Luminescence based enzyme-labeled phage (Phazyme) assays for rapid detection of Shiga toxin producing Escherichia coli serogroups

doi:10.4161/bact.1.2.15666

Bacteriophage. 2011 Mar-Apr; 1(2): 101–110.

PMCID: PMC3278647

Luminescence based enzyme-labeled phage (Phazyme) assays for rapid detection of Shiga toxin producing Escherichia coli serogroups

John D Willford,¹ Bledar Bisha,² Kyle E Bolenbaugh,¹ and Lawrence D Goodridge²

¹Department of Animal Science; University of Wyoming; Laramie, WY USA

²Center for Meat Safety and Quality; Food safety Cluster; Department of Animal Sciences; Colorado State University; Fort Collins, CO USA

Corresponding author.

Correspondence to: Lawrence D. Goodridge; Email: Lawrence.Goodridge/at/colostate.edu

Received January 21, 2011; Revised March 23, 2011; Accepted March 31, 2011.

Abstract

Most diagnostic approaches for Shiga toxin producing Escherichia coli (STEC) have been designed to detect only serogroup O157 that causes a majority, but not all STEC related outbreaks in the United States. Therefore, there is a need to develop methodology that would enable the detection of other STEC serogroups that cause disease. Three bacteriophages (phages) that infect STEC serogroups O26, O103, O111, O145 and O157 were chemically labeled with horseradish peroxidase (HRP). The enzyme-labeled phages (Phazymes) were individually combined with a sampling device (a swab), STEC serogroup-specific immunomagnetic separation (IMS) beads, bacterial enrichment broth and luminescent HRP substrate, in a self-contained test device, while luminescence was measured in a hand-held luminometer.

The O26 and O157 Phazyme assays correctly identified more than 93% of the bacteria tested during this study, the O123 Phazyme assay identified 89.6%, while the O111 and O145 Phazyme assays correctly detected 82.4% and 75.9%, respectively. The decreased specificity of the O111 and O145 assays was related to the broad host ranges of the phages used in both assays. The Phazyme assays were capable of directly detecting between 10⁵ and 10⁶ CFU/ml in pure culture, depending on the serogroup. In food trials, the O157 Phazyme assay was able to detect E. coli O157:H7 in spinach consistently at levels of 1 CFU/g and occasionally at levels of 0.1 CFU/g. The assay detected 10⁰ CFU/100 cm² on swabbed meat samples and 10² CFU/100 ml in water samples. The Phazyme assay effectively detects most STEC in a simple and rapid manner, with minimal need for instrumentation to interpret the test result.

Key words: Shiga toxin producing Escherichia coli (STEC), rapid detection, enzyme-labeled phages (Phazymes), horseradish peroxidase (HRP), swab, hand held luminometer

Introduction

Shiga toxin producing Escherichia coli (STEC) are important agents of foodborne illness, causing an estimated 175,905 cases of illness, 2,409 hospitalizations and 20 deaths each year.¹ In the United States, E. coli O157:H7 is the most reported serotype of STEC; however, at least 100 other STEC serotypes have been implicated in disease.²^–⁴ While the prevalence of these serotypes has been considered to be low in the US, it has been reported that a significant proportion of STEC illness is caused by non-O157 STEC.¹ For example, it has been recently estimated that almost two thirds of STEC associated disease is caused by non-O157 serotypes,¹ and the Centers for Disease Control and Prevention (CDC) has previously advocated testing for non-O157 STEC strains.²

While many methods have been developed to detect STEC in food and water, most of the tests are only designed to detect bacteria that belong to STEC serogroup O157. Many diagnostic methods have been developed for the detection of E. coli O157 including culture on solid media, immunoassays and molecular based (polymerase chain reaction) methods.⁵^–⁹ Cultural based methods are slow, requiring 24–48 hours for results, while immunoassays and molecular techniques can be labor intensive, time consuming, and/or require extensive training of laboratory personnel and expensive equipment to read results. There is an acute need to develop improved detection assays for STEC, and these diagnostic tests should be targeted at STEC serogroups O26, O103, O111, O145 and O157 that have been the main serogroups implicated in US food and waterborne disease outbreaks.¹⁰^,¹¹ In addition, a number of these serogroups including O26, O103, O111 and O145 have been implicated as important agents of foodborne disease worldwide.¹²^–¹⁵ Such detection methods are especially required, since it has been suggested that the non-O157 STEC incidence is underestimated due to the lack of detection methods for these pathogens.¹⁶^,¹⁷

An ideal field-based, detection method should be sensitive, specific and rapid while being easy to complete with little or no instrumentation. The binding properties of bacteriophages (phages) have often been exploited to create rapid bacterial detection assays.¹⁸^–²¹ The purpose of this study was to develop field-based diagnostic assays with the ability to detect five main STEC serogroups: O26, O103, O111, O145 and O157. To achieve this, luminescent assays, based on enzyme-labeled phages (Phazymes), were developed by combining the Phazymes with a sampling device (swab), STEC specific immunomagnetic (IMS) beads, bacterial enrichment broth and a luminescent substrate to form an integrated assay that detected STEC in food and water samples within 9 hours.

Results

STEC serogroup specific Phazyme assays were created and used to detect STEC belonging to serogroups O26, O103, O111, O145 and O157 in pure culture and in inoculated food and water samples. The creation of the enzyme-labeled phages was a straightforward and easy process, and labeling of the phages with the HRP kit caused a reduction in infective phage titer by 1.16 (±0.28) logs, regardless of the phage that was labeled. The reduction in infective phage titer did not affect the sensitivity of the Phazyme assays.

Each Phazyme assay was tested for specificity by testing STEC strains belonging to each respective serogroup as well as non-STEC bacteria. A positive result was defined as a luminescent reaction which produced at least 100 RLU and was significantly higher (p < 0.05) than the Phazyme-only control. The results for pure cultures are shown in Tables 1 and and22. The results indicated that the O26 and O157 Phazyme assays were capable of correctly identifying (as a true positive or negative) greater than 93% of the bacterial strains tested (Table 3). The O103 Phazyme assay identified 89.6%, while the O111 and O145 Phazyme assays correctly identified 82.4% and 75.9% of the bacterial strains tested (Table 3).

Table 1

STEC serogroups screened using the Phazyme assay

Table 2

Non STEC strains used as negative controls to determine the specificity of each Phazyme assay

Table 3

Specificity of each assay

The sensitivity of each Phazyme assay was evaluated, using pure, overnight cultured STEC strains from each of the five serogroups. The results are shown in Figures 2 and and33. The Phazyme assays had direct (without enrichment) detection limits of 1.18 × 10⁵ CFU/ml (O26), 3.85 × 10⁵ CFU/ml (O103), 1.32 × 10⁶ CFU/ml (O111), 6.82 × 10⁴ CFU/ml (O145) and 2.91 × 10⁵ CFU/ml (O157) (Fig. 2). With an eight hour enrichment step, the detection limit for the O26, O103, O111 and O157 Phazyme assays was determined to be an initial inoculum of 10⁰ CFU/ml (Fig. 3). The O145 Phazyme assay demonstrated a significantly higher (p < 0.0001) detection limit of 10⁴ CFU/ml (Fig. 3).

Figure 2

Direct detection limit of each Phazyme assay. The detection threshold is set as the Phazyme-only average plus 5 times the standard error. A logarithmic regression line (powered for O103) is included along with its equation and R²-value for each serogroup. (more ...)

Figure 3

Detection limit (with enrichment) of each Phazyme assay. With an eight hour enrichment step, the detection limit for the O26 (A), O103 (B), O111 (C) and O157 (E) Phazyme assays was determined to be an initial inoculum of 10⁰ CFU/ml. The O145 Phazyme assay (more ...)

Phage adsorption assays were conducted in order to investigate the ability of the phages to bind to the strains from each STEC serogroup (Fig. 4). Logarithmic growth phase cultures of STEC (10⁷ CFU/ml) were mixed with phages at a MOI of 0.1 and free phages in the mixture were plotted against the incubation time to estimate ka values. The results are shown in Figure 4. After 20 minutes, 93.1% of phage CBA120 was adsorbed to its host E. coli O157 cell. In contrast, only 69% of phage 56 was adsorbed to the E. coli O111 strain (Fig. 4).

Figure 4

Phage adsorption assay. Phage and E. coli serogroup combinations: (A) CBA120 mixed with E. coli O157 (diamond), (B) AR1 mixed with E. coli O103 (square), E. coli O145 (triangle) and E. coli O26 (cross), (C) phage 56 mixed with E. coli O111 (circle). Initial (more ...)

The O157 Phazyme assay was evaluated for its ability to detect low levels of E. coli O157 in artificially contaminated food and water samples. In spinach samples, the Phazyme assay was able to consistently detect 1 CFU/g of E. coli O157:H7 and detected 0.1 CFU/g of E. coli in one of three samples (Fig. 5A). The initial inoculum level of 1 CFU/g or higher produced significantly higher (p < 0.0055) relative light unit (RLU) readings than spinach-only controls. An initial inoculum level of 0.1 CFU/g produced moderately higher (p = 0.0804) RLU readings as compared to the spinach-only control. The average RLU level for the 0.1 CFU/g inoculum sample was largely elevated by the single positive result of the three samples.

Figure 5

Evaluation of the Phazyme assay in spinach, meat and water. With stomaching and 8 h enrichment in TSBCT, the O157 Phazyme assay was able to detect 1 CFU/g of spinach consistently and 0.1 CFU/g of spinach in 1 of 3 trials (A). For statistical purposes, (more ...)

The Phazyme assay consistently detected E. coli O157:H7 at levels of 1.76 CFU/100 cm² of beef carcass tissue (Fig. 5B). The RLU levels for samples with this level of contamination were significantly higher (p = 0.0358) for both meat types. The background level provided by the food matrix was significantly higher (p < 0.0001) for rib steak than for top round steak. A clearly noticeable difference between the two meat samples is the fat content; with the rib steak containing both more marbling and fat content, and this may have contributed to the higher background readings in the rib steak (Fig. 5B).

In water samples spiked with E. coli O157:H7, the Phazyme assay had a detection limit of 10² CFU/100 ml (Fig. 5C). At this inoculation level, the RLU level was significantly higher (p = 0.0040) than phage-only controls. The Phazyme assay was unable to distinguish spiked from unspiked water samples at E. coli O157:H7 concentrations below 10² CFU/100 ml (p = 0.988).

Discussion

The host specificity of phages makes them ideal candidates for use as recognition elements in bacterial diagnostic assays. Several phage-based methods have been developed to effect rapid detection of bacteria. These methods include the creation of genetically modified reporter phages and detection methods in which unmodified phages infect their host and amplify themselves to a detectable level which is measured by an end point assay such as the plaque assay. Alternatively, phages can be used to specifically lyse their host bacteria, releasing intracellular components which can then be detected.

Another phage-based approach to bacterial detection is to directly label the infecting phage itself. The resulting phages can then specifically tag bacterial cells, in a manner similar to antibodies, and may then be detected by several methods including epifluorescence microscopy, flow cytometry, confocal microscopy and fluorescent plate readers. Hennes and coworkers were the first to utilize labeled phages to detect bacteria. In their work, fluorescently stained phages were used as probes to label, identify and enumerate specific strains of marine bacteria and cyanobacteria. The results of the study indicated that the fluorescently labeled phages could be effectively used to detect and quantify specific groups of bacteria within mixed microbial communities.¹⁹

Following up on this work, Goodridge and coworkers created fluorescently labeled phages that could detect E. coli O157:H7.¹⁸ In this approach, the double stranded DNA (dsDNA) contained within phage LG1 was labeled with the fluorescent nucleic acid stain YOYO-1™. Immunomagnetic separation was used to initially concentrate the E. coli O157:H7 cells, which were then immersed in a suspension of the labeled phage. The suspension was subsequently filtered onto a black membrane and viewed under an epifluorescence confocal microscope, which showed that the target E. coli O157:H7 cells had a “halo” like appearance, due to the bound fluorescent phages. When used in combination with flow cytometry, the assay was capable of detecting 10⁴ CFU/ml in pure culture. In spiked food, the assay had a detection limit of 2.2 CFU/g in artificially contaminated ground beef following 6 hours enrichment, while between 10¹ and 10² CFU/ml of artificially contaminated raw milk were detectable after a 10 hour enrichment step. Other researchers used similar methods to effect detection of other bacterial species. For example, Mosier-Boss and coworkers labeled the dsDNA of phage P22 and used it to detect Salmonella enterica serovar Typhimurium. Following phage infection, the researchers were able to visualize the labeled DNA inside of infected cells with the use of a fluorescent microscopic imaging system.²⁰ Similarly, several researchers combined the reporter phage concept and labeling of phages to create genetically modified labeled phages, which displayed the green fluorescent reporter protein on the capsid surface.²¹^,²² This technique allowed the detection of viable but non-culturable bacteria (VBNC) within one hour, if sufficient levels of bacteria were present.²¹^,²² Phages have also been labeled with quantum dots and used to detect bacteria.²³

One disadvantage of the above studies is the fact that they all required expensive instrumentation to detect the fluorescently bound phages. The use of such equipment, while allowing for extremely sensitive detection of bacterial cells, limits the use of such methods to a laboratory setting.

In this study, we attempted to address some of the limitations of previous labeled phage assays by creating several labeled phages by decoration of the external structural proteins of the phage with HRP, using a simple chemical process. The methodology was rapid and simple, and it is theoretically possible to label any phage using this technique. When compared to fluorescent labels, the use of an enzyme (HRP) to label the phages increased the sensitivity of the assay, especially when a luminescent substrate was used. Luminescent substrates have the widest dynamic range of all three (colorimetric, fluorescent and luminescent) classes of substrates.²⁴ The Phazyme assay also required minimal equipment to read the test results. The Snap Valve™ devices that house the reagents for each Phazyme assay are designed to fit into a number of hand-held luminometers, and these devices are already widely used in the food industry for hygiene monitoring.

STEC infection may result from the ingestion of contaminated food or water or may be associated with animal contact.²⁵ Reduction of food, water and environmental contamination by STEC is an important part of infection control, and detection of these pathogens in food and water samples represents the first step of an integrated control plan. In this study, Phazyme assays were created to detect E. coli isolates from STEC serogroups O26, O103, O111, O145 and O157. The rationale for focusing on these serogroups is due to the fact that STEC is associated with a wide spectrum of illness ranging from uncomplicated watery diarrhea to hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS), which may result in death and many studies have confirmed that serogroups O26, O103, O111, O145 and O157 are highly associated with HC and HUS.²⁶^–³¹

In pure culture, the direct detection limits of the Phazyme assays ranged from 6.82 × 10⁴ CFU/ml for serogroup O145 to 1.32 × 10⁶ CFU/ml for serogroup O111 (Fig. 2). The direct detection limits of 4 of the 5 serogroups (O26, O103, O145 and O157) were within 0.7 log units, ranging from 6.82 × 10⁴ CFU/ml for O145 to 3.85 × 105 CFU/ml for O103. The serogroup O111 Phazyme assay was the least sensitive of all of the assays, with a direct detection limit of 1.32 × 10⁶ CFU/ml. One of the reasons for the differences in sensitivity between the O111 Phazyme assay and the rest of the assays may be due to the fact that the phage that was used in the O111 assay, phage 56, adsorbed with the lowest efficiency to O111 cells, when compared to bacterial cells from other serogroups (Fig. 4). For example, after 20 minutes, 69% of phage 56 was adsorbed to the O111 host, which corresponds to almost 9% less binding efficiency than the next nearest phage (ARI binding to O103 cells; 77.9%). Phage CBA120 adsorbed with the best efficiency to its host O157 cells with an efficiency of 93.1%. The binding efficiency of each phage to its host cells is directly relevant to the sensitivity of the assay, since the higher amount of target cell-phage will increase the amount of HRP that is present for detection. The isolation of phages that adsorb with higher efficiency to STEC serogroup O111 cells should increase the direct sensitivity of the assay.

When an 8 hour enrichment step was included, the sensitivity of 4 of the 5 Phazyme assays (serogroups O26, O103, O111 and O157) was 10⁰ CFU/ml (Fig. 3). The one exception was the assay for serogroup O145, which was approximately 4 orders of magnitude less sensitive, with a detection limit of 10⁴ CFU/ml. The reason for this is unknown. The lack in sensitivity was not due to inefficient adsorption of phage to the O145 bacterial surface, since after 20 minutes, 81% of phage AR1 adsorbed to the O145 cells, which is similar to the adsorption rates of AR1 for the O26 and O103 cells, 82.6% and 77.9% respectively (Fig. 4). The lack of sensitivity may have been due to the fact that the O145 IMS beads did not seem to be as effective at concentrating the bacterial cells as the other serogroup-specific IMS beads. For example, when IMS was conducted on E. coli cells belonging to STEC serogroups O26, O103, O111 and O157, a visible agglutination of the beads (due to multiple bacterial cells binding to more than one bead in solution) was observed. However, when IMS was conducted on O145 cells, the visible agglutination was not observed (data not shown). If the O145 IMS beads are inefficient at concentrating the bacterial cells, this would result in less bacteria present that could be labeled with phage, which would explain the lower sensitivity of the O145 Phazyme assay.

Interestingly, the O157 Phazyme assay displayed a distinct hook effect (Fig. 3E). For any binding assay to give accurate results there must be an excess of the recognition element (RE) (antibodies, phages, aptamers, etc.,) relative to the analyte being detected. It is only under the conditions of RE excess that the dose response curve is positively sloped and the assay provides accurate quantitation. As the concentration of analyte begins to exceed the amount of the RE, the dose response curve will flatten (plateau) and with further increases may paradoxically become negatively sloped in a phenomenon termed the high concentration hook effect.³² Because the possibility exists that some samples may have analyte concentrations in excess of the RE, it is necessary to validate all samples by dilutional linearity analysis to establish if they are on the valid, positively sloped region of the curve or on the negatively sloped hook region of the curve. In this work, the fact that the labeling of the phages resulted in greater than a 1 log decrease in the concentration of infective phages, indicates that there is a high probability that the high concentration hook effect phenomena occurred at high bacterial concentrations. However, the hook effect only becomes an issue if the assay will be used to quantify the concentration of the target analyte. The Phazyme assays are designed to only detect and not quantitate STEC; as such, the hook effect should not constitute a problem as even the highest concentration of cells (103 CFU/ml) produced a signal that was much higher than the background (Fig. 3E).

The specificity of the O26, O103 and O157 Phazyme assays were observed to be greater than 93% (Table 3). The O111 and O145 assays were less specific with 82.4% and 75.9% of the bacteria tested correctly identified. The lower specificity observed for these assays is directly related to the host range of the bacteriophages used in these tests. For example, both phage AR1 and phage 56 attached to several non-STEC isolates (Table 2), and this contributed to the lower specificity. In the future, phages that have less broad host ranges will be isolated and used in Phazyme assays for STEC serogroups O111 and O145.

The results of the food trials showed that the O157 assay was capable of very sensitive detection of STEC O157 cells in a variety of food and water samples. The results of the inoculated beef studies demonstrated the effects that food matrices can have on detection sensitivity. For example, the background of the rib steak samples was much higher than the background of the top round steak samples, which was probably due to the high fat content on the rib steak. Although this did not affect the sensitivity of the test (the Phazyme assay detected 100 CFU/100 cm² in both samples), it is clear that the sample matrix must be taken into account when conducting the Phazyme assay.

Materials and Methods

Bacterial strains.

A total of 93 microbial strains (Tables 1 and and22) were used for specificity and sensitivity studies. The bacteria studied comprised 10 strains of E. coli O26, 5 strains of E. coli O103, 10 strains of E. coli O111, 5 strains of E. coli O145 and 33 strains of E. coli O157. In addition, seven non-typed Shiga toxin-producing E. coli (STEC) strains and 6 generic E. coli strains were included as negative controls. Seventeen other negative controls, representing 12 bacterial genera and 1 yeast strain, were also employed in testing. Stock cultures were maintained in 40% glycerol and kept frozen at −80°C until use. Fresh microbial cultures for use in experiments were produced by inoculating frozen stock cultures onto tryptic soy agar (TSA) (Difco, BD, 236930), and incubating the plates overnight at 37°C. For growth experiments, the inocula consisted of stationary-phase cells that were obtained by inoculating tryptic soy broth (TSB) (Difco, 211823) with cells from an overnight TSA plate and incubating the preparations overnight with shaking at 37°C.

Phages and host bacteria.

Three phages were used in this study. Phage CBA120 (infects STEC O157 isolates) was obtained from Dr. Betty Kutter at Evergreen State College in Olympia, Washington. Phages AR1 (infects O26, O103, O145 and O157 isolates) and 56 (infects O111 isolates) were from our phage collection. E. coli O157:H7 strain 920332 was used in the propagation of phages AR1 and CBA120. E. coli O111:H8 strain LG22 was used in the propagation of phage 56. The phages were propagated on their host bacteria by adding one ml of overnight cultured cells and 10 ml of phage (multiplicity of infection [MOI] = 10) to 500 ml of TSB followed by incubation at 37°C with shaking overnight. Ten ml of chloroform was added to the flask to release any progeny phage within the host cells, and the suspension was incubated with shaking at 37°C for an additional 10 minutes, followed by the addition of 29.2 g of NaCl (1 M concentration) and incubation on ice for 2 hours. The phage lysate was centrifuged for 10 minutes at 8,000x g and the pellet was discarded. Phages were isolated from the supernatant fluid by addition of 50 g of polyethylene glycol (PEG) 8,000 (10% w/v), followed by incubation on ice for 2 hours and centrifugation as described. The supernatant was discarded and the phage/PEG complexes were isolated from the sides of the centrifuge tube by resuspension in lambda diluent (pH 7.5). Following chloroform extraction of the PEG, the partially purified phage lysates were stored at 4°C until ready for use.

Production of Phazymes.

The phage lysates were removed from the refrigerator and ultracentrifuged at 247,355x g for 60 minutes in a Beckman SW41Ti rotor. Following ultracentrifugation, the pellet was resuspended in 500 µl of phosphate buffered saline (PBS) (pH 7.4) overnight at 4°C. The suspension was subsequently chloroform-treated and centrifuged for 15 minutes at 8,000x g to further separate any remaining bacterial debris.

The protein concentration of each phage suspension was determined using the BCA Protein Assay Kit (Pierce Biotechnology Inc., 23225). Approximately 200 µl (corresponding to 100 µg of protein) of each phage suspension (10⁸–10⁹ PFU/ml) was labeled with horseradish peroxidase (HRP) with the use of the Lightning-Link™ horseradish peroxidase (LL-HRP) kit (Innova BioSciences, 701-0002). HRP labeling was performed according to the manufacturer's instructions. At the conclusion of the labeling reaction, the Phazymes were diluted into 2 ml of PBS and stored at 4°C.

Completion of the Phazyme assay.

A total of 5 individual Phazyme assays were created, which corresponded to one Phazyme assay for each individual STEC serogroup. The Phazyme assays were developed based on the use of Snap Valve™ devices (Hygiena). Individual Phazymes, suspended in either TSB (AR1) or blocking buffer (0.1% (w/v) skim milk in PBS) (CBA120 and 56), were added to the cap of the device. The bottom of each Snap Valve™ device contained a sampling device (swab), selective growth media [TSB supplemented with cefixime (0.0125 mg/ml) and tellurite (0.625 mg/ml) (TSBCT)] and STEC (O26, O103, O111, O145 or O157) serogroup specific immunomagnetic separation (IMS) beads (Invitrogen, 710.13, 710.11, 710.09, 710.07, 710.04) (Fig. 1A). The IMS beads were added to each Snap ValveTM device, such that their specificity corresponded to the Phazyme in the cap of the device (i.e., O157 IMS beads were added to Snap Valves that contained the O157 specific Phazyme). To complete the Phazyme swab method, the swab was removed, the surface of the food to be tested was swabbed, and the swab was returned to the Snap Valve™ device, followed by 8-hour enrichment, with shaking at 37°C. Following enrichment, the IMS beads (with any STEC cells attached) were concentrated by placing the Snap Valve™ device into a portable device (Fig. 1B and E), that contained a magnet which attracted the IMS beads and any attached bacteria, allowing the growth media to be removed (Fig. 1B). Following one wash step with PBS Tween 20 (0.1% v/v), the cap of the Snap Valve™ device was broken, releasing the Phazyme (10⁴–10⁵ PFU/ml) into the bottom of the device, where it bound to any STEC cells present (Fig. 1C and D). Following incubation at 37°C for 20 minutes, the IMS beads (with any STEC/Phazyme attached) were concentrated and washed twice with PBS Tween 20 (0.1% v/v) (Fig. 1E). Finally, the substrate was added to the bottom of the device where it reacted with any Phazyme present to produce a signal (Fig. 1F). Two classes of substrates were evaluated, including a colorimetric substrate (1-Step™ Ultra TMB-ELISA, Pierce Biotechnology, Inc., 34028) and a luminescent substrate [SuperSignal^® ELISA Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Inc., 37074)]. For colorimetric detection, a positive test was determined visually and indicated by the development of a yellow color, while negative tests remained colorless. For luminescent detection, the Snap Valve™ devices were placed into a SystemSure II handheld luminometer (Hygiena, SS2) and luminescence (relative light units) was measured (Fig. 1G).

Figure 1

The Phazyme assay. The swab is removed, the surface to be tested is swabbed, and the swab is returned to the device, followed by an eight hour enrichment (A). Following enrichment, the IMS beads (with E. coli cells attached) are concentrated, the growth (more ...)

Evaluation of the Phazyme assay.

Each serogroup-specific Phazyme assay was evaluated for specificity (ability to correctly distinguish between STEC and non-STEC bacteria) and for sensitivity (determination of the lowest number of detectable cells). A total of 86 strains of bacteria and 1 yeast (Tables 1 and and22) were used to determine specificity and sensitivity of the developed assays. The specificity studies were conducted using bacterial growth inocula (10⁸–10⁹ CFU/ml) which consisted of stationary phase cells that were obtained by inoculating TSB with a single colony from an overnight TSA plate and incubating overnight with shaking at 37°C. Each bacterial strain was evaluated using the Phazyme assay by employing the methodology described above, with the exception that the enrichment step was replaced with an incubation step at room temperature for 25 minutes to allow time for the IMS beads to bind any cells present. Two controls were included for each bacterial strain tested. The controls consisted of a cell-only control (the Phazyme assay was conducted, but no Phazyme was added to the assay) and a Phazymeonly control (the Phazyme assay was conducted in the absence of bacteria). A positive luminescent result was determined statistically using a SAS Proc GLM to verify that samples producing at least 100 relative light units (RLU) were significantly higher (p < 0.05) than their cell-only and Phazyme-only control equivalents. Any sample that was determined to be a false positive was screened with the entire assay, including the eight hour selective enrichment in TSBCT.

Individual E. coli strains representing each STEC serogroup were used to determine the detection limit of the Phazyme assay. Two methods were used to evaluate the detection limit of the assays. In the first batch of experiments, the direct detection limit (the lowest number of cells that each Phazyme assay could detect without an enrichment step) was determined by serially (10-fold) diluting an overnight culture of each bacterial strain. For a given STEC serogroup, the dilutions of the corresponding strain were added to individual Snap ValveTM Phazyme devices, followed by incubation at room temperature for 25 minutes to allow time for the IMS beads to bind any cells present. The rest of the Phazyme assay was performed as described. Results were determined using generated luminescence and the RLU output was graphed against the corresponding bacterial concentration. A linear regression line of best fit was used to determine the lower detection limit of each assay.

In the second batch of experiments, the lower detection limit of each assay was determined by incorporating an 8 hour enrichment step in the assay. Each strain was enriched overnight in TSB, serially (10-fold) diluted in lambda diluent [100 mM NaCl, 8 mM MgSO₄·7H₂O, 50 mM Tris-HCl (pH 7.5)], and enumerated by spread plating the 10-fold serial dilutions onto TSA plates, followed by overnight incubation at 37°C. Following enumeration, 100 µl of each dilution was added to the corresponding Phazyme assay, which was completed as described above. For luminescent detection, measurements were taken in triplicate.

For both experiments, a positive luminescent result was determined statistically using a SAS Proc GLM to verify that samples were significantly higher (p < 0.05) than their Phazyme-only control equivalents. All data were analyzed via Analysis of Variance (ANOVA) to elucidate any possible differences. Additionally, each inoculation level was analyzed by t-test, which was then compared against one another and against the phage-only control for the specific serogroup. As being significantly different than the phage-only control was the threshold for determining a positive test result, all p-values reported were the t-test results against the phage-only control for the specific serogroup. Cell-only controls were not utilized as the specificity assay showed minimal levels of horseradish peroxidase enzyme activity in bacteria. For determination of the direct detection limit, the detection threshold was set at the Phazyme-only average plus 5 times the standard error.

Phage binding efficiency.

Adsorption assays were conducted in order to assess the ability of the phages to bind to their target bacteria. The assays, with minor modifications, were conducted according to the method described by Yoichi and coworkers.³³ Briefly, 5 exponential growth phase E. coli strains representing each STEC serogroup were diluted in TSB medium to a final cell concentration of 10⁷ CFU/ml. The bacterial cell cultures (10 ml) were pre-warmed at 37°C for 10 minutes and mixed with their corresponding phage (CBA120 for E. coli O157, AR1 for E. coli O26, O103 and O145, and 56 for E. coli O111) at a MOI of 0.1. The mixture was incubated at 37°C with shaking. Following the addition of phages, 500 µl aliquots of each mixture were removed at 5, 10 and 20 minute time points and the aliquots were centrifuged (17,400x g, 1 minute, 4°C). The phage titer of the supernatant was determined by plaque assay using an appropriate bacterial strain for each phage, and the phage titer at time 0 was defined as 100%.³³ The time course of the free phage concentration (i.e., the amount of phages remaining in solution over time) in the culture provided an adsorption rate constant (ka), which represented phage adsorption affinity toward the host cell.

Food and water trials.

The O157 Phazyme assay was evaluated for its ability to detect E. coli O157 in artificially contaminated samples including spinach, ground beef and water. E. coli O157:H7 strain 2 (EC2) (tet^R) was used in all experiments. For all experiments, 10-fold serial dilutions of an overnight stationary phase culture of strain EC2 were prepared in PBS and enumerated by plate count as described. Each sample to be tested was analyzed in triplicate.

For spinach samples, 10 ml aliquots of EC2 were added to 170 g (6 oz.) samples of store bought, pre-packaged baby spinach at final concentrations of 10⁻¹, 10⁰, 10¹ and 10² CFU/g. The inoculated spinach was shaken in a zip lock bag for 2–3 minutes and allowed to dry on absorbent paper for 2 hours in a biological safety cabinet. After the spinach was dried, three 25 g sub-samples were obtained for each dilution and placed in a filter stomacher bag (3 M, 6469), and 75 ml of TSBCT was added to each bag, which was homogenized in a Tekmar STO-400 stomacher (Tekmar, STO-400) on full speed for 2 minutes. The homogenized samples were incubated at 37°C without shaking for 8 hours. Following incubation, 1 ml of sample was removed from each bag and added to the bottom of the SnapValve™ Phazyme device, which contained 2 ml of TSBCT and 20 µl of O157 IMS beads. The sample was allowed to incubate at room temperature for 25 minutes to allow time for the IMS beads to bind any O157 cells present. The rest of the Phazyme assay was performed as described. In addition to Phazyme-only controls, a spinach only control was included which consisted of uninoculated spinach that was processed in an identical manner as the inoculated spinach samples.

To simulate beef carcass tissue, 100 cm² portions of beef (top round steak and rib steak) were inoculated by pipetting and spreading 1 ml of diluted E. coli O157:H7 strain EC2 over the entire surface of the meat with a glass spreader (hockey stick), to provide final bacterial concentrations of 10⁰, 10¹, 10², 10³ and 10⁴ CFU/100 cm². Three samples (two top round steak and one rib steak) were inoculated for each dilution. The meat was allowed to dry for one hour in a biohazard cabinet and the entire surface of each meat sample was swabbed with an individual device. The rest of the assay was performed as described. In addition to Phazyme-only controls, uninoculated meat controls were included for each type of steak.

Tap water was spiked with strain EC2 to provide final levels of 10⁰, 10¹, 10², 10³ and 10⁴ CFU/100 ml water. One hundred ml of each spiked water sample was added to 100 ml of double strength (2x) TSBCT and incubated at 37°C without shaking for 8 hours. Following enrichment, 2 ml of the culture was added directly to the bottom of the SnapValve™ Phazyme device which contained 20 µl of O157 IMS beads. The sample was allowed to incubate at room temperature for 25 minutes to allow time for the IMS beads to bind any O157 cells present. The rest of the Phazyme assay was performed as described.

Conclusions

In conclusion, the results of this study indicate that Phazyme assays were able to detect low levels of STEC in pure culture and artificially contaminated food and water samples following an 8 hour enrichment. The components of the entire assay system were combined in a single tube device and test results were read using a hand-held luminometer, which allowed the development of a rapid sensitive field-based detection system for STEC. Future research will focus on increasing the specificity of the O111 and O145 assays, and increasing the sensitivity of the O145 assay. Further studies will also focus on the development of Phazyme assays for other foodborne pathogens, including Salmonella spp. and Campylobacter jejuni. The Phazyme assay is an integrated field-based diagnostic that shows great potential to rapidly detect foodborne pathogens in the food processing environment with minimal need for equipment.

Acknowledgements

This research was supported by a USDA National Research Initiative grant (2005-01879) and a National Oceanic and Atmospheric Administration grant (NA07OAR170428), and a USDA Specialty Crop Research Initiative grant (2009-01208). The authors would like to thank Dr. Betty Kutter at Evergreen State College for supplying phage CBA120, Dr. Jennifer A. Ritchie at Harvard Medical School for supplying E. coli strains TEA026 and EDL933, and Katie Kessler in the University of Wyoming Meat Laboratory for supplying the meat samples.

Abbreviations


STEC	Shiga toxin producing Escherichia coli

HRP	horseradish peroxidase

Phazymes	enzyme-labeled phages

IMS	immunomagnetic separation

RLU	relative luminescence units

TSA	tryptic soy agar

TSB	tryptic soy broth

PEG	polyethylene glycol

MOI	multiplicity of infection

PBS	phosphate buffered saline

PFU	plaque forming units

TSBCT	TSB supplemented with cefixime and tellurite

ka	adsorption rate constant

VBNC	viable but non-culturable bacteria

RE	recognition element

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