several serotypes,
as well as O157:H7, have been reported in countries around the
world (1, 3, 8, 21, 32, 33).
Traditional culture methods typically lack the sensitivity
needed for direct detection of environmental pathogens.
Thus, enrichment cultures are used so that low numbers of pathogens can
multiply to detectable numbers before confirmatory tests are applied,
and there is a considerable time delay from sampling until the results are available. IMS is being used more often for the detection of
pathogens in food and other specimens following enrichment culture
(9, 25, 32, 33, 35). One objective of the research reported
here was to adapt IMS for direct detection of pathogens such as
E. coli O157:H7 from food and water without
enrichment culture.
FAbs have been used to confirm and enumerate E. coli O157:H7 bacteria by a direct epifluorescent filter
technique (31). FAbs have also been used in conjunction with
IMS (12, 24) for detection of pathogens, including
E. coli O157:H7, in meat and other samples. Without
enrichment culture, there is no proven way of determining the viability
or metabolic activity of bacteria detected by IMS and FAb staining. We
have incorporated incubation with a respiratory fluorochrome,
cyanoditolyl tetrazolium chloride (CTC) (22, 27, 29), to
determine the respiratory activity of cells captured by IMS. Our hybrid
technique includes IMS of target bacteria, incubation with CTC to
determine respiratory activity, staining with a direct FAb to
identify the captured cells, and enumeration by epifluorescence
microscopy or solid-phase laser scanning. To facilitate rapid
enumeration and increase sensitivity, we used a laser scanning
cytometer (Scan RDI; Chemunex, Inc.). This is based on direct
solid-phase laser scanning to detect fluorescently stained bacteria on
a membrane filter (19, 34). The system has been
validated for routine microbiology quality control analysis of
pharmaceutical-grade water (14).
The novel approach reported here for the direct detection of respiring
E. coli O157:H7 can be completed in less than an
8-h working day. This is a significant advantage over existing
techniques for detecting active EHEC which typically require a 24-h
incubation for preenrichment, followed by confirmatory tests
(15).
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MATERIALS AND METHODS |
Bacterial strains and culture conditions.
A human isolate of
E. coli O157:H7, strain 3A-3299/85, was obtained
from R. Wilson, Pennsylvania State University, University Park, Pa.
Strain 932 was supplied by the U.S. Environmental Protection Agency in
Cincinnati, Ohio.
Unless stated otherwise, dehydrated media were obtained from Difco and
chemicals were obtained from Sigma. Culture slopes were subcultured on
tryptone lactose yeast extract (TLY) agar and incubated overnight at
35°C. Growth was harvested in 20% glycerol-2% peptone and stored
at 
85°C. Cultures were streaked on MacConkey agar, sorbitol
MacConkey (SMAC) agar, and sheep blood agar plates to confirm purity.
Identification was confirmed by using the API 20E system
(bioMérieux).
Yeast tryptone medium (
4) was inoculated with a frozen stock
culture and incubated for 20 h at 35°C and 150 rpm. Cells
were
collected by microcentrifugation (2,500 rpm for 2 min; DuPont
Sorvall
MC12V microcentrifuge), resuspended in one half of the
volume of 0.1%
peptone, and vortexed for 1 min. The suspension
was passed through a
5-µm-pore-size nylon mesh filter to remove
clumps, vortexed for 1 min, and diluted to ca. 10
8 CFU/ml by adjusting the optical
density to 50 Klett units (Klett-Summerson
Photoelectric Colorimeter).
The final suspension was diluted to
obtain the required numbers of
cells for inoculation of liquid
or meat
samples.
Immunomagnetic beads.
Superparamagnetic beads with a nominal
diameter of 0.6 µm (Bangs Laboratories, Inc.) were coated with
anti-O157 rabbit serum (Difco). The adsorption procedure was adapted
from the sodium phosphate-phosphate-buffered saline (PBS) method
(5). The beads (0.1 ml of a 2% suspension) were removed
from suspension with a magnet and resuspended in 0.344 ml of phosphate
buffer (pH 5.5) containing 0.056 ml of antiserum. The suspension was
incubated for 1 h while being mixed at 60 rpm at room temperature.
After being coated, the beads were rinsed by removal from the solution with a magnet and resuspension in 0.4 ml of phosphate buffer, rinsed
again in 0.1 ml of PBS (pH 7.5), and stored in 0.1 ml of PBS with 3%
bovine serum albumin (BSA) at 4°C. Coated beads were refrigerated for
up to 1 month before use.
Sample preparation.
Suspensions prepared as described above
were used to inoculate liquid suspensions of water, 0.1% peptone, PBS,
PBS-BSA, and enzyme-detergent solution. Beef hamburger was freshly
ground on request at local supermarkets, transported to the laboratory
within 10 min at ambient temperature, and refrigerated for up to 4 h before use. A 1-g sample of ground beef was weighed into a stomacher '80' bag (4 by 6 in.; Seward), and 0.1 ml of an O157 suspension (101 to 107 CFU/ml) was added. The meat and
suspension were mixed by massaging the bag manually for 5 min. Peptone
(20 ml of a 0.1% solution) was added before the bag was sealed with a
heated pouch sealer (Kapak Corp.). The meat and peptone were
homogenized in a stomacher (Seward) for 2 min. The bag was opened
aseptically, and 20 ml of enzyme-detergent solution (26) was
added. The bag was resealed and incubated for 20 min at room
temperature with mixing at 120 rpm on an orbital shaker. After
homogenization for 2 min in the stomacher, the contents were passed
through a 25-mm-diameter coarse plastic Swinnex filter screen
(Millipore) without filter paper to remove fibrous material.
Uninoculated control samples were also tested to confirm the absence of
detectable numbers of E. coli O157 bacteria.
IMS.
A sample of liquid or meat homogenate suspension was
removed, and 0.01 ml of immunomagnetic beads coated with anti-O157
antibody per ml of sample was added. The vial was incubated at room
temperature horizontally at 60 rpm for 1 h. An aliquot was
transferred to a sterile vial, and the remaining suspension was
retained for plating.
Superparamagnetic particles were removed from the samples by using a
magnetic particle concentrator (Dynal) and locating the
magnetic
concentrator and tube horizontally for 10 min for liquid
samples and 30 min for meat suspensions. The supernatant was removed
with a long
Pasteur pipette, taking care not to disturb the tube
contents and
retaining the
supernatant.
Plating.
Beads were resuspended in the original volume of
PBS or 0.1% peptone for plating. The original inoculum, suspensions
retained prior to bead removal, resuspended beads, and supernatant
removed from the beads were plated on SMAC, TLY, and TLYD (TLY with
0.1% deoxycholate) agar. Plates were incubated for 24 h at
35°C. Sorbitol-negative colonies on SMAC agar were enumerated as
E. coli O157:H7, and the results from TLY and TLYD
plates were compared to determine injury (28).
CTC incubation and filtration.
For liquid samples, beads
were resuspended to the original volume and 1 ml was filtered through a
25-mm-diameter black polycarbonate membrane with a 0.2-µm pore size
(Millipore) and incubated for 3 h in the dark at room temperature
on an absorbent pad (Millipore) saturated with 0.6 ml of 5 mM CTC
(Polysciences) in 0.1% peptone. The filter was transferred to a pad
saturated with 0.6 ml of 3.7% formalin and incubated for at least 5 min at room temperature or stored in a refrigerator overnight. Beads
removed from a hamburger mixture were either resuspended and incubated
on pads as described above or suspended in 0.5 ml of 5 mM CTC and
incubated in solution for 3 h.
FAb staining.
The filters incubated on a pad saturated with
CTC solution were transferred onto a drop (0.025 ml) of PBS in a petri
dish. A drop (0.025 ml) of fluorescein isothiocyanate (FITC)-conjugated anti-E. coli O157 antibody (Kirkegaard and Perry
Laboratories), diluted 1:100 in PBS, was spread over the membrane
filter. A pad saturated with water was added to the lid of the dish,
which was incubated at room temperature for 20 min. The filter was
transferred to a filter apparatus and rinsed three times with 1 ml of PBS.
Beads incubated in CTC solution were removed by IMS, resuspended in 0.5 ml of FITC conjugate diluted 1:100, and incubated
for 20 min. The beads
were removed from FITC solution by IMS,
reconstituted to the original
volume with peptone, filtered through
a polycarbonate filter, and
rinsed three times with 1 ml of PBS.
The inoculum suspension was also
incubated with CTC and FITC,
stained, and enumerated for comparison
with the numbers of cells
removed by
IMS.
Epifluorescence examination.
A pad containing calcium buffer
(pH 9.0) with 2% diazabicyclo[2.2.2]octane (DABCO; Sigma) added to
retard photobleaching was mounted on a 1-in. circular magnet attached
to a slide. The filter was placed on the pad, an additional drop of
buffer was added, and a coverslip was placed on top. The sample was
examined by using a 100× oil immersion objective with epifluorescence
filters appropriate for FITC and CTC (22).
Solid-phase laser cytometer enumeration.
For the enumeration
of membrane filters with less than 103 cells on them, a
Scan RDI rapid solid-phase laser scanning system (Chemunex) was
used. Each membrane filter was loaded onto a stainless steel carrier on
top of a cellulose acetate membrane (Millipore) saturated with 0.1 ml
of buffer. The carrier was placed in the instrument, and the entire
filter surface was scanned within about 3 min. The carrier with the
filter was transferred to a microscope (Nikon) with a
computer-controlled stage (Prion). Under the control of the Scan
RDI software, each object identified as a bacterial cell was examined
to confirm that it was stained with the anti-O157 antibody-FITC
conjugate. The presence of CTC-formazan crystals was also recorded.
The IMS-CTC-FAb method is summarized in Fig.
1.
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FIG. 1.
Flow chart of the steps involved in performing the
IMS-CTC-FAb ground beef assay. RT, room temperature.
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Micrography.
E. coli O157:H7 cells inoculated
into ground beef and enumerated by the IMS-CTC-FAb procedure were
photographed by using a Nikon Optiphot microscope with B2A
epifluorescence filters, a Nikon N70 camera, and Fujichrome Provia 400 professional 35-mm slide film. Internegatives were made from slides and
printed in monochrome on Panalure Select paper (Kodak).
For electron micrographs,
E. coli O157:H7 cells
were extracted from peptone by IMS, filtered through a polycarbonate
membrane,
and fixed with 2.5% glutaraldehyde. The filters were
examined
by using a scanning electron microscope (JEOL), and
micrographs
were taken on monochrome film (Polaroid) in the Image and
Chemical
Analysis Laboratory at Montana State
University.
Data analysis.
The recovery of added cells was determined
for each experiment by comparing the number of E. coli
O157 bacteria detected with the number added to the samples.
Variability was calculated and reported as the standard error of the
mean. Results for replicated experiments were analyzed and plotted by
using SigmaPlot version 4.0 for Windows (SPSS, Inc.) to determine and
display the linear regressions.
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RESULTS |
IMS.
The superparamagnetic beads used in these experiments
were smaller than bacteria (0.6 µm in diameter) and were coated by
incubation with commercial polyclonal rabbit O157-specific antiserum.
Electron microscopy showed that many beads attached to each target
E. coli O157:H7 cell (Fig.
2). It is probable that this binding of
multiple beads per cell and the resultant strong magnetic force during IMS contributed significantly to the extremely high rates of recovery of target bacteria reported below. In addition, epifluorescence microphotography indicated that some target cells had a halo around them (Fig. 3), which probably represents
the numerous attached beads seen in Fig. 2.
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FIG. 2.
Electron micrographs of immunomagnetic beads attached to
E. coli O157:H7 cells. Original magnifications: a,
×8,000; b, ×20,000. Cells appear as faint rods with five or more
white beads attached. The pores in the polycarbonate nuclear etched
membrane can be seen in the background. WD, working distance.
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FIG. 3.
Photograph of E. coli O157:H7 cells
after IMS, CTC incubation, and the FAb reaction. The cells were stained
with FITC-conjugated O157-specific antibody and bright spots of
CTC-formazan, which indicates respiratory activity.
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The IMS procedure resulted in both cell injury and recovery,
depending on the suspension medium. Initial suspensions made
from broth cultures were moderately injured, as determined by
their differential growth on TLYD and TLY agar media. With water
suspensions, before IMS, 46.8% ± 7.8% (
n = 16)
of the bacteria
were injured, compared with 40.0% ± 16.6%
(
n = 6) with 0.1% peptone.
After IMS, comparable
injury values for the target bacteria isolated
with beads were
82.5% ± 3.0% for water and 53.8% ± 13.2% for peptone
suspensions. Similarly, 30.3% ± 8.4% (
n = 11) of the
cells inoculated
into hamburger and 54.4% ± 17.4%
(
n = 7) of the cells recovered
by IMS were injured. In
all cases, the IMS procedure contributed
to membrane injury
(
17), although the effect was less pronounced
in peptone
suspensions and hamburger than in water
suspensions.
Recovery of E. coli O157:H7.
For all
three sample types, more respiring, FAb-positive cells were detected
than were enumerated by SMAC plating of the beads after IMS (Table
1). The extremely large and highly
variable recovery ratio for the water suspensions was observed mainly
because of two instances with small inocula (ca. 100
CFU/ml) when the Scan RDI results for CTC-positive, FAb-positive cells
captured by immunomagnetic beads were more than 40 times higher than
the corresponding plate counts of the captured cells. If these two
results were omitted from the analysis, the mean recovery ratio was
3.18 ± 0.55 (n = 7), which is more comparable to
the results for meat and peptone suspensions.
Comparison of the numbers of
E. coli O157:H7
bacteria detected by the IMS-CTC-FAb technique with the SMAC
plate counts of
the inoculum in ground beef shows that the novel
method was 6.0
times more productive (Table
1). With artificially
contaminated
peptone and water, the new method was 3.0 and 2.4 times
more productive,
respectively. The samples had been inoculated with
10
0 to 10
6 CFU of
E. coli
O157:H7 per g or per ml.
E. coli O157:H7 cells
were not detected in uninoculated control samples of hamburger
(data
not shown). Microscopic analyses gave no evidence of clumped
cells in
the inoculum, which suggested that the apparent increase
in cell
numbers during the enzyme-detergent treatment, IMS, CTC
incubation, and
FAb reaction was not due to the dispersion of
clumped bacteria. As the
increase was observed when using the
microscopic methods, as well as
when using agar plating, it was
attributed to growth of bacteria from
the time of sample preparation
through to fixation prior to FAb
staining. However, the numbers
of FAb-positive cells recovered by IMS
compared with those in
the inoculum did not increase for the
peptone or water suspensions,
although there was a 4.5-times
increase with ground beef samples
(Table
1).
The relative numbers of CTC-reducing, FAb-positive target cells
observed after capture on beads, compared with those in the
inoculum
(Table
1), also indicated that more than four times
the numbers
in the inoculum were detected in hamburger, approximately
equal numbers
were detected in peptone, and lower numbers were
detected when the
cells were suspended in sterile water. The lower
result for the
water suspensions was caused by the failure of
many cells to
reduce CTC. The results for FAb-positive cells recovered
on beads
compared to those in the inoculum provide further evidence
for growth
of
E. coli O157:H7 in ground beef preparations but
not in peptone or
water.
With respect to SMAC plate counts, slightly higher numbers of target
E. coli cells were detected by IMS with inoculated
hamburger
or peptone and lower numbers were detected with water
suspensions
(Table
1). This result suggests that the conditions in the
meat
homogenate and peptone suspensions were more favorable to
detection
by plate counting than those in the water suspensions. This
may
have been related to the additional cell injury which was observed
in the water suspensions compared to the preparations in
peptone.
Regression comparison with plating.
The majority of
regression coefficients exceeded 0.93 (Tables
2 and 3).
Plots show that most of the data points fell within the 95% confidence
intervals of the regression (Fig. 4 and
5). For the smaller inocula
(<104/g or ml), the counts were performed by using the
Scan RDI system, and when these results were combined with the
manual microscope counts for the larger inocula, the data
appeared to be linear. This suggested that the Scan RDI results are
comparable to direct microscope counts, although the Scan RDI system
permits detection to a much lower level, i.e., <10 cells/g of
hamburger and ca. 1 cell/ml in suspension.
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TABLE 3.
Combined IMS-CTC-FAb results for samples of sterile
water or 0.1% peptone inoculated with 100 to
106 CFU of E. coli O157:H7
per mla
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FIG. 4.
Linear regression plots of enumeration results for
artificially inoculated hamburger. (a) Respiring O157 cells (CTC
positive, FAb positive) enumerated by IMS versus the number used as the
inoculum. (b) Respiring O157 cells enumerated by IMS versus SMAC plate
counts (PC) of inoculated cells. (c) SMAC plate counts of cells
enumerated by IMS versus SMAC plate counts of inoculated cells. The
solid line represents the linear regression, and the dotted lines
indicate the 95% confidence interval.
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FIG. 5.
Linear regression plots of composite enumeration results
for artificially inoculated peptone (0.1%) and water. (a) Respiring
O157 cells (CTC positive, FAb positive) enumerated by IMS versus the
number used as the inoculum. (b) Respiring O157 cells enumerated by IMS
versus SMAC plate counts of inoculated cells. (c) SMAC plate counts of
cells enumerated by IMS versus SMAC plate counts of inoculated cells.
The solid line represents the linear regression, and the dotted lines
indicate the 95% confidence interval.
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DISCUSSION |
For current direct microscope techniques which utilize
membrane filtration, we find that the lower limits of
detection are ca. 104 cells/g of sample, using our IMS
technique followed by CTC incubation and FITC-labeled antibody
staining. Others have reported detection limits as low as 16 cells/g
for manual microscopy following filtration and FAb staining
(31). However, we have been unable to repeat those results
because of the inherent difficulty in filtering the ground beef
suspensions by that method (data not shown). With >104
cells/g of hamburger, using a 1-ml sample after IMS, it is necessary to
count at least 20 microscope fields to obtain a statistically reliable
count of at least 300 cells.
With smaller numbers of target bacteria, the lower detection limit of
microscopy could be reduced by increasing the number of fields
examined, but this would result in a significant increase in the time
taken for enumeration and also in the stress on the operator.
Alternatively, multiple filters could be compared and enumerated,
which would also increase the time input. Application of improved
concentration techniques in addition to IMS could also be
employed to increase sensitivity. The Scan RDI system obviates the need
for increased microscopy time by first enumerating and locating each
particle detected and then permitting the operator to examine and
validate each particle counted as a bacterial cell. It is also
possible to record the presence of CTC-formazan in each cell, as a
measure of respiratory activity, when the particles are being validated
microscopically after Scan RDI analysis.
Most current techniques that use IMS for the detection of food-borne
pathogens incorporate an incubation to enrich the target organism
(e.g., see references 9, 10, 12, 24, 25, and 35). The direct detection of E. coli
O157 has been demonstrated in samples of water, animal feces, and soil
(21) by using commercially prepared immunomagnetic beads
(Dynal). The investigators processed samples for IMS in PBS containing
0.1% BSA and 0.1% Tween 20 for extraction and plated captured cells
on SMAC agar containing
4-methylumbelliferyl-
-D-glucuronide. Using laboratory
cultures, we have demonstrated that 77 to 121% of the inoculum
was recovered on the beads with inocula of <10 to
105 CFU/ml. The high recoveries were attributed to cell
division, possibly as a result of completion of replication, during the IMS procedure. It is possible, but less likely, that injured
target cells within the inoculum which were not able to reduce CTC or react with the FAb recovered during the preparation procedures so that
they were observed as respiring, FAb-positive cells after the recovery
and incubation steps.
The data presented here suggest that cell numbers increased during IMS,
particularly in samples of ground beef. These differences were
observed with both agar plating and FAb procedures, which show that the
organisms detected react strongly with the confirmatory anti-O157 FAb.
This FAb was obtained commercially (Kirkegaard and Perry Laboratories)
and had been extensively adsorbed by using a non-O157:H7
E. coli serotype. Reactions with serotypes
O55:K59:NM and O124:H30 were demonstrated only at high antibody protein
concentrations. E. coli O157:H7 bacteria were
not detected in control samples of uninoculated hamburger. Thus, it is
unlikely that the increases in cell numbers during IMS resulted
from reactions with nontarget bacteria.
The IMS-CTC-FAb procedure described here represents a prototype for the
development of techniques for the detection of other pathogenic
bacteria in environmental samples. We have demonstrated the detection
of Salmonella typhimurium by the CTC-FAb method (22), and the use of a suitable capture antibody for coating beads would be expected to function for that organism. It is also possible to utilize alternative fluorochromes, other than CTC, to
assess a variety of physiological functions in combination with
specific FAb methods for identification (16). Techniques such as these will have applications both in the timely analysis of
food and water samples and in ecological studies (17, 21). With more reliable, rapid procedures such as these, which do not require cultivation of environmentally stressed bacteria, better approaches to the control of water- and food-borne bacterial pathogens will be attainable.
| |
ACKNOWLEDGMENTS |
We thank John Lisle for reviewing the manuscript, in addition to
Scott Burnett and Patricia Kraft for technical assistance. Rob Bargatze
and John Jutila, Montana ImmunoTech, Inc., contributed helpful
discussions and advice. Nancy Equall, Image and Chemical Analysis
Laboratory at Montana State University, assisted with electron
microscopy. Martin Rollefson (Flying Photo, Bozeman, Mont.) and Rick
Harrison (Rikshots, Bozeman, Mont.) processed and printed the photographs.
Funding from the Program for the Development of Applied Biotechnology
at Montana State University, the National Institutes of Health,
Environmental Health Sciences, phase I Small Business Innovative
Research program, and from the NASA Life Sciences program is gratefully
acknowledged. This material is based on work supported, in part, by the
U.S. Army Research Office under contract G-DAAH0496-01-0442.
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FOOTNOTES |
*
Corresponding author. Mailing address:
Microbiology Department, Montana State University
Bozeman,
Bozeman, MT 59717. Phone: (406) 994-3041. Fax: (406) 994-4926. E-mail: barryp{at}montana.edu.
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Applied and Environmental Microbiology, May 1999, p. 1966-1972, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
