1999, 65(4):1397. Appl. Environ. Microbiol. 

Lawrence Goodridge, Jinru Chen and Mansel Griffiths 

O157:H7Escherichia coliDetection of Fluorescent-Bacteriophage Assay for Development and Characterization of a

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

Apr. 1999, p. 1397–1404 Vol. 65, No. 4

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Development and Characterization of a Fluorescent-BacteriophageAssay 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 immunomagneticseparation and a fluorescently stained bacteriophage for detection of Escherichia coli O157:H7 in broth. Whenit 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 estimatebacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between102 and 103 cells/ml; however, the modified DEFT was found to be an unreliable method for determiningbacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, isa sensitive technique for presumptive detection of E. coli O157:H7 in broth cultures.

Since Escherichia coli O157:H7 was first identified as a causeof hemorrhagic colitis in 1982 (30), this pathogen has beenrecognized as an important agent of food-borne disease with aworldwide distribution (25). Although many other means oftransmission have been documented, the most common sourceof outbreaks of E. coli O157:H7 infection has been ground beef(15). The largest reported outbreak of E. coli O157:H7 infec-tion in North America to date occurred in the northwesternUnited States in late 1992 and early 1993 and was associatedwith the consumption of undercooked ground beef at multipleoutlets of a fast food chain (5).

Other foods that have been epidemiologically implicated inoutbreaks 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 de-crease the importance of this organism in foods such as groundbeef. Still, fruits and vegetables are often consumed uncooked.Also, food products such as salami, in which raw ground meatis preserved by a process of fermentation and drying, are con-sidered ready to eat and are not generally cooked before con-sumption (34). E. coli O157:H7 poses a significant threat inthese foods. Obviously, rapid, sensitive, and simple techniquesthat can detect E. coli O157:H7 in foods need to be establishedso that the risk of E. coli O157:H7 outbreaks can be reduced.Many genetic and immunological techniques have been devel-oped for detection of serotype O157:H7. For example, Meng etal. (21) described a PCR technique that could detect as few as25 CFU of E. coli O157:H7 within 3 h. Also, there are manynucleic acid-based assays which can specifically detect all ve-rotoxigenic E. coli (VTEC) types, including E. coli O157:H7.These assays utilize primers and probes that hybridize specif-ically to complementary sequences found in the VTEC toxingenes (32). However, while these assays are specific, sensitive,and rapid, they are often expensive. Additionally, genetic as-says are often labor-intensive, because sample manipulation is

often required to liberate the bacteria from complex foodmatrices.

There are many immunological tests for detection of E. coliO157:H7. A rapid latex agglutination assay is commerciallyavailable for rapid presumptive identification of E. coli O157:H7 (24). The latex test has been found to be specific forserotype O157:H7 alone. The major drawback of the latexagglutination assay seems to be the time that it takes to obtainpure, isolated colonies that can be tested. A solid-phase fluo-rescence-based immunoassay has been developed for detectionof E. coli O157:H7 (9). In this assay, a soft glass capillary tubeserves as a solid support to which heat-killed E. coli O157:H7cells are absorbed. Biotin-conjugated polyclonal anti-E. coliO157:H7 antibody is used, and the antigen-antibody complexband is detected by using avidin molecules labeled with thefluorescent dye Cy5. When this assay is used, it is possible todetect 1 CFU/10 g of ground beef following a 7-h enrichmentstep. Immunoassays are widely used for detection of E. coliO157:H7 and are simple and rapid. However, immunoassaysoften require the use of appropriate controls in order to obtainmaximum specificity, since it has been reported that severalbacteria belonging to the genus Escherichia, including Esche-richia hermanii, E. coli O148:NM, and E. coli O117:H27, aswell as Salmonella urbana (group N salmonellae), cross-reactwith polyclonal antiserum raised against E. coli O157 (2, 4, 27).

Alternatively, immunomagnetic separation (IMS) per-formed with magnetic beads coated with anti-O157 antibodieshas been investigated in conjunction with assay methods, andthis technique shows promise for rapid detection of E. coliO157 in foods and environmental samples. IMS can easilyseparate bacteria from complex matrices, and when combinedwith other assay systems, IMS can decrease the occurrence offalse-positive results observed with immunoassays. The exis-tence of bacteriophages specific for E. coli O157:H7 (31) pre-sents opportunities to develop methods for O157:H7 detectionin which the separation capabilities of IMS and the specifichost ranges of the bacteriophages are used. Therefore, theobjectives of this study were to develop a rapid, simple, andsensitive IMS-bacteriophage-based assay that could detectE. coli O157:H7; to evaluate the specificity of the assay forE. coli O157:H7 and other E. coli strains, as well as otherbacteria; and to evaluate the threshold for detection of E. coliO157:H7 in pure culture.

* Corresponding author. Mailing address: Department of Food Sci-ence, 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.

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MATERIALS AND METHODS

Bacterial strains. A total of 51 strains of bacteria (Table 1) were used forenumeration, recovery, and specificity studies. The bacteria studied comprised 27strains 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 otherstrains were gifts from the Department of Pathobiology, University of Guelph.One strain, Shigella dysenteriae ATCC 27345, was obtained from the Departmentof Microbiology, University of Guelph. Of the 27 E. coli O157 strains used, 21were 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 O157strains of other H types. Stock cultures were maintained in 30% glycerol andwere frozen at 220°C. Fresh bacterial host cultures for use in experiments wereproduced by inoculating frozen stock cultures onto Luria-Bertani (LB) agarplates (LB broth [Difco Laboratories, Detroit, Mich.] containing 1.5% agar[Difco]) and incubating the plates overnight at 37°C. For growth experiments, theinocula consisted of stationary-phase cells that were obtained by inoculating LB

broth with cells from an overnight LB agar plate and incubating the preparationsovernight with shaking at 37°C.

Bacteriophage and host. The bacteriophage used in this study, designatedLG1, was isolated in our laboratory from chicken feces and was amplified incultures with its host, E. coli O157:H7 strain EC920333. Ten milliliters of anovernight culture of EC920333 and 1.5 ml of phage LG1 (1011 PFU/ml) wereadded to 250 ml of LB broth in a 500-ml Erlenmeyer flask and incubated withshaking at 37°C for 7 h. Following lysis, 10 ml of chloroform was added to theflask to release any progeny phage which may still have been in the host cells, andthe suspension was incubated with shaking at 37°C for an additional 10 min. Thebroth suspension was centrifuged at 5,000 3 g for 15 min, and the supernatantwas 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 thedark at 4°C.

Preparation of fluorescently labeled bacteriophage. Fluorescently stained bac-teriophage were produced by using a modification of the procedure originallydescribed by Hennes and Suttle (16). Following bacteriophage amplification, thephage were centrifuged at 186,000 3 g for 1 h in a model L8-M ultracentrifuge(Beckman Instruments, Mississauga, Ontario, Canada). The phage pellets wereresuspended 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 over-night. The next day, the phage pellets were gently resuspended and pooled inmicrocentrifuge tubes. To resuspend any bacteriophage which remained, thecentrifuge tubes were washed with approximately 1 ml of lambda buffer, and theresulting preparation was added to the pooled pellets. To remove any bacterialnucleic acid that adhered to the phage, the samples were treated with 1 mg ofDNase (Boehringer, Mannheim, Germany) per ml and 1 mg of RNase (Boehr-inger) per ml and incubated at room temperature for 30 min. Approximately 200ml of chloroform was added to each microcentrifuge tube, and the samples werecentrifuged in a model 5415 C Eppendorf centrifuge (Brinkman Instruments,Rexdale, Ontario, Canada) at 16,000 3 g for 5 min. The aqueous phase contain-ing the phage was removed and added to new microcentrifuge tubes, and 15 mlof the fluorescent nucleic acid dye YOYO-1 (Molecular Probes, Inc., Eugene,Oreg.) was added to each tube. The tubes were inverted several times and in-cubated in the dark at 4°C for 72 h. Following phage staining, the microcentri-fuge tubes were suspended in ultracentrifuge tubes filled with water and centri-fuged at 186,000 3 g for 1 h. The microcentrifuge tubes were floated out of theultracentrifuge 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 thesamples. Fluorescent-phage samples were placed in a model 8MC MicroUltra-filtration device (Amicon, Inc., Beverly, Mass.) and filtered through a 10-mm-diameter Diaflo ultrafiltration membrane (Amicon) with a molecular weightcutoff of 300,000. Lambda buffer was used to wash the samples as they were fil-tered. The samples were filtered at 25 lb/in2 for 1 h and then concentrated untilthe 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 boundphage. The retentate and the filter effluent were pooled in a microcentrifugetube, and chloroform purification was performed as described above. The fluo-rescent-phage samples were filtered through 0.2-mm-pore-size syringe filters andstored in the dark at 4°C. The final concentration of fluorescent phage wasbetween 1011 and 1012 PFU/ml.

IMS and labeling of bacteria with fluorescent phage. One-milliliter portions ofstationary-phase cultures were added to microcentrifuge tubes containing 20 mlof 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 anOrbitron Rotator II (Fisher Scientific, Mississauga, Ontario, Canada), they wereplaced in a magnetic rack (Dynal) so that the magnetic beads could be separatedfrom the enrichment broth. The beads were washed twice in lambda buffer andresuspended in 200 ml of the fluorescent-phage suspension. The samples wererotated at 30 rpm for 15 min at room temperature. The magnetic beads wereseparated from the fluorescent-phage suspension, washed once, and resuspendedin 1 ml of lambda buffer.

Epifluorescence microscopy. Fifty-one bacterial isolates (Table 1) were sub-jected to the fluorescent-bacteriophage assay (FBA) and examined by epifluo-rescence microscopy. Each bacterial isolate was tested twice. Controls consistingof immunomagnetic beads incubated with the fluorescent phage were included inorder 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-sizeblack membrane filters (Fisher Scientific, Nepean, Ontario, Canada) which wereoverlaid on prewetted 0.4-mm-pore-size membrane filters. Both filters werehoused in a Swinnex filtration device (Fisher Scientific, Nepean, Ontario, Can-ada). Following filtration, the membrane filters were removed from the filtrationdevice, placed on glass slides, and covered with coverslips. The slides wereviewed at a magnification of 31,000 with an Olympus model BH-2 epifluo-rescence microscope equipped with a filter set capable of illuminating YOYO-1-stained bacteriophage (excitation wavelength, 491 nm; emission wavelength,509 nm). Pictures were taken with a scanning laser confocal microscope (Bio-Rad, Mississauga, Ontario, Canada) equipped with a krypton-argon mixed-gaslaser at a magnification of 31,600.

TABLE 1. Bacterial strains used in the specificity study

Taxon Strain Origin

Escherichia coli O157:H7 EC920333 Health CanadaEscherichia coli O157:H7 EC920005 Health CanadaEscherichia coli O157:H7 EC920026 Health CanadaEscherichia coli O157:H7 EC920027 Health CanadaEscherichia coli O157:H7 EC920037 Health CanadaEscherichia coli O157:H7 EC920081 Health CanadaEscherichia coli O157:H7 EC920192 Health CanadaEscherichia coli O157:H7 EC920321 Health CanadaEscherichia coli O157:H7 EC950050 Health CanadaEscherichia coli O157:H7 EC960275 Health CanadaEscherichia coli O157:H7 EC920267 Health CanadaEscherichia coli O157:H7 EC920283 Health CanadaEscherichia coli O157:H7 EC920409 Health CanadaEscherichia coli O157:H7 EC940235 Health CanadaEscherichia coli O157:H7 EC940239 Health CanadaEscherichia coli O157:H7 EC940340 Health CanadaEscherichia coli O157:H7 EC940435 Health CanadaEscherichia coli O157:H7 EC940468 Health CanadaEscherichia coli O157:H7 EC960313 Health CanadaEscherichia coli O157:H7a EC960279 Health CanadaEscherichia coli O157:H7a EC960274 Health CanadaEscherichia coli O157:NM EC930195 Health CanadaEscherichia coli O157:H7 E32511 University of Guelphb

Escherichia coli O157:H7 933W University of Guelphb

Escherichia coli O6:H34 EC910051 Health CanadaEscherichia coli O103:H2 EC910037 Health CanadaEscherichia coli O145:NM EC910040 Health CanadaEscherichia coli O126:H8 EC910061 Health CanadaEscherichia coli O2:H6 EC920229 Health CanadaEscherichia coli O2:H5 EC920232 Health CanadaEscherichia coli O2:H1 EC950209 Health CanadaEscherichia coli O4:H5 EC950211 Health CanadaEscherichia coli O6:H5 EC950212 Health CanadaEscherichia coli O157:HI9 EC940076 Health CanadaEscherichia coli O157:H25 EC960282 Health CanadaEscherichia coli O157:HI2 EC960308 Health CanadaEscherichia coli 2-4-77 EC2477 University of Guelphb

Escherichia coli 2-1-214 EC21214 University of Guelphb

Escherichia coli O26:H1I H19 University of Guelphb

Escherichia coli O139:K82 412 University of Guelphb

Escherichia coli O128:BI2 H.1.8 University of Guelphb

Salmonella enteritidis University of Guelphc

Salmonella typhimurium SA941256 University of Guelphc

Listeria monocytogenes University of Guelphc

Shigella dysenteriae ATCC 27345 University of Guelphd

Shigella dysenteriae N5 University of Guelphc

Enterobacter aerogenes N8 University of Guelphb

Serratia marcescens N9 University of Guelphb

Klebsiella pneumoniae University of Guelphb

Escherichia hermanii Health CanadaSalmonella urbana Health Canada

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

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Flow cytometry. Forward light scatter and side scatter fluorescence were mea-sured simultaneously with a Coulter Elite flow cytometer (Coulter Electronic,Burlington, Ontario, Canada). Samples were added to the sample introductiontube at a pressure of 9.79 lb/in2. The sheath fluid used was Isoton II (CoulterElectronic), which had been prefiltered through a 0.22-mm-pore-size filter (Gel-man Sciences, Ann Arbor, Mich.). A constant pressure of 10.5 lb/in2 was appliedto the sheath fluid-sample stream, which emerged from the flow chamberthrough an orifice having a diameter of 100 mm. The stream was interrogateddirectly beneath the orifice in quartz by a focused argon ion laser operating at awavelength of 488 nm and an output power of 50 mW. To detect forward-anglelight scatter, a neutral-density filter was placed in front of the forward-angle lightscatter detector. Side-angle light scatter was collected by using a 488-nm dichroicfilter and a band pass filter (488 6 5 nm). A 600-nm dichroic filter was placed infront of the photomultiplier to detect green fluorescence. Photomultiplier volt-ages were fixed for each set of experiments. Samples to be examined by flowcytometry were prepared as described above and were analyzed in triplicate, andcontrols consisting of immunomagnetic beads and labeled phage were includedin each group of samples in order to determine the level of background fluores-cence.

Bacterial counts. The accuracy of using fluorescent bacteriophage to estimatethe abundance of E. coli O157:H7 in a particular sample was evaluated. Anovernight culture of E. coli O157:H7 strain EC920333 was diluted by preparinga series of 1021 dilutions in lambda buffer until the final dilution was 1028. Thedifferent dilutions were concentrated and labeled as described above and wereanalyzed by using epifluorescence microscopy and flow cytometry. For analysis byepifluorescence microscopy, a modification of the direct epifluorescent-filtertechnique (DEFT), as described by Pettipher (28), was employed. After sampleswere examined with the Olympus model BH-2 epifluorescence microscope, asdescribed above, the numbers of tagged cells in 15 microscope fields of view weredetermined, and the values were averaged. The microscope fields were selectedat random. The DEFT count per milliliter was calculated by multiplying theaverage number of cells per microscope field by the microscope factor. Themicroscope factor was calculated by the following formula: microscope factor 5area 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 filtermembrane. The area of the microscope field of view was calculated from theradius of the field of view, as determined with a stage micrometer. Samplesexamined by the DEFT and flow cytometry were analyzed in triplicate. Platecounts were determined in duplicate with the overnight sample in order todetermine the original number of CFU per milliliter by spread plating 10-foldserial dilutions of a stationary-phase culture onto LB agar plates and thenincubating the preparations overnight at 37°C.

Efficiency of IMS. An overnight culture of E. coli O157:H7 strain EC920333was serially diluted 100-fold in lambda buffer so that four different cell popula-tions were created. The concentrations of the 100-fold dilutions were estimatedto be 108, 106, 104, and 102 CFU/ml. Just after the dilutions were prepared(without enrichment), the efficiency of magnetic capture was tested with eachdilution. To determine the cell numbers in the dilutions, duplicate plate countswere obtained. After magnetic capture, the beads were resuspended in 1 ml oflambda buffer, and duplicate plate counts of each sample were obtained. Afterincubation at 37°C overnight, colonies were counted, and the values were com-pared with the numbers of CFU per milliliter in the original dilutions.

Bacteriophage specificity. In order to determine its host range, bacteriophageLG1 was tested with 51 bacterial strains (Table 1). Bacteriophage lysis assayswere performed by using a modification of the traditional double-layer plaqueformation technique described by Hershey et al. (17). Lysis assays were con-ducted in 10-cm-diameter petri plates (Fisher Scientific, Nepean, Ontario, Can-ada). The bottom agar layer consisted of LB agar. The top agar layer consistedof 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 topagar was steamed for approximately 10 min and allowed to cool to 47°C. Onehundred microliters of an overnight culture of the bacterium to be tested wasadded to the top agar, which was then vortexed and poured onto the bottomlayer. The top agar was allowed to solidify, and 20 ml of a bacteriophage sus-pension (1011 PFU/ml) was pipetted onto the top agar layer. The plates wereincubated at 37°C overnight and then examined for the presence of clear zonesof lysis. The ability of the fluorescent phage to bind to host cells was alsoevaluated. IMS and fluorescent-phage labeling of overnight cultures of all 51bacterial strains were performed as described above. The samples were examinedby epifluorescence microscopy.

Scanning electron microscopy. Bacteria and immunomagnetic beads wereincubated with shaking at 30 rpm on an Orbitron Rotator II for 30 min at roomtemperature, washed twice, and resuspended in 1 ml of lambda buffer. Thesuspension was filtered onto a polycarbonate 0.2-mm-diameter filter (PoreticsCorp., Livermore, Calif.) in a 13-mm syringe apparatus by using a Buchnervacuum filtration unit. The filter was transferred to phosphate-buffered saline(PBS) (pH 7.5) containing 2% glutaraldehyde and incubated for 1 h at roomtemperature in order to fix the bacterial cells and immunomagnetic beads.Samples were rinsed in several changes of PBS for 30 min at room temperatureand 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

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 VIIsputter coater (Anatech Corp., Alexandria, Va.), and scanned with a modelS-570 scanning electron microscope (Hitachi Corp., Danbury, Conn.) with anaccelerating voltage of 10 keV.

RESULTS

Epifluorescence microscopy. Phage LG1 was stained withthe dimeric nucleic acid dye YOYO-1 and examined by epi-fluorescence microscopy. The phage appeared as green specksthat were easily distinguished from the black background. IMSof E. coli O157:H7 on magnetic beads and addition of thestained bacteriophage to the separated beads resulted in re-moval of detritus and other particulate material from the sam-ple, and the fluorescent bacteriophage attached to the hostcells, giving the cells a “halolike” appearance (Fig. 1). Non-specific binding of LG1 to the immunomagnetic beads wasnegligible in the control samples.

Flow cytometry. Overnight cultures of the LG1 host, E. coliO157:H7 strain EC920333, were concentrated with O157-spe-cific magnetic beads, labeled with fluorescent phage, and ana-lyzed by flow cytometry. Each overnight culture was testedtwice. In each test, all of the cultures were analyzed in tripli-cate, and the means and standard deviations were plotted.Figure 2 shows the expected number of cells, as determined byplate counting, and the percent fluorescence for each individ-ual cell culture, as detected by flow cytometry, for a single test.Bacterial concentrations of 104 CFU/ml or more gave positiveresults. At concentrations below 104 CFU/ml, the percent flu-orescence readings were indistinguishable from the back-ground readings. The percent fluorescence values did not cor-relate 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 datawere organized as individual histograms showing the spread offorward scatter and fluorescence over the population of cellsand beads and also as two-parameter dot plots (Fig. 3). Exam-ination of the dot plots revealed three regions. Region D hadthe largest particles and greatest fluorescence, followed byregion C. Region E consisted of nonfluorescent debris.

Bacterial counts. To evaluate the accuracy of using fluores-cent bacteriophage to estimate bacterial counts, a modifiedDEFT was employed. Figure 4 shows the average number ofCFU per milliliter as determined by plate counting and theaverage number of CFU per milliliter as calculated by theDEFT. DEFT counting was performed three times. The platecounts 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:H7strain EC920333 was diluted to final concentrations of 108, 105,104, and 102 CFU/ml, as confirmed by plate counting. Just afterthe dilutions were prepared, the efficiency of magnetic capturewas determined with each dilution. Following IMS, each dilu-tion was plated onto LB agar in duplicate. The results obtainedare presented in Fig. 5, which shows that the numbers ofbacteria recovered were within 1 log10 value of the numbers ofcells in the original culture.

Specificity of fluorescent phage LG1. LG1 was tested with 51strains of bacteria to determine its host range and specificity.LG1 formed plaques on all VT-positive E. coli O157 strainsand on the two VT-negative E. coli O157 strains tested, as wellas on E. hermanii, type 1 S. dysenteriae ATCC 27345, and threenon-O157:H7 VTEC strains. The FBA was also tested forspecificity, and we found that the assay identified all of theO157:H7 strains, as well as type 1 S. dysenteriae ATCC 27345,

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one strain of E. coli O157:NM, and E. coli O157:HI9. There-fore, using immunomagnetic beads increased the specificity ofthe FBA.

DISCUSSION

The general patterns of E. coli O157:H7 transmission sug-gest that the infectious dose of this pathogen is low (15). There-fore, 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 ofthe microbial communities on earth (26). Bacteriophage areeasy to isolate and inexpensive to purify. Isolation of a bacte-riophage that initially was reported to be specific for E. coliO157:H7 (31) suggested that fluorescent labeling of this bac-teriophage might be used as a rapid method for detection ofE. coli O157:H7. Fifty-one bacterial isolates representing sevengenera were used to evaluate the use of fluorescent phage LG1for detection of E. coli O157:H7. Most of these bacteria arecommon food-borne pathogens. E. hermanii and S. urbana(group N salmonellae) were included and were used to deter-mine the specificity of the immunomagnetic beads because it isknown that these organisms cross-react with polyclonal anti-serum raised against E. coli O157 (2, 27).

The results obtained in this study show that bacteriophageLG1 is specific for a subset of VTEC and other closely relatedbacteria, such as S. dysenteriae and E. hermanii, and has a hostrange similar to that described for bacteriophage AR1 de-scribed by Ronner and Cliver (31). The susceptibility of a bac-terium to bacteriophage infection is primarily dependent onwhether the bacteriophage can attach to receptors on the cell(18). Based on morphological characteristics, bacteriophageLG1 belongs to the family Myoviridae and is probably mostclosely related to bacteriophage T4. T4 uses the lipopolysac-charide (LPS) of E. coli B as a receptor whenever it possessesat least one of the two terminal glucose residues (29). T4 alsouses 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 theseresults, since some E. coli strains produce mutant phenotypes.For example, E. coli K-12 sometimes does not produce LPSwith a complete core oligosaccharide (35). It is unclear whatexactly on the bacterial surface constitutes a receptor for LG1.There are bacteriophage that attack only motile strains ofbacteria (18). It is unlikely that the H antigen of E. coli O157is the receptor for bacteriophage LG1, because this phage wasfound to infect several serotypes of E. coli O157, each possess-ing different H antigens, including H7, H12, and H19. In ad-dition, LG1 lysed one strain of nonmotile E. coli O157.

If the characteristics of the T-even phages are used as amodel, it is possible that LPS (specifically, the O side chain)and an outer membrane protein (although not necessarily out-er membrane protein C) are receptors for phage LG1. How-ever, the results of this work show that this may not be so. LG1was 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. coliO139:K82, E. coli O128:B12, and E. coli O157:H12. However,it failed to infect E. coli O157:H25. In addition, phage LG1 waslytic on E. coli O26:H1, S. dysenteriae (type 1), and E. hermanii,

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

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

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indicating that the receptor is not O antigen specific. It isprobable that the receptor for phage LG1 is a component ofthe O antigen, but further studies are needed to determine thereceptor 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 antibody-coated paramagnetic polymer beads and is considered fast,safe, sensitive, and reproducible (23). The results obtainedwith commercially available paramagnetic beads coated withan O157 polyclonal antibody were compared with plate count

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 bacteriophageLG1. 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 mainlyunbound 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, whichexhibits 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.

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results in order to determine the efficiency of recovery ofE. coli O157:H7. The IMS count results and plate count resultscorrelated 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 magneticbead method was better at higher dilutions than at lower di-lutions. With the 1028 dilution (containing approximately 102

CFU/ml), the immunomagnetic beads captured 61.5% of theE. coli O157:H7 cells, whereas with the 1022 dilution (contain-ing approximately 108 CFU/ml) only 11.5% of E. coli O157:H7cells were captured. In a similar study, Mortlock (23) foundthat as the numbers of E. coli O157:H7 cells in suspensiondecreased, the level of recovery increased from a minimum of23% at a concentration of 107 CFU/ml to a maximum of 51%at a concentration of 103 CFU/ml. This could have been due tothe bead-to-organism ratio not being correct at higher concen-trations of organisms. It has previously been shown that thereshould be a 3:1 bead-to-organism ratio for maximum recovery(23). This explains the low recovery rate obtained with largercell populations and is an important point to consider whendense bacterial suspensions are evaluated.

The main problem with IMS to date seems to be the num-bers of organisms other than E. coli O157 that attach nonspe-cifically to the magnetic beads (6). Other researchers havefound that incorporating a washing step consisting of washingwith the detergent Tween 20 and PBS into the IMS methoddecreases, but does not eliminate, nonspecific binding of or-ganisms to the beads (8). In addition to the nonspecific bindingof the bacteria to the beads, there is the problem of cross-reactions of E. coli O157 polyclonal antibodies. Antisera pre-pared against the O157 antigen are known to cross-react withO antigens of 12 other serogroups of E. coli, as well as with Oantigens 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 poly-clonal antisera to the antigenic relationships between the LPSof 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 epitopesinvolving the presence of N-acyl derivatives of the 4-amino-4,6-dideoxy-a-D-mannopyranosyl residues occurred in the O-polysaccharide portion of the LPS.

In this study, the FBA was evaluated to determine whetherthere were cross-reactions due to the O157 magnetic beads andnonspecific binding of bacteriophage LG1 to the captured non-O157 cells. Twenty-eight non-E. coli O157:H7 bacteria belong-ing to several genera were tested; these organisms includedS. dysenteriae, E. hermanii, and S. urbana, each of which is known

to cross-react with E. coli O157 polyclonal antisera. Only 2 ofthe 28 non-E. coli O157:H7 bacteria, S. dysenteriae (type 1) andE. 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 de-spite being susceptible to LG1. Therefore, the use of E. coliO157 magnetic beads in conjunction with bacteriophage LG1conferred a level of specificity on the FBA that neither the beadsnor LG1 possessed on its own. Although LG1 induced lysis ofseven non-E. coli O157:H7 bacteria in this study and, as men-tioned above, the E. coli O157 magnetic beads can bind manydifferent bacteria, the FBA gave positive results with only threenon-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 advanta-geous, since both E. coli O157:NM and S. dysenteriae causehemorrhagic colitis and hemolytic-uremic syndrome (33). Re-garding the issue of assay specificity, it would be of some valueto isolate a diverse range of phages and extensively character-ize their host ranges, since there may be phages which aremore specific for E. coli O157:H7 than LG1 is; such phageswould increase the accuracy of the FBA. Still, the fact that LG1is specific for a subset of VTEC instead of E. coli O157:H7does not invalidate the principle of this assay.

An effort was made to correlate the DEFT count and platecount results. The DEFT did not adequately enumerate E. coliO157:H7 over the range from 102 to 108 CFU/ml. There was agood correlation between DEFT count and plate count results(n 5 8; r 5 0.93). However, this correlation was not significantat the 95% confidence level (P 5 0.07). The DEFT was able toestimate plate counts more reliably at higher dilutions than atlower dilutions. There were several reasons for this. One rea-son was the inability of the magnetic beads to pull 100% of thebacterial population out of suspension. As mentioned above, athigher dilutions the magnetic beads were more efficient atconcentrating bacteria, while at lower dilutions IMS failed tocapture a large percentage of the bacteria. Similarly and pre-dictably, this trend was observed with DEFT. The DEFTcounts were closer to the plate counts with smaller populationsof cells than with larger populations of cells. Another reasonfor the inability of the DEFT to accurately predict plate countswas the counting method employed. The DEFT counting meth-od required that any cell or group of cells separated by adistance that was less than twice the smallest diameter of the

FIG. 5. Relationship between plate counts following IMS and plate countsfor different dilutions of E. coli O157:H7 in broth. Two replicates from each100-fold dilution (1022, 1024, 1026, and 1028) were counted. The line is thefitted regression line (y 5 1.063x 2 0.077).

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

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cells nearest each other must be counted as one clump or cell.Under ideal conditions, in any given field of view there shouldbe groups of bacterial cells close enough together to be count-ed as one clump and bacteria far enough apart from one an-other 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 tomagnetic beads and therefore had to be counted as a clumpinstead of individual cells. Thus, it was not the number of bac-terial cells but rather the number of magnetic beads with bac-teria attached in a given field of view that was counted. Thisresulted in substantial underestimation of the number of bac-teria in a given sample. This counting inadequacy, combinedwith less than 100% recovery of bacteria by IMS, resulted inthe DEFT counts that were less accurate as the bacterial num-bers increased. In the future, new counting methods that takeinto account these two factors will have to be devised.

Due to the problems and labor intensity of the DEFT, flowcytometry was used as an alternative method for fluorescencedetection. Different dilutions of E. coli O157:H7 were labeledby 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 fluorescentvalues obtained were unreliable, probably because of laserscattering by the immunomagnetic beads. A sensitivity of 104

CFU/ml is consistent with previously published results. Mc-Clelland and Pinder (20) detected 104 Salmonella cells per mlby using flow cytometry and fluorescent antibodies. In thepresent study, when the data were analyzed as a dot plot, threedistinct regions were observed, regions D, C, and E (Fig. 3).Region D exhibited the largest amount of fluorescence andalso contained the largest particles. The magnetic beads andbacterial cells formed large complexes (Fig. 3D), and thesecomplexes were the large particles observed in region D. Re-gion C consisted mostly of magnetic beads, and region E con-tained nonfluorescent debris. These results show that flow cy-tometry offers food microbiologists numerous possibilities fordetection and enumeration and, most importantly, a relativelysensitive and rapid method for identification of microorgan-isms.

Many rapid methods for detection of E. coli O157:H7 havebeen described. These methods include immunological meth-ods, such as enzyme immunoassays and immunoblot tests. IMShas been used to identify serotype O157:H7. Yu and Bruno(36) evaluated a commercial sensor that combined IMS andelectrochemiluminescence to detect E. coli O157 in food. Theresults indicated that the detection limits for E. coli O157:H7were between 102 and 104 CFU/ml in buffer and between 1 3103 and 2 3 103 CFU/ml in food samples. Alternatively, ge-netic techniques, such as the PCR, have been described assensitive 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 genesencoding VT1 and VT2 irrespective of the bacterial serotype.Using a direct sample protocol, they were able to detect 110CFU of VTEC/10 g of ground beef. When a 6-h enrichmentstep was included, the detection limit was 1 to 10 cells/10 g ofground beef. These methods are rapid and sensitive but can becomplex, labor-intensive, and expensive. The FBA is rapid,technically simple, and specific for E. coli O157:H7, providedthat there are at least 104 cells/ml present prior to flow cytom-etry. This assay takes 8 h to complete and consists of a 7-henrichment step, followed by immunomagnetic capture andphage labeling, which takes 1 h. In the future, the FBA willneed to be adapted for maximum throughput. This would bebest achieved by converting the assay to a microtiter format.

Such a format should allow many samples to be tested simul-taneously. One problem envisioned is the effect that the mag-netic beads would have in terms of signal interference or flu-orescent light scatter. This problem could be solved by coatingthe inside walls of the microtiter wells with an E. coli O157:H7polyclonal antibody, which would eliminate the need for im-munomagnetic beads. The FBA produced several false-posi-tive results. Due to the presence of false-positive results, cul-ture results must be confirmed in order to ensure that allpositive test results are E. coli O157:H7 results. Since the FBAemploys separation technology in the form of IMS (perhaps inthe future it will employ separation technology in the form ofantibody-coated microtiter wells), any samples suspected to beE. coli O157:H7 positive could easily be plated onto a selectivemedium, such as cefixime-tellurite-sorbitol MacConkey agar.The successful use of the FBA to detect E. coli O157:H7 inpure cultures indicates that it may be possible to use this assayfor presumptive detection of serotype O157:H7 in foods, suchas ground beef and milk.

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