Determination of the Sensitivity of a Rapid Escherichia coli O157:H7 Assay for Testing 375-Gram Composite Samples

Abstract

Both 25-g single-size ground beef samples and 375-g composite ground beef samples were tested by a method combining an immunomagnetic separation (IMS) technique with a sandwich enzyme-linked immunosorbent assay (ELISA) system (IMS-ELISA). The results demonstrated that IMS-ELISA could detect the target, Escherichia coli O157:H7, at the level of 10⁻¹ CFU/g of sample in either the 25- or 375-g sample size.

Escherichia coli O157:H7 has been the most frequently isolated serotype of enterohemorrhagic E. coli in North America (6, 19, 22). People infected with enterohemorrhagic E. coli often develop hemorrhagic colitis and/or hemolytic-uremic syndrome (12, 13, 20, 22); children, the elderly, and immunocompromised people are the most susceptible to this type of infection (10, 19). The sources of contamination include raw ground beef (3, 4, 6), unpasteurized milk, untreated water, and apple cider (2, 5), while person-to-person transmission is often observed in outbreaks. Among all of the possible contaminated food products, undercooked ground beef has been linked to most of the E. coli O157:H7 outbreaks. The U.S. food industry has responded to this emerging pathogen by conducting testing programs for E. coli O157:H7, especially in raw ground beef.

Most manufacturers of commercial detection systems have developed their assays based on a 25-g single sample in a 250-ml enrichment broth format. This sampling plan does not necessarily provide meaningful results for the large quantities of ground beef produced by wholesale meat suppliers unless a substantial number of assays are performed. A composite sampling plan combines randomly collected samples into one large composite sample and tests the entire unit at once. This statistical sampling plan is used extensively for Salmonella detection in the food industry (1). Instead of 15 25-g samples, testing a single 375-g composite sample provides an economic advantage to the industry.

A technique for cell separation using immunomagnetic beads has been developed for the detection of E. coli O157:H7 (7, 11, 18, 21). This relatively simple and rapid method uses iron-impregnated latex beads coated with an antibody against E. coli O157 which are added to samples that have already been enriched. After repeated washing and concentration, the majority of nontarget organisms can be removed, thus leaving the target, E. coli O157, in the sample container. In the case of raw ground beef, the target organism (E. coli O157) is often present at very low levels (<100 CFU/g), while the level of other organisms may be as high as 10⁴ to 10⁶ CFU/g. In this situation, the majority of the non-E. coli O157 organisms in the meat could serve as competitors which may prevent the growth of the target organism to the assay detection limit (e.g., 10⁶ CFU/ml after enrichment). To increase the sensitivity of the rapid detection assay for E. coli O157 in raw ground beef and to eliminate the inhibitory effect of potential competing meat organisms, immunomagnetic separation (IMS) is a favored technique to be applied to the detection system.

In this study, IMS was combined with a sandwich enzyme-linked immunosorbent assay (ELISA) system (IMS-ELISA) (14) to analyze E. coli O157:H7-inoculated raw ground beef in both 25-g single samples and 375-g composite samples and to determine the sensitivity of this detection system.

Raw ground beef samples were obtained from a local wholesale meat supplier. Aerobic plate counts (APC) and total coliform counts of the raw ground beef samples were obtained by the following method. First, 25 g of meat sample was homogenized in Butterfield's phosphate buffer (1:10) by a stomacher (Lab-Blender 400; Tekmar Co., Cincinnati, Ohio) for 2 min. One-milliliter aliquots of the homogenized samples were serially diluted and plated on plate-count agar and violet bile red agar (BBL, Becton Dickinson Microbiology Systems, Cockeysville, Md.) for APC and total coliform counts (16), respectively. The plates of both media were incubated at 35 to 37°C for 48 h. The reason for performing the coliforms counts was to obtain the concentration of enteric microorganisms that would possibly grow along with the target, E. coli O157:H7, in the biochemical selective media used for the confirmation.

A total of 50 single (25-g) and 25 composite (375-g) samples were aseptically weighed at the time the raw ground beef samples were received. Samples were diluted 1:10 in prewarmed (35 to 37°C) Trypticase soy broth (TSB) (BBL, Becton Dickinson Microbiology Systems) containing acriflavine (final concentration of 10 μg/ml) (TSB+a) (8).

A mixture of two E. coli O157:H7 strains was used for the inoculation. Isolate R100, from the culture collection of Silliker Laboratories of Ohio, Inc. (Columbus, Ohio) (isolated from an infected individual), and E. coli O157:H7 ATCC 35150, provided by Organon Teknika (Durham, N.C.), were grown separately in 10 ml of brain heart infusion (BHI) broth (BBL, Becton Dickinson Microbiology Systems) overnight at 37°C and then combined in equal volumes. The culture density was first estimated by direct microscopic count at the time of inoculation. The inoculation concentrations (Table 1) were achieved by serially diluting the culture in Butterfield's phosphate buffer. One milliliter of diluted culture solution was then added into the raw ground beef enrichment mixture, while the actual inoculated culture concentrations were confirmed by plating another 1 ml of the inoculum onto plate-count agar. The plates were incubated at 37°C for 24 h. After inoculation, the content of each enrichment meat sample was homogenized manually by thoroughly stirring it with a sterile plastic rod.

TABLE 1.

Experimental design

Sample size (g)	Sizes of E. coli O157:H7 inocula (CFU)/g of meat (CFU/container)	Incubation condition
25	0.04, 0.4, 1 (1, 10, 25)	Agitation (100 rpm)
25	0.04, 0.4, 1 (1, 10, 25)	Static
375	0.04, 0.4, 1 (15, 150, 375)	Static

The raw ground beef samples inoculated with E. coli O157:H7 at the designated concentrations were then incubated at 37°C for 6 h. For each incubation condition, 5 replicates of each inoculation concentration and 10 uninoculated samples were prepared. A positive control was also analyzed by inoculating E. coli O157:H7 in TSB+a without ground beef (Table 1). After 6 h of enrichment, 1.0 ml of sample broth was removed for IMS.

The IMS procedure was performed according to the manufacturer's (Dynal Inc., Lake Success, N.Y.) instructions and is briefly described here. First, E. coli O157-specific antibody-coated magnetic beads (Dynabead anti-O157) were prepared by pipetting 20 μl of immunomagnetic beads for each sample into a 1-ml microcentrifuge tube which was placed in a magnetic particle concentrator (MPC) (Dynal MPC-M). Beads were concentrated for 2 min at the inner wall of the microcentrifuge tube that was in contact with the magnet. The supernatant (containing preservation solution) was then removed and replaced with the same volume of bead washing solution (prepared by diluting the bead-washing buffer concentrate provided by the manufacturer 1:25, with storage at 2 to 8°C). The magnet on the MPC was removed and the prepared immunobeads were thoroughly mixed with the washing solution. Twenty microliters of the mixture was then distributed into each microcentrifuge tube. One milliliter of the 6-h enrichment sample broth was pipetted into the microcentrifuge tube containing the immunobeads and incubated at room temperature with agitation at 50 rpm for 30 min. After incubation, the magnetic concentration procedure was performed by placing the tubes in the MPC for 2 min; at the same time, the MPC was inverted several times to enhance the concentration of the immunobeads. At the end of the 2 min, the supernatant was discarded, followed by removal of the magnet from the MPC. The mixture was resuspended in 0.5 ml of TSB+a and incubated at 37°C overnight.

After overnight (10 to 12 h) incubation, 0.25 ml of the 0.5-ml IMS enrichment broth was used for the ELISA; the rest was saved for biochemical and serological confirmation. A sandwich ELISA system (EHEC-Tek; Organon Teknika, Inc.) was used to detect the presence of E. coli O157:H7 in ground beef samples. The ELISA procedures were performed according to the manufacturer's instructions.

Final confirmation for E. coli O157:H7 was determined by first streaking the unboiled IMS samples that were ELISA-positive onto sorbitol-MacConkey agar plates and incubating the plates at 42°C overnight (15). Up to 12 white colonies representing non-sorbitol fermenters were subcultured on a set of plates with eosin-methylene blue (EMB) (BBL, Becton Dickinson Microbiology Systems) or phenol red-sorbitol (PRS) with 4-methylumbelliferyl-β-d-glucuronide (MUG) agar (PRS-MUG) (15.0 g of phenol red broth base [BBL], 5.0 g of sorbitol, 20.0 g of Bacto agar [Difco Laboratories], and 0.05 g of MUG [Sigma Chemical Co., St. Louis, Mo.], all per liter) (17). Gridlines were drawn on the plates to create 12 areas. Suspected E. coli O157:H7 colonies picked from sorbitol-MacConkey agar were streaked onto the center of the EMB agar and stabbed into the corresponding section of a PRS-MUG plate. Both plates were incubated at 37°C overnight. Colonies on PRS-MUG plates were observed for sorbitol fermentation (with yellow being positive and pink being negative) and under UV light for MUG reaction (with fluorescence being positive and a lack of fluorescence being negative). EMB plates were observed for dark purple colonies with or without a green metallic sheen, the typical reaction of E. coli. Only those colonies demonstrating sorbitol-negative, MUG-negative characteristics on PRS-MUG and E. coli-positive traits on EMB agar were considered suspect for E. coli O157:H7 (9) and used for the subsequent serological confirmation.

Presumptive E. coli O157:H7 colonies were picked from PRS-MUG plates for the E. coli O157 latex test (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom). These colonies demonstrating true antibody-antigen agglutination were confirmed as E. coli O157 colonies. These colonies were then selected and grown in 3 ml of BHI broth at 37°C overnight for E. coli H7 serotyping. A 3-ml sample of 1.62% formalinized saline (1.62 ml of 37% formaldehyde in 100 ml of 85% NaCl solution) was added to the overnight BHI culture broth. A 0.5-ml aliquot of the formalinized culture broth was then mixed with an equal amount of 1.2% formalinized E. coli H7 antiserum (Difco Laboratories) and incubated in a 48°C water bath for at least 2 h. Samples demonstrating typical tube agglutination were confirmed as E. coli H7.

None of the uninoculated raw ground beef samples used in the study was determined to be positive for E. coli O157:H7. These data indicated that there was no naturally E. coli O157:H7-contaminated meat sample which could be identified by the IMS-ELISA system that would interfere with the interpretation of the results of inoculated samples.

The concentration of meat microflora played an important role in determining the sensitivity of the pathogen detection assay. The microflora counts of the meat samples used in this study were a mean APC of 4.1 × 10³ CFU/g and a mean total coliform count of 1.0 × 10² CFU/g.

Results of the IMS-ELISA tests for inoculated samples are summarized in Table 2. The actual culture concentrations presented in Table 2 were obtained from total plate counts for 1 ml of diluted culture, with the assumption that the concentration of culture inoculated into the meat enrichment broth was at the same log level as that plated onto plate count agar, since they were both taken from the same diluted culture solution. Regardless of the size of the sample and the incubation condition during the first 6 h of enrichment, true positive results were observed at inoculation concentrations of 3 × 10⁻¹ and 8 × 10⁻¹ CFU/g, where all five replicated samples under these inoculation conditions were positive by the IMS-ELISA and were confirmed both biochemically and serologically. Compared with a previous investigation that did not incorporate IMS into the assay, the detection limit of an E. coli O157 ELISA was approximately 10¹ CFU/g of raw ground beef sample (data not shown). The IMS procedure used in this study resulted in an increase in sensitivity approximately 100-fold above that of the ELISA.

TABLE 2.

IMS-ELISA results for ground beef samples inoculated with E. coli O157:H7

Sample size (g)	Agitation	CFU inoculated/g (CFU/container)a	No. of positive results (n = 5)	No. with confirmation (both O157 and H7)
25	No	0.25 (3)	3	2
25	No	0.32 (8)	5	5
25	No	0.84 (21)	5	5
25	Yes	0.25 (3)	2	1
25	Yes	0.32 (8)	5	5
25	Yes	0.84 (21)	5	5
375	No	0.032 (12)	3	2
375	No	0.032 (120)	5	5
375	No	0.8 (300)	5	5

^aAPC after plating 1 ml of inoculum 

Several E. coli O157:H7 IMS-ELISA-positive samples could not be confirmed by the methods employed in this study (only in samples with the lowest inoculation concentration). Comparing the concentration of inoculated target organism to the APC and coliform counts from the raw ground beef sample, there is reason to believe that the differences in inoculation concentration (2.5 × 10⁻¹ CFU of target organism versus 4 × 10³ APC and 1.0 × 10² total coliforms/g of meat sample) would prevent the target organism from becoming the predominant organism growing in the medium. This competition would later result in an unsuccessful isolation of target E. coli O157 colonies from the agar plates used for biochemical confirmation. Although IMS, by design, could reduce the concentration of non-E. coli O157, this study demonstrated that the concentration of organisms in the meat flora remained higher than that of the target organism at the level of at least 2 logs (unpublished data).

In conclusion, sample size (25-g single versus 375-g composite) and incubation status (shaken versus static) did not affect the sensitivity of the rapid assay for detecting the target organism (ca. 0.3 E. coli O157:H7 cells/g of sample). When testing high-microbial-content food samples, such as raw ground beef, for a single microorganism (e.g., E. coli O157:H7), the target/nontarget ratio becomes a critical factor for assay sensitivity. IMS is one alternative for removing nontarget organisms during enrichment without affecting the full potential for growth of the target organism, compared to highly selective enrichment media containing inhibitory agents (e.g., modified EC with novobiocin) (unpublished data). With today's heavy demands on testing ground beef for E. coli O157 by all meat producers, especially those applying hazard analysis and critical control point programs, a sampling plan using a 375-g composite sample should be considered.