Development and Characterization of a Fluorescent-Bacteriophage ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1999, p. 1397–1404 0099-2240/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 65, No. 4

Development and Characterization of a Fluorescent-Bacteriophage Assay for Detection of Escherichia coli O157:H7 LAWRENCE GOODRIDGE, JINRU CHEN,†

AND

MANSEL GRIFFITHS*

Department of Food Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 Received 2 June 1998/Accepted 9 November 1998

In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coli O157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 104 cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 102 and 103 cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection of E. coli O157:H7 in broth cultures. Since Escherichia coli O157:H7 was first identified as a cause of hemorrhagic colitis in 1982 (30), this pathogen has been recognized as an important agent of food-borne disease with a worldwide distribution (25). Although many other means of transmission have been documented, the most common source of outbreaks of E. coli O157:H7 infection has been ground beef (15). The largest reported outbreak of E. coli O157:H7 infection in North America to date occurred in the northwestern United States in late 1992 and early 1993 and was associated with the consumption of undercooked ground beef at multiple outlets of a fast food chain (5). Other foods that have been epidemiologically implicated in outbreaks of E. coli O157:H7 infection include vegetables, salad bar items, fruits, and salami (10, 15). Since E. coli O157: H7 is not heat resistant, proper cooking practices should decrease the importance of this organism in foods such as ground beef. Still, fruits and vegetables are often consumed uncooked. Also, food products such as salami, in which raw ground meat is preserved by a process of fermentation and drying, are considered ready to eat and are not generally cooked before consumption (34). E. coli O157:H7 poses a significant threat in these foods. Obviously, rapid, sensitive, and simple techniques that can detect E. coli O157:H7 in foods need to be established so that the risk of E. coli O157:H7 outbreaks can be reduced. Many genetic and immunological techniques have been developed for detection of serotype O157:H7. For example, Meng et al. (21) described a PCR technique that could detect as few as 25 CFU of E. coli O157:H7 within 3 h. Also, there are many nucleic acid-based assays which can specifically detect all verotoxigenic E. coli (VTEC) types, including E. coli O157:H7. These assays utilize primers and probes that hybridize specifically to complementary sequences found in the VTEC toxin genes (32). However, while these assays are specific, sensitive, and rapid, they are often expensive. Additionally, genetic assays are often labor-intensive, because sample manipulation is

often required to liberate the bacteria from complex food matrices. There are many immunological tests for detection of E. coli O157:H7. A rapid latex agglutination assay is commercially available for rapid presumptive identification of E. coli O157: H7 (24). The latex test has been found to be specific for serotype O157:H7 alone. The major drawback of the latex agglutination assay seems to be the time that it takes to obtain pure, isolated colonies that can be tested. A solid-phase fluorescence-based immunoassay has been developed for detection of E. coli O157:H7 (9). In this assay, a soft glass capillary tube serves as a solid support to which heat-killed E. coli O157:H7 cells are absorbed. Biotin-conjugated polyclonal anti-E. coli O157:H7 antibody is used, and the antigen-antibody complex band is detected by using avidin molecules labeled with the fluorescent dye Cy5. When this assay is used, it is possible to detect 1 CFU/10 g of ground beef following a 7-h enrichment step. Immunoassays are widely used for detection of E. coli O157:H7 and are simple and rapid. However, immunoassays often require the use of appropriate controls in order to obtain maximum specificity, since it has been reported that several bacteria belonging to the genus Escherichia, including Escherichia hermanii, E. coli O148:NM, and E. coli O117:H27, as well as Salmonella urbana (group N salmonellae), cross-react with polyclonal antiserum raised against E. coli O157 (2, 4, 27). Alternatively, immunomagnetic separation (IMS) performed with magnetic beads coated with anti-O157 antibodies has been investigated in conjunction with assay methods, and this technique shows promise for rapid detection of E. coli O157 in foods and environmental samples. IMS can easily separate bacteria from complex matrices, and when combined with other assay systems, IMS can decrease the occurrence of false-positive results observed with immunoassays. The existence of bacteriophages specific for E. coli O157:H7 (31) presents opportunities to develop methods for O157:H7 detection in which the separation capabilities of IMS and the specific host ranges of the bacteriophages are used. Therefore, the objectives of this study were to develop a rapid, simple, and sensitive IMS-bacteriophage-based assay that could detect E. coli O157:H7; to evaluate the specificity of the assay for E. coli O157:H7 and other E. coli strains, as well as other bacteria; and to evaluate the threshold for detection of E. coli O157:H7 in pure culture.

* Corresponding author. Mailing address: Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext. 2269. Fax: (519) 824-6631. E-mail: mgriffit @uoguelph.ca. † Present address: Center for Food Safety & Quality Enhancement, University of Georgia, Griffin, GA 30223-1797. 1397

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TABLE 1. Bacterial strains used in the specificity study Taxon

Strain

Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O157:H7a Escherichia coli O157:H7a Escherichia coli O157:NM Escherichia coli O157:H7 Escherichia coli O157:H7 Escherichia coli O6:H34 Escherichia coli O103:H2 Escherichia coli O145:NM Escherichia coli O126:H8 Escherichia coli O2:H6 Escherichia coli O2:H5 Escherichia coli O2:H1 Escherichia coli O4:H5 Escherichia coli O6:H5 Escherichia coli O157:HI9 Escherichia coli O157:H25 Escherichia coli O157:HI2 Escherichia coli 2-4-77 Escherichia coli 2-1-214 Escherichia coli O26:H1I Escherichia coli O139:K82 Escherichia coli O128:BI2 Salmonella enteritidis Salmonella typhimurium Listeria monocytogenes Shigella dysenteriae Shigella dysenteriae Enterobacter aerogenes Serratia marcescens Klebsiella pneumoniae Escherichia hermanii Salmonella urbana

EC920333 EC920005 EC920026 EC920027 EC920037 EC920081 EC920192 EC920321 EC950050 EC960275 EC920267 EC920283 EC920409 EC940235 EC940239 EC940340 EC940435 EC940468 EC960313 EC960279 EC960274 EC930195 E32511 933W EC910051 EC910037 EC910040 EC910061 EC920229 EC920232 EC950209 EC950211 EC950212 EC940076 EC960282 EC960308 EC2477 EC21214 H19 412 H.1.8 SA941256 ATCC 27345 N5 N8 N9

Origin

Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada University of Guelphb University of Guelphb Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada Health Canada University of Guelphb University of Guelphb University of Guelphb University of Guelphb University of Guelphb University of Guelphc University of Guelphc University of Guelphc University of Guelphd University of Guelphc University of Guelphb University of Guelphb University of Guelphb Health Canada Health Canada

a

Toxin-negative strain. Department of Pathobiology. Department of Food Science. d Department of Microbiology. b c

MATERIALS AND METHODS Bacterial strains. A total of 51 strains of bacteria (Table 1) were used for enumeration, recovery, and specificity studies. The bacteria studied comprised 27 strains of E. coli O157 and 24 strains of other bacteria representing eight genera. The E. coli O157 strains were obtained from Health Canada, and the other strains were gifts from the Department of Pathobiology, University of Guelph. One strain, Shigella dysenteriae ATCC 27345, was obtained from the Department of Microbiology, University of Guelph. Of the 27 E. coli O157 strains used, 21 were verotoxin (VT)-producing O157:H7 strains, 1 was a VT-producing O157: NM strain, 2 were VT-negative O157:H7 strains, and 3 were VT-negative O157 strains of other H types. Stock cultures were maintained in 30% glycerol and were frozen at 220°C. Fresh bacterial host cultures for use in experiments were produced by inoculating frozen stock cultures onto Luria-Bertani (LB) agar plates (LB broth [Difco Laboratories, Detroit, Mich.] containing 1.5% agar [Difco]) and incubating the plates overnight at 37°C. For growth experiments, the inocula consisted of stationary-phase cells that were obtained by inoculating LB

broth with cells from an overnight LB agar plate and incubating the preparations overnight with shaking at 37°C. Bacteriophage and host. The bacteriophage used in this study, designated LG1, was isolated in our laboratory from chicken feces and was amplified in cultures with its host, E. coli O157:H7 strain EC920333. Ten milliliters of an overnight culture of EC920333 and 1.5 ml of phage LG1 (1011 PFU/ml) were added to 250 ml of LB broth in a 500-ml Erlenmeyer flask and incubated with shaking at 37°C for 7 h. Following lysis, 10 ml of chloroform was added to the flask to release any progeny phage which may still have been in the host cells, and the suspension was incubated with shaking at 37°C for an additional 10 min. The broth suspension was centrifuged at 5,000 3 g for 15 min, and the supernatant was withdrawn and filtered through 0.2-mm-pore-size syringe filters (Nalgene, Rochester, N.Y.) to remove the bacterial debris. The phage were stored in the dark at 4°C. Preparation of fluorescently labeled bacteriophage. Fluorescently stained bacteriophage were produced by using a modification of the procedure originally described by Hennes and Suttle (16). Following bacteriophage amplification, the phage were centrifuged at 186,000 3 g for 1 h in a model L8-M ultracentrifuge (Beckman Instruments, Mississauga, Ontario, Canada). The phage pellets were resuspended in 400 ml of lambda buffer (0.58% [wt/vol] sodium chloride, 0.2% [wt/vol] magnesium sulfate, 5.0% [vol/vol] Tris-HCl [pH 7.5], 0.01% [wt/vol] gelatin; pH 7.5) per centrifuge tube and incubated at room temperature overnight. The next day, the phage pellets were gently resuspended and pooled in microcentrifuge tubes. To resuspend any bacteriophage which remained, the centrifuge tubes were washed with approximately 1 ml of lambda buffer, and the resulting preparation was added to the pooled pellets. To remove any bacterial nucleic acid that adhered to the phage, the samples were treated with 1 mg of DNase (Boehringer, Mannheim, Germany) per ml and 1 mg of RNase (Boehringer) per ml and incubated at room temperature for 30 min. Approximately 200 ml of chloroform was added to each microcentrifuge tube, and the samples were centrifuged in a model 5415 C Eppendorf centrifuge (Brinkman Instruments, Rexdale, Ontario, Canada) at 16,000 3 g for 5 min. The aqueous phase containing the phage was removed and added to new microcentrifuge tubes, and 15 ml of the fluorescent nucleic acid dye YOYO-1 (Molecular Probes, Inc., Eugene, Oreg.) was added to each tube. The tubes were inverted several times and incubated in the dark at 4°C for 72 h. Following phage staining, the microcentrifuge tubes were suspended in ultracentrifuge tubes filled with water and centrifuged at 186,000 3 g for 1 h. The microcentrifuge tubes were floated out of the ultracentrifuge tubes by adding water, the supernatant was carefully withdrawn, and the fluorescent phage pellets were resuspended in 1 ml of lambda buffer. Ultrafiltration was performed to remove any excess dye that remained in the samples. Fluorescent-phage samples were placed in a model 8MC MicroUltrafiltration device (Amicon, Inc., Beverly, Mass.) and filtered through a 10-mmdiameter Diaflo ultrafiltration membrane (Amicon) with a molecular weight cutoff of 300,000. Lambda buffer was used to wash the samples as they were filtered. The samples were filtered at 25 lb/in2 for 1 h and then concentrated until the final retentate volume was approximately 1 ml. The retentate was removed, and the filter was washed with 800 ml of lambda buffer to remove any bound phage. The retentate and the filter effluent were pooled in a microcentrifuge tube, and chloroform purification was performed as described above. The fluorescent-phage samples were filtered through 0.2-mm-pore-size syringe filters and stored in the dark at 4°C. The final concentration of fluorescent phage was between 1011 and 1012 PFU/ml. IMS and labeling of bacteria with fluorescent phage. One-milliliter portions of stationary-phase cultures were added to microcentrifuge tubes containing 20 ml of E. coli O157-specific immunomagnetic beads (Dynal Inc., Lake Success, N.Y.). After the tubes were rotated at 30 rpm for 30 min at room temperature with an Orbitron Rotator II (Fisher Scientific, Mississauga, Ontario, Canada), they were placed in a magnetic rack (Dynal) so that the magnetic beads could be separated from the enrichment broth. The beads were washed twice in lambda buffer and resuspended in 200 ml of the fluorescent-phage suspension. The samples were rotated at 30 rpm for 15 min at room temperature. The magnetic beads were separated from the fluorescent-phage suspension, washed once, and resuspended in 1 ml of lambda buffer. Epifluorescence microscopy. Fifty-one bacterial isolates (Table 1) were subjected to the fluorescent-bacteriophage assay (FBA) and examined by epifluorescence microscopy. Each bacterial isolate was tested twice. Controls consisting of immunomagnetic beads incubated with the fluorescent phage were included in order to determine the amount of nonspecific binding of the phage to the beads. For analysis, 1-ml samples were filtered onto 25-mm-diameter, 0.2-mm-pore-size black membrane filters (Fisher Scientific, Nepean, Ontario, Canada) which were overlaid on prewetted 0.4-mm-pore-size membrane filters. Both filters were housed in a Swinnex filtration device (Fisher Scientific, Nepean, Ontario, Canada). Following filtration, the membrane filters were removed from the filtration device, placed on glass slides, and covered with coverslips. The slides were viewed at a magnification of 31,000 with an Olympus model BH-2 epifluorescence microscope equipped with a filter set capable of illuminating YOYO1-stained bacteriophage (excitation wavelength, 491 nm; emission wavelength, 509 nm). Pictures were taken with a scanning laser confocal microscope (BioRad, Mississauga, Ontario, Canada) equipped with a krypton-argon mixed-gas laser at a magnification of 31,600.

VOL. 65, 1999 Flow cytometry. Forward light scatter and side scatter fluorescence were measured simultaneously with a Coulter Elite flow cytometer (Coulter Electronic, Burlington, Ontario, Canada). Samples were added to the sample introduction tube at a pressure of 9.79 lb/in2. The sheath fluid used was Isoton II (Coulter Electronic), which had been prefiltered through a 0.22-mm-pore-size filter (Gelman Sciences, Ann Arbor, Mich.). A constant pressure of 10.5 lb/in2 was applied to the sheath fluid-sample stream, which emerged from the flow chamber through an orifice having a diameter of 100 mm. The stream was interrogated directly beneath the orifice in quartz by a focused argon ion laser operating at a wavelength of 488 nm and an output power of 50 mW. To detect forward-angle light scatter, a neutral-density filter was placed in front of the forward-angle light scatter detector. Side-angle light scatter was collected by using a 488-nm dichroic filter and a band pass filter (488 6 5 nm). A 600-nm dichroic filter was placed in front of the photomultiplier to detect green fluorescence. Photomultiplier voltages were fixed for each set of experiments. Samples to be examined by flow cytometry were prepared as described above and were analyzed in triplicate, and controls consisting of immunomagnetic beads and labeled phage were included in each group of samples in order to determine the level of background fluorescence. Bacterial counts. The accuracy of using fluorescent bacteriophage to estimate the abundance of E. coli O157:H7 in a particular sample was evaluated. An overnight culture of E. coli O157:H7 strain EC920333 was diluted by preparing a series of 1021 dilutions in lambda buffer until the final dilution was 1028. The different dilutions were concentrated and labeled as described above and were analyzed by using epifluorescence microscopy and flow cytometry. For analysis by epifluorescence microscopy, a modification of the direct epifluorescent-filter technique (DEFT), as described by Pettipher (28), was employed. After samples were examined with the Olympus model BH-2 epifluorescence microscope, as described above, the numbers of tagged cells in 15 microscope fields of view were determined, and the values were averaged. The microscope fields were selected at random. The DEFT count per milliliter was calculated by multiplying the average number of cells per microscope field by the microscope factor. The microscope factor was calculated by the following formula: microscope factor 5 area of membrane through which sample is filtered (in square millimeters)/ [microscope field area (in square millimeters) 3 sample volume (in milliliters)]. The area of the filter used was calculated from the internal radius of the filter membrane. The area of the microscope field of view was calculated from the radius of the field of view, as determined with a stage micrometer. Samples examined by the DEFT and flow cytometry were analyzed in triplicate. Plate counts were determined in duplicate with the overnight sample in order to determine the original number of CFU per milliliter by spread plating 10-fold serial dilutions of a stationary-phase culture onto LB agar plates and then incubating the preparations overnight at 37°C. Efficiency of IMS. An overnight culture of E. coli O157:H7 strain EC920333 was serially diluted 100-fold in lambda buffer so that four different cell populations were created. The concentrations of the 100-fold dilutions were estimated to be 108, 106, 104, and 102 CFU/ml. Just after the dilutions were prepared (without enrichment), the efficiency of magnetic capture was tested with each dilution. To determine the cell numbers in the dilutions, duplicate plate counts were obtained. After magnetic capture, the beads were resuspended in 1 ml of lambda buffer, and duplicate plate counts of each sample were obtained. After incubation at 37°C overnight, colonies were counted, and the values were compared with the numbers of CFU per milliliter in the original dilutions. Bacteriophage specificity. In order to determine its host range, bacteriophage LG1 was tested with 51 bacterial strains (Table 1). Bacteriophage lysis assays were performed by using a modification of the traditional double-layer plaque formation technique described by Hershey et al. (17). Lysis assays were conducted in 10-cm-diameter petri plates (Fisher Scientific, Nepean, Ontario, Canada). The bottom agar layer consisted of LB agar. The top agar layer consisted of 1% (wt/vol) tryptone (Difco), 0.8% (wt/vol) sodium chloride (Fisher Scientific, Nepean, Ontario, Canada), and 0.5% agar. For each strain tested, 3 ml of top agar was steamed for approximately 10 min and allowed to cool to 47°C. One hundred microliters of an overnight culture of the bacterium to be tested was added to the top agar, which was then vortexed and poured onto the bottom layer. The top agar was allowed to solidify, and 20 ml of a bacteriophage suspension (1011 PFU/ml) was pipetted onto the top agar layer. The plates were incubated at 37°C overnight and then examined for the presence of clear zones of lysis. The ability of the fluorescent phage to bind to host cells was also evaluated. IMS and fluorescent-phage labeling of overnight cultures of all 51 bacterial strains were performed as described above. The samples were examined by epifluorescence microscopy. Scanning electron microscopy. Bacteria and immunomagnetic beads were incubated with shaking at 30 rpm on an Orbitron Rotator II for 30 min at room temperature, washed twice, and resuspended in 1 ml of lambda buffer. The suspension was filtered onto a polycarbonate 0.2-mm-diameter filter (Poretics Corp., Livermore, Calif.) in a 13-mm syringe apparatus by using a Buchner vacuum filtration unit. The filter was transferred to phosphate-buffered saline (PBS) (pH 7.5) containing 2% glutaraldehyde and incubated for 1 h at room temperature in order to fix the bacterial cells and immunomagnetic beads. Samples were rinsed in several changes of PBS for 30 min at room temperature and dehydrated with a graded ethanol series (50, 70, 80, 95, and 100% ethanol). The samples were critical point dried for 3 min with CO2 by using a Samdri

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model 780 critical point dryer. The samples were mounted on specimen stubs, sputter coated with 20 nm of gold-palladium by using a model Hummer VII sputter coater (Anatech Corp., Alexandria, Va.), and scanned with a model S-570 scanning electron microscope (Hitachi Corp., Danbury, Conn.) with an accelerating voltage of 10 keV.

RESULTS Epifluorescence microscopy. Phage LG1 was stained with the dimeric nucleic acid dye YOYO-1 and examined by epifluorescence microscopy. The phage appeared as green specks that were easily distinguished from the black background. IMS of E. coli O157:H7 on magnetic beads and addition of the stained bacteriophage to the separated beads resulted in removal of detritus and other particulate material from the sample, and the fluorescent bacteriophage attached to the host cells, giving the cells a “halolike” appearance (Fig. 1). Nonspecific binding of LG1 to the immunomagnetic beads was negligible in the control samples. Flow cytometry. Overnight cultures of the LG1 host, E. coli O157:H7 strain EC920333, were concentrated with O157-specific magnetic beads, labeled with fluorescent phage, and analyzed by flow cytometry. Each overnight culture was tested twice. In each test, all of the cultures were analyzed in triplicate, and the means and standard deviations were plotted. Figure 2 shows the expected number of cells, as determined by plate counting, and the percent fluorescence for each individual cell culture, as detected by flow cytometry, for a single test. Bacterial concentrations of 104 CFU/ml or more gave positive results. At concentrations below 104 CFU/ml, the percent fluorescence readings were indistinguishable from the background readings. The percent fluorescence values did not correlate with plate count values over the range from 106 to 102 CFU/ml (n 5 6; r 5 0.74; P 5 0.15). The flow cytometry data were organized as individual histograms showing the spread of forward scatter and fluorescence over the population of cells and beads and also as two-parameter dot plots (Fig. 3). Examination of the dot plots revealed three regions. Region D had the largest particles and greatest fluorescence, followed by region C. Region E consisted of nonfluorescent debris. Bacterial counts. To evaluate the accuracy of using fluorescent bacteriophage to estimate bacterial counts, a modified DEFT was employed. Figure 4 shows the average number of CFU per milliliter as determined by plate counting and the average number of CFU per milliliter as calculated by the DEFT. DEFT counting was performed three times. The plate counts and the DEFT counts correlated well for E. coli O157: H7 in the range from 108 to 102 CFU/ml (r 5 0.93); however, this correlation was not significant at the 95% confidence level (P 5 0.07). Efficiency of IMS. An overnight culture of E. coli O157:H7 strain EC920333 was diluted to final concentrations of 108, 105, 104, and 102 CFU/ml, as confirmed by plate counting. Just after the dilutions were prepared, the efficiency of magnetic capture was determined with each dilution. Following IMS, each dilution was plated onto LB agar in duplicate. The results obtained are presented in Fig. 5, which shows that the numbers of bacteria recovered were within 1 log10 value of the numbers of cells in the original culture. Specificity of fluorescent phage LG1. LG1 was tested with 51 strains of bacteria to determine its host range and specificity. LG1 formed plaques on all VT-positive E. coli O157 strains and on the two VT-negative E. coli O157 strains tested, as well as on E. hermanii, type 1 S. dysenteriae ATCC 27345, and three non-O157:H7 VTEC strains. The FBA was also tested for specificity, and we found that the assay identified all of the O157:H7 strains, as well as type 1 S. dysenteriae ATCC 27345,

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FIG. 1. E. coli O157:H7 cells labeled with fluorescent bacteriophage LG1. Bar 5 10 mm. Magnification, 31,000.

one strain of E. coli O157:NM, and E. coli O157:HI9. Therefore, using immunomagnetic beads increased the specificity of the FBA. DISCUSSION The general patterns of E. coli O157:H7 transmission suggest that the infectious dose of this pathogen is low (15). Therefore, the objective of this research was to develop a sensitive, rapid, and inexpensive method for detection of E. coli O157: H7. Bacteriophage are abundant and ubiquitous components of the microbial communities on earth (26). Bacteriophage are easy to isolate and inexpensive to purify. Isolation of a bacteriophage that initially was reported to be specific for E. coli O157:H7 (31) suggested that fluorescent labeling of this bacteriophage might be used as a rapid method for detection of E. coli O157:H7. Fifty-one bacterial isolates representing seven genera were used to evaluate the use of fluorescent phage LG1 for detection of E. coli O157:H7. Most of these bacteria are common food-borne pathogens. E. hermanii and S. urbana (group N salmonellae) were included and were used to determine the specificity of the immunomagnetic beads because it is known that these organisms cross-react with polyclonal antiserum raised against E. coli O157 (2, 27). The results obtained in this study show that bacteriophage LG1 is specific for a subset of VTEC and other closely related bacteria, such as S. dysenteriae and E. hermanii, and has a host range similar to that described for bacteriophage AR1 described by Ronner and Cliver (31). The susceptibility of a bacterium to bacteriophage infection is primarily dependent on whether the bacteriophage can attach to receptors on the cell (18). Based on morphological characteristics, bacteriophage LG1 belongs to the family Myoviridae and is probably most closely related to bacteriophage T4. T4 uses the lipopolysaccharide (LPS) of E. coli B as a receptor whenever it possesses at least one of the two terminal glucose residues (29). T4 also uses outer membrane protein C as a receptor. The bacterial receptor or receptors for LG1 are not known. LG1 infects E. coli C but cannot infect E. coli K-12 or B (14a),

although care must be taken in the interpretation of these results, since some E. coli strains produce mutant phenotypes. For example, E. coli K-12 sometimes does not produce LPS with a complete core oligosaccharide (35). It is unclear what exactly on the bacterial surface constitutes a receptor for LG1. There are bacteriophage that attack only motile strains of bacteria (18). It is unlikely that the H antigen of E. coli O157 is the receptor for bacteriophage LG1, because this phage was found to infect several serotypes of E. coli O157, each possessing different H antigens, including H7, H12, and H19. In addition, LG1 lysed one strain of nonmotile E. coli O157. If the characteristics of the T-even phages are used as a model, it is possible that LPS (specifically, the O side chain) and an outer membrane protein (although not necessarily outer membrane protein C) are receptors for phage LG1. However, the results of this work show that this may not be so. LG1 was able to infect 100% of the E. coli O157:H7 strains tested, as well as one E. coli O157:NM strain, E. coli O157:HI9, E. coli O139:K82, E. coli O128:B12, and E. coli O157:H12. However, it failed to infect E. coli O157:H25. In addition, phage LG1 was lytic on E. coli O26:H1, S. dysenteriae (type 1), and E. hermanii,

FIG. 2. Flow cytometry graph showing mean percent fluorescence values and standard deviations obtained for different numbers of E. coli O157:H7 cells analyzed in triplicate. The shaded area at the bottom indicates the background fluorescence. The clear area above the background fluorescence area represents the standard deviation of the background fluorescence.

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FIG. 3. (A) Two-parameter dot plot of forward scatter and side scatter for a pure culture of E. coli O157:H7 (107 CFU/ml) labeled with fluorescent bacteriophage LG1. There are three regions. Region D (large circle) contains large aggregates of magnetic beads and E. coli O157:H7 cells; region C (small circle) contains mainly unbound magnetic beads; and region E contains cellular debris. (B) One-parameter histogram of green fluorescence data for a pure culture of E. coli O157:H7 (107 CFU/ml) labeled with fluorescent bacteriophage LG1. There are two main peaks of fluorescence. Region D exhibits the most fluorescence, followed by region C, which exhibits little fluorescence. Region E is not fluorescent. (C and D) Scanning electron micrographs of E. coli O157:H7 cells attached to immunomagnetic beads. (C) E. coli O157:H7 cells attached to a single bead. Bar 5 2 mm. Magnification, 312,600. (D) E. coli O157:H7 cells and immunomagnetic beads forming a large complex. Bar 5 8.6 mm. Magnification, 32,940.

indicating that the receptor is not O antigen specific. It is probable that the receptor for phage LG1 is a component of the O antigen, but further studies are needed to determine the receptor for this bacteriophage. Prior to the FBA, IMS was used as a method for concen-

trating E. coli O157:H7 in a pure form. IMS utilizes antibodycoated paramagnetic polymer beads and is considered fast, safe, sensitive, and reproducible (23). The results obtained with commercially available paramagnetic beads coated with an O157 polyclonal antibody were compared with plate count

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FIG. 4. Relationship between FBA-DEFT counts and plate counts for different dilutions of E. coli O157:H7 in broth. Two replicates from each 100-fold dilution (1022, 1024, 1026, and 1028) were counted. The line is the fitted regression line (y 5 0.994x 1 2.570).

results in order to determine the efficiency of recovery of E. coli O157:H7. The IMS count results and plate count results correlated well over the range from 108 to 102 CFU/ml (n 5 8; r 5 0.97; P 5 0.03). Generally, the efficiency of the magnetic bead method was better at higher dilutions than at lower dilutions. With the 1028 dilution (containing approximately 102 CFU/ml), the immunomagnetic beads captured 61.5% of the E. coli O157:H7 cells, whereas with the 1022 dilution (containing approximately 108 CFU/ml) only 11.5% of E. coli O157:H7 cells were captured. In a similar study, Mortlock (23) found that as the numbers of E. coli O157:H7 cells in suspension decreased, the level of recovery increased from a minimum of 23% at a concentration of 107 CFU/ml to a maximum of 51% at a concentration of 103 CFU/ml. This could have been due to the bead-to-organism ratio not being correct at higher concentrations of organisms. It has previously been shown that there should be a 3:1 bead-to-organism ratio for maximum recovery (23). This explains the low recovery rate obtained with larger cell populations and is an important point to consider when dense bacterial suspensions are evaluated. The main problem with IMS to date seems to be the numbers of organisms other than E. coli O157 that attach nonspecifically to the magnetic beads (6). Other researchers have found that incorporating a washing step consisting of washing with the detergent Tween 20 and PBS into the IMS method decreases, but does not eliminate, nonspecific binding of organisms to the beads (8). In addition to the nonspecific binding of the bacteria to the beads, there is the problem of crossreactions of E. coli O157 polyclonal antibodies. Antisera prepared against the O157 antigen are known to cross-react with O antigens of 12 other serogroups of E. coli, as well as with O antigens of E. hermanii, S. dysenteriae, Citrobacter freundii, group N salmonellae (S. urbana), and other bacteria (2, 3, 27). Perry and Bundle (27) have linked the cross-reactivity of polyclonal antisera to the antigenic relationships between the LPS of E. coli O157:H7 strains and the LPS of other bacteria, including E. hermanii, Brucella melitensis, and Brucella abortus. These authors found that in all of these bacteria epitopes involving the presence of N-acyl derivatives of the 4-amino4,6-dideoxy-a-D-mannopyranosyl residues occurred in the Opolysaccharide portion of the LPS. In this study, the FBA was evaluated to determine whether there were cross-reactions due to the O157 magnetic beads and nonspecific binding of bacteriophage LG1 to the captured nonO157 cells. Twenty-eight non-E. coli O157:H7 bacteria belonging to several genera were tested; these organisms included S. dysenteriae, E. hermanii, and S. urbana, each of which is known


to cross-react with E. coli O157 polyclonal antisera. Only 2 of the 28 non-E. coli O157:H7 bacteria, S. dysenteriae (type 1) and E. coli O157:H19, were positive as determined by the assay. E. coli O157:NM was also positive as determined by the FBA; however, E. coli O157:NM may actually be E. coli O157:H7 (13). Also, E. hermanii did not give a false-positive result despite being susceptible to LG1. Therefore, the use of E. coli O157 magnetic beads in conjunction with bacteriophage LG1 conferred a level of specificity on the FBA that neither the beads nor LG1 possessed on its own. Although LG1 induced lysis of seven non-E. coli O157:H7 bacteria in this study and, as mentioned above, the E. coli O157 magnetic beads can bind many different bacteria, the FBA gave positive results with only three non-E. coli O157:H7 organisms, S. dysenteriae, E. coli O157: HI9, and E. coli O157:NM. With the exception of E. coli O157: HI9, detection of these false-positive organisms is advantageous, since both E. coli O157:NM and S. dysenteriae cause hemorrhagic colitis and hemolytic-uremic syndrome (33). Regarding the issue of assay specificity, it would be of some value to isolate a diverse range of phages and extensively characterize their host ranges, since there may be phages which are more specific for E. coli O157:H7 than LG1 is; such phages would increase the accuracy of the FBA. Still, the fact that LG1 is specific for a subset of VTEC instead of E. coli O157:H7 does not invalidate the principle of this assay. An effort was made to correlate the DEFT count and plate count results. The DEFT did not adequately enumerate E. coli O157:H7 over the range from 102 to 108 CFU/ml. There was a good correlation between DEFT count and plate count results (n 5 8; r 5 0.93). However, this correlation was not significant at the 95% confidence level (P 5 0.07). The DEFT was able to estimate plate counts more reliably at higher dilutions than at lower dilutions. There were several reasons for this. One reason was the inability of the magnetic beads to pull 100% of the bacterial population out of suspension. As mentioned above, at higher dilutions the magnetic beads were more efficient at concentrating bacteria, while at lower dilutions IMS failed to capture a large percentage of the bacteria. Similarly and predictably, this trend was observed with DEFT. The DEFT counts were closer to the plate counts with smaller populations of cells than with larger populations of cells. Another reason for the inability of the DEFT to accurately predict plate counts was the counting method employed. The DEFT counting method required that any cell or group of cells separated by a distance that was less than twice the smallest diameter of the

FIG. 5. Relationship between plate counts following IMS and plate counts for different dilutions of E. coli O157:H7 in broth. Two replicates from each 100-fold dilution (1022, 1024, 1026, and 1028) were counted. The line is the fitted regression line (y 5 1.063x 2 0.077).

VOL. 65, 1999

cells nearest each other must be counted as one clump or cell. Under ideal conditions, in any given field of view there should be groups of bacterial cells close enough together to be counted as one clump and bacteria far enough apart from one another to be counted separately. In the present study, however, because magnetic beads were used to concentrate the bacteria, most of the bacteria present in a field of view were attached to magnetic beads and therefore had to be counted as a clump instead of individual cells. Thus, it was not the number of bacterial cells but rather the number of magnetic beads with bacteria attached in a given field of view that was counted. This resulted in substantial underestimation of the number of bacteria in a given sample. This counting inadequacy, combined with less than 100% recovery of bacteria by IMS, resulted in the DEFT counts that were less accurate as the bacterial numbers increased. In the future, new counting methods that take into account these two factors will have to be devised. Due to the problems and labor intensity of the DEFT, flow cytometry was used as an alternative method for fluorescence detection. Different dilutions of E. coli O157:H7 were labeled by using the FBA and were analyzed with a flow cytometer. The detection limit when this technique was used was 104 CFU/ml. At concentrations below this level, the fluorescent values obtained were unreliable, probably because of laser scattering by the immunomagnetic beads. A sensitivity of 104 CFU/ml is consistent with previously published results. McClelland and Pinder (20) detected 104 Salmonella cells per ml by using flow cytometry and fluorescent antibodies. In the present study, when the data were analyzed as a dot plot, three distinct regions were observed, regions D, C, and E (Fig. 3). Region D exhibited the largest amount of fluorescence and also contained the largest particles. The magnetic beads and bacterial cells formed large complexes (Fig. 3D), and these complexes were the large particles observed in region D. Region C consisted mostly of magnetic beads, and region E contained nonfluorescent debris. These results show that flow cytometry offers food microbiologists numerous possibilities for detection and enumeration and, most importantly, a relatively sensitive and rapid method for identification of microorganisms. Many rapid methods for detection of E. coli O157:H7 have been described. These methods include immunological methods, such as enzyme immunoassays and immunoblot tests. IMS has been used to identify serotype O157:H7. Yu and Bruno (36) evaluated a commercial sensor that combined IMS and electrochemiluminescence to detect E. coli O157 in food. The results indicated that the detection limits for E. coli O157:H7 were between 102 and 104 CFU/ml in buffer and between 1 3 103 and 2 3 103 CFU/ml in food samples. Alternatively, genetic techniques, such as the PCR, have been described as sensitive methods for detecting E. coli O157 in food (7, 14). Gilgen et al. (14) used PCR to detect VTEC in ground beef. These researchers used two nested PCR assays to detect genes encoding VT1 and VT2 irrespective of the bacterial serotype. Using a direct sample protocol, they were able to detect 110 CFU of VTEC/10 g of ground beef. When a 6-h enrichment step was included, the detection limit was 1 to 10 cells/10 g of ground beef. These methods are rapid and sensitive but can be complex, labor-intensive, and expensive. The FBA is rapid, technically simple, and specific for E. coli O157:H7, provided that there are at least 104 cells/ml present prior to flow cytometry. This assay takes 8 h to complete and consists of a 7-h enrichment step, followed by immunomagnetic capture and phage labeling, which takes 1 h. In the future, the FBA will need to be adapted for maximum throughput. This would be best achieved by converting the assay to a microtiter format.


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Such a format should allow many samples to be tested simultaneously. One problem envisioned is the effect that the magnetic beads would have in terms of signal interference or fluorescent light scatter. This problem could be solved by coating the inside walls of the microtiter wells with an E. coli O157:H7 polyclonal antibody, which would eliminate the need for immunomagnetic beads. The FBA produced several false-positive results. Due to the presence of false-positive results, culture results must be confirmed in order to ensure that all positive test results are E. coli O157:H7 results. Since the FBA employs separation technology in the form of IMS (perhaps in the future it will employ separation technology in the form of antibody-coated microtiter wells), any samples suspected to be E. coli O157:H7 positive could easily be plated onto a selective medium, such as cefixime-tellurite-sorbitol MacConkey agar. The successful use of the FBA to detect E. coli O157:H7 in pure cultures indicates that it may be possible to use this assay for presumptive detection of serotype O157:H7 in foods, such as ground beef and milk. REFERENCES 1. Ahmed, S., and J. Cowden. 1997. An outbreak of E. coli O157 in central Scotland, abstr. V179/1. Presented at the 3rd International Symposium and Workshop on Shiga Toxin (Verocytotoxin)-Producing Escherichia coli Infections, Baltimore, Md. 22 to 26 June, 1997. 2. Aleksic, S., H. Karch, and J. Bockemu ¨hl. 1992. A biotyping scheme for Shiga-like (Vero) toxin producing Escherichia coli O157 and a list of serological cross-reactions between O157 and other gram-negative bacteria. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 276:221–230. 3. Bettelheim, K. A., H. Evangelidis, J. L. Pearce, E. Sowers, and N. A. Strockbine. 1993. Isolation of a Citrobacter freundii strain which carries the Escherichia coli O157 antigen. J. Clin. Microbiol. 31:760–761. 4. Borczyk, A. A., N. Harnett, M. Lombos, and H. Lior. 1990. False positive identification of Escherichia coli O157 by commercial latex agglutination test. Lancet 336:946–947. 5. Centers for Disease Control and Prevention. 1993. Update: multistate outbreak of Escherichia coli O157:H7 infections from hamburgers—eastern United States, 1992–1993. Morbid. Mortal. Weekly Rep. 42:258–263. 6. Chapman, P. A., D. J. Wright, and C. A. Siddons. 1994. A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157:H7 from bovine feces. J. Med. Microbiol. 40:424–427. 7. Clark, C. 1995. Escherichia coli and other verotoxigenic E. coli. Report for the Canadian Meat Council. Guelph Group for Research in Food Safety, Guelph, Ontario, Canada. 8. Cubbon, M. D., J. E. Coia, M. F. Hanson, and F. M. Thomson-Carter. 1996. A comparison of immunomagnetic separation, direct culture and polymerase chain reaction for the detection of verocytotoxin-producing Escherichia coli O157 in human faeces. J. Med. Microbiol. 44:219–222. 9. Czajka, J., and C. A. Batt. 1996. A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli O157:H7 in ground beef and apple cider. J. Appl. Bacteriol. 81:601–607. 10. Del Rosario, B. A., and L. R. Beuchat. 1995. Survival and growth of enterohemorrhagic Escherichia coli O157:H7 in cantaloupe and watermelon. J. Food Prot. 58:105–107. 11. Dimmock, N. J., and S. B. Primrose. 1987. Introduction to modern virology. Blackwell Scientific Publications, Oxford, Great Britain. 12. Duncan, L., V. Mai, A. Carter, A. K. Carlson, A. Borczyk, and M. A. Karmali. 1987. Outbreak of gastrointestinal disease—Ontario. Can. Dis. Weekly Rep. 13:5–8. 13. Feng, P., P. I. Fields, B. Swaminathan, and T. S. Whittman. 1996. Characterization of nonmotile variants of Escherichia coli O157:H7 and other serotypes by using an antiflagellin monoclonal antibody. J. Clin. Microbiol. 34: 2856–2859. 14. Gilgen, M., P. Hubner, C. Hofelein, J. Luthy, and U. Candrian. 1998. PCRbased detection of verotoxin-producing Escherichia coli (VTEC) in ground beef. Res. Microbiol. 149:145–154. 14a.Goodridge, L. Unpublished data. 15. Griffin, P. M., and R. V. Tauxe. 1991. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13:60–98. 16. Hennes, K. P., and C. A. Suttle. 1995. Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy. Limnol. Oceanogr. 40:1054–1059. 17. Hershey, A. D., G. Kalmanson, and J. Brofenbrenner. 1943. Quantitative methods in the study of the phage-antiphage reaction. J. Immunol. 46:267– 279.

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