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Detection of Escherichia coli O157:H7 in ground beef in eight hours
Journal of Microbiological Methods 34 (1998) 89–98
Journal of Microbiological Methods
Detection of Escherichia coli O157:H7 in ground beef in eight hours Christopher M. Gooding, Prabhakara V. Choudary* Antibody Engineering Laboratory and the Department of Entomology, University of California, Davis, CA 95616 -8584, USA Received 28 April 1998; received in revised form 10 July 1998; accepted 19 July 1998
Abstract A new method involving enrichment and immuno-polymerase chain reaction is presented for rapid and sensitive detection of Escherichia coli O157:H7 in ground beef. The bacteria in the spiked beef sample were enriched in a non-selective medium for 250 min and the E. coli O157:H7 cells were captured rapidly from the culture medium, using magnetic beads coated with E. coli O157-specific antibody. Partial stretches of sequences encoding Shiga toxins (Stx1 and Stx2) were amplified by the polymerase chain reaction and detected by agarose gel electrophoresis. Detection of a 215 / 212-base pair amplicon indicated contamination of the test sample with E. coli O157:H7. The method enabled detection of a single colony-forming unit of E. coli O157:H7 conclusively in 8 h. The suitability of the method to real life pathogen monitoring applications was demonstrated by accurate confirmation of E. coli O157:H7 in coded culture-positive samples of naturally contaminated ground beef and hamburger patties. 1998 Elsevier Science B.V. All rights reserved. Keywords: EHEC; Food safety; Hamburger; HACCP; Immuno detection; Infectious disease; PCR
1. Introduction Escherichia coli O157:H7 has been identified conclusively as a food pathogen in 1982 with the demonstration of its causal association with two outbreaks of hemorrhagic colitis, following the consumption of hamburgers (Riley et al., 1983; Wells et al., 1983). Escherichia coli O157:H7 has since been linked with many a foodborne bacterial outbreak in the USA, Canada and the UK and is now the most frequently isolated diarrheagenic type of E. coli in North America (Qadri and Kayali, 1998). An epidemic of E. coli O157:H7 with over 6000 cases has *Corresponding author. Tel.: 11-530-752-5563; fax: 11-530752-1537; e-mail: [email protected]
occurred recently in Japan (Izumiya et al., 1997; Swinbanks, 1996; Watanabe and Ozasa, 1997). In 1996, an E. coli O157:H7 outbreak associated with cooked beef pies has caused the death of 16 people in Scotland (Coia et al., 1998). Most E. coli O157:H7 outbreaks have been associated with the consumption of undercooked beef or raw milk (Centers for Disease Control, 1997; Cieslak et al., 1997; Keene et al., 1997). To address epidemiological issues and develop effective management strategies such as HACCP (hazard analysis critical control points) system for the prevention and control of this pathogen, a rapid and sensitive method of identification is required (Chapman et al., 1997). Traditionally, selective microbiological culture
0167-7012 / 98 / $ – see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S0167-7012( 98 )00070-0
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techniques are the primary methods of isolation and identification of E. coli O157:H7, with confirmation obtained from biochemical and serological tests (Johnson et al., 1995). Recently, however, there has been a surge of interest in more rapid and sensitive techniques, especially the polymerase chain reaction (PCR). Using a thermostable DNA polymerase and specific oligonucleotide primers in an iterative cycle of DNA synthesis, PCR amplifies a target stretch of DNA sequence exponentially. PCR procedures of multiple formats and combinations have been developed and used by several investigators for the detection of E. coli O157:H7 (Fields et al., 1997; Meng et al., 1997; Scheu et al., 1998). Gannon et al. (1992) had used Stx 1 and Stx 2 genes (previously known as Shiga-like toxin, SLT-I or verotoxin, VT1 and SLT-II or VT2, respectively) as templates in PCR to detect 1 cfu of E. coli O157:H7 in 1 g of ground beef in 15 h. It involved a 6-h culture enrichment step, followed by a 9-h step of isolation of DNA, PCR and agarose gel electrophoresis. Others have used immunomagnetic separation (IMS) for rapid isolation of E. coli O157:H7 from enrichment broth (Blanco et al., 1996; Weagent et al., 1995). Commercial tests with comparable sensitivities have also become available. Each of these methods has individual merits over the culture methods, but they are still too time-consuming for wide application in effective prevention and control strategies. For instance, the Tecra E. coli O157:H7 kit with 2-h assay time requires 18-h pre-enrichment (Flint and Hartley, 1995). The Ampcor Diagnostics MicroScreen test kit with 15-min detection time requires a minimum enrichment of 20 h (Czajka and Batt, 1996). Similarly, several PCR-based methods are also making their debut in the market. The Bax system from Qualicon is one such method without IMS, but requires enrichment overnight. Thus, there is a strong need for a sensitive and specific method that can be completed within a working day (Gooding and Choudary, 1997). To address this gap, we devised and optimized an approach using a combination of the above-described steps as modules of an integrated procedure. It can be readily assembled in a desired format to fit a specific contamination situation, by combining only the required modules. The suitability of the method
for contamination-monitoring purposes was demonstrated by accurate detection of E. coli O157:H7 in coded, culture-positive beef samples from previous outbreaks. This paper reports the method development, optimization and evaluation.
2. Materials and methods
2.1. Strains, media and growth conditions Twenty-four strains of bacteria were tested in this study as listed in Table 1. Ten strains were E. coli O157:H7, one was E. coli O111, and the rest were non-E. coli control bacteria. Only E. coli O157:H7 carried the Stx genes. The bacterial stocks were stored frozen at 2 708C in broth containing 15% glycerol and resuscitated by plating on agar (1.5%, w / v) plates and incubating overnight at optimal temperatures. E. coli O157:H7 stocks were streaked on sorbitol MacConkey (SMAC) (March and Ratnam, 1986) agar plates and incubated overnight at 378C, and the non-E. coli control strains on TYS (tryptone, 1%; yeast extract, 1%; sodium chloride, 0.5%) agar plates and incubated overnight on a lab bench (25–288C). Different strains of bacteria were each cultured in 20 ml of broth in a 250-ml Erlenmeyer flask inoculated with a single colony from the agar plates and shaking at 378C at 120 rpm on a gyrotory shaker (Model G24; New Brunswick Scientific, Edison, NJ, USA). Tryptose soy broth (TSB; DIFCO, Detroit, MI, USA) or modified EC broth (mEC; DIFCO, Detroit, MI, USA) with or without novobiocin (20 mg l 21 ) (Sigma Chemical, St. Louis, MO, USA) was used for growing E. coli strains and TYS broth for the rest. The identity of E. coli O157:H7 was confirmed by spread-plating on sorbitol MacConkey agar, containing cefimide (0.05 mg / l) and potassium tellurite (2.5 mg / l) (SMACCT) (Zadik et al., 1993). The non-E. coli control strains, CG1, CG2 and CG4, were grown at 308C, and CG5 at 378C in Brain Heart Infusion broth (DIFCO, Detroit, MI, USA). Serial dilutions of these cultures prepared in broth were used as inoculum to spike the ground beef samples. The inoculum size (cfu / ml) was determined by counting the number of colonies obtained from plating a portion (100 ml) of each dilution on SMAC
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Table 1 List of the bacterial strains used in the study, their Stx gene status and sources UCD number
Strain
Stx genotype
Original source
HF 1 HF 2 HF 3 HF 4 HF 5 HF 13 HF 14 HF 15 HF 51 HF 52 J5 CG 1 CG 2 CG 3 CG 4 CG 5 CG 6 CG 7 CG 8 CG 9 S 36 CG 10 CG 11
Escherichia coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O111 Aeromonas hydrophila Bacillus cereus Citrobacter freundii Enterobacter cloacae Enterococcus faecalis Hafnia alvei Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa Salmonella enteritidis Shigella sonnei Staphylococcus aureus
1&2 1&2 0e 2 1 NK NK NK 2 1&2 0 0 0 0 0 0 0 0 0 0 0 0 0
ATCC a 42895 ATCC 43894 ATCC 43888 ATCC 43889 ATCC 43890 CMDL b (Beef) CMDL (Beef) CMDL (Beef) USUHS c USUHS NK d ATCC 7965 ATCC 14579 ATCC 8090 ATCC 13047 ATCC 19433 ATCC 29926 ATCC 13883 ATCC 13315 ATCC 10145 ATCC 13076 ATCC 29930 ATCC 12600
a
ATCC, American Type Culture Collection, Rockville, MD. CMDL, California Department of Health Services Microbial Diseases Laboratory, Berkeley, CA. c USUHS, Uniformed Services University of Health Sciences, Bethesda, MD. d NK, not known. e 0, Stx gene(s) absent. b
agar plates and incubating at 378C. Unspiked beef samples were included as negative controls. To examine the effect of cold-stress (48C) on the viability and recovery of E. coli O157:H7, cultures with different inoculum sizes were each grown in TSB / mEC in Erlenmeyer flasks, shaking at 120 rpm for 7 days at 48C, followed by overnight at 378C and were tested using the new assay. The growth rates of the test cultures were monitored as a function of the A 600 values of the cultures at various time points.
a decimal serial dilution (in the range of 10 6 to 10 9 ) of each test culture in TSB (100 ml), using a pipetman (P-200) tip. The inoculated meatballs, in triplicates, were held at room temperature for 1 h to allow the inoculum to diffuse and were then frozen at 2 208C for 24 h or until further processing.
2.2. Experimental contamination of ground beef samples with E. coli 0157: H7
The frozen meatballs were each transferred aseptically to the mesh insert of a stomacher bag (Applied Biosolutions, New York, NY, USA) and thawed at room temperature for 30 min. Then, TSB (225 ml) was added to each bag, and the meatballs were homogenized for 1 min at room temperature in a stomacher (Tekmar, Cincinnati, OH, USA). The beef homogenate was incubated in the bag at 378C for
Ground beef (20% fat), purchased from a local grocery, was divided aseptically into 25-g portions, individually patted into balls and placed in an ethanol-sterilized plastic box. The meatballs were each inoculated in the center (ca. 1.5-cm deep) with
2.3. Pre-enrichment of the bacteria present in the spiked ground beef
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250 min, without shaking. The controls were processed without the 250-min incubation step.
2.4. Immunocapture of E. coli O157: H7 cells After the 250-min enrichment step, 1 ml-samples of each culture were removed from the stomacher bag and mixed with 20 ml of Dynabeads anti-E. coli O157 (Dynal, Lake Success, NY, USA) in a 1.8-ml microfuge tube. The tubes were shaken gently (150 rpm, Vortex Genie II, Fisher Scientific, Pittsburgh, PA, USA) for 30 min at room temperature, and the culture medium was aspirated, holding the beads in place using a magnetic particle concentrator (Promega, Madison, WI, USA). The beads were washed 3 3 with phosphate-buffered saline (PBS; 0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.4) containing 0.05% Tween 20. As a negative control, a 1-ml portion of the enriched culture was centrifuged in a microfuge at 14 000 3 g for 2 min, the supernatant was removed, and the PCR reaction mixture added directly to the pellet. The immunocapture step was optimized with respect to the incubation time, capacity and saturation. For optimization, the parameter under test was examined in a selected range, maintaining the rest constant. The optimal bead concentration was determined by incubating 1 ml-samples of each enriched culture for 30 min with 10, 20 or 50 ml of Dynabeads Anti-E. coli O157, washing and subjecting to PCR, followed by gel electrophoresis. To determine the optimal length of incubation time, the tubes containing immunobeads and the meat homogenate were shaken gently at room temperature for 15, 30, 45 or 60 min, washed and subjected to PCR, followed by gel electrophoresis. To estimate the capacity of the beads to capture E. coli O157:H7 cells, different dilutions of E. coli O157:H7 in TSB were treated with the beads as described above, and plate counts of the residual bacteria in the supernatant were determined.
2.5. PCR The PCR amplifications were carried out as described earlier (Gooding and Choudary, 1997), using a primer set derived from the Shiga toxin genes, Stx 1
and Stx 2 (Paton et al., 1993). This primer pair amplifies a 215-bp from the 586–800-bp stretch of the Stx 1 coding sequence and / or a 212-bp segment off the 583–794 bp portion of the Stx 2 coding sequence, respectively. Paton et al. (1993) designed these primer sequences originally and demonstrated their E. coli O157:H7-specificity in the PCR examination of human fecal cultures. The primers, with a GC content of 34% and a T m of 568C, were as follows: Forward: 59 . ATACAGAG(GA)G(GA)ATTTCGT , 39 Reverse: 59 . TGATGATG(AG)CAATTCAGTAT , 39 Amplification of the target bacterial DNA (Stx 1 and / or Stx 2 sequences) was performed in 0.5-ml PCR tubes (Fisher Scientific, Pittsburgh, PA, USA) using 100-ml total reaction volumes, containing 20 ml of the Dynabeads-bound bacteria as the source of template DNA. The reaction mixture consisted of (final concentrations): 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl 2 , 1 ml / l Triton X-100, 200 mM each of the deoxynucleoside triphosphates (Pharmacia Biotech, Piscataway, NJ, USA), 1.5 U of Taq polymerase (Promega, Madison, WI, USA) and 0.5 mM each of the primers, overlaid with 70 ml of sterile mineral oil (Sigma Chemical, St. Louis, MO, USA). The PCR was carried out in a programmable thermocycler (Model PTC150; M.J. Research, Watertown, MA, USA) using the following conditions: an initial cell lysis-cum-denaturation step at 958C for 5 min, followed by 35 cycles of denaturation at 928C for 1 min, primer annealing at 508C for 1 min and primer extension at 728C for 1 min. Experimental parameters of the PCR were each optimized in a separate set of experiments. For optimization of Mg 21 concentration, MgCl 2 was varied between 0.5 and 2.5 mM, with 1 to 100 target bacterial cells as template source, and the size and intensity of the amplicons were monitored and compared for all the concentrations of MgCl 2 tested. Using 1.5 mM MgCl 2 in the assay, the sensitivity of PCR was analyzed in terms of the maximum number of cycles required to yield a detectable PCR
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product from a minimal number of target bacterial cells. A target range of 2 to 165 cells was evaluated in 30, 40 or 50 cycles. The specificity of the primers was ascertained by testing a panel of target and non-target bacterial strains for PCR products of the expected size (215 / 212 bp). The products of PCR amplification from each reaction were analyzed by agarose gel electrophoresis (Sambrook et al., 1989). Typically, 10 ml of the amplicons were mixed with 2 ml of the loading solution with marker dyes (Sigma Chemical, St. Louis, MO, USA) and resolved by electrophoresis for 30 min at a constant power of 100 V in a 1.5% horizontal slab agarose (FMC Bioproducts, Rockland, ME, USA) gel in Tris–acetate–EDTA buffer (Sambrook et al., 1989), containing 0.5 mg / ml ethidium bromide (Sigma Chemical, St. Louis, MO, USA). HaeIII-digested fX 174 RF DNA (Life Technologies, Gaithersburg, MD, USA) was used as markers for DNA size. The DNA bands in the gels were visualized by UV transillumination and were photographed using a Polaroid camera system (Photo / PrepI, Fotodyne, Hartland, WI, USA). Each experiment was repeated twice with two replicates of each sample.
2.6. Determination of sensitivity limits of the method The detection limits of the method were established by assaying for the target-specific PCR amplification products of the Stx sequences (215 / 212 bp) from a range of inoculum sizes of the E. coli O157:H7 strains. The strain HF1 was tested in the range of 2 to 10 4 cfu / g of sample (Fig. 1), and HF4, HF5 and HF14 were tested in the range of 0.1 to .10 4 cfu / g sample (Table 2).
2.7. Detection of E. coli O157: H7 in naturally contaminated beef Five coded frozen samples (MDL001-MDL005) of naturally-contaminated beef from previous outbreaks of E. coli O157:H7 (Table 3) were obtained from the California Department of Health Services
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Fig. 1. Agarose gel electrophoresis of the immuno-PCR products of Stx 1 and Stx 2 genes from ground beef samples, spiked with Escherichia coli O157:H7. Ground beef samples (25 g) were each inoculated with 100 ml of serial dilutions, each comprising a different inoculum size (ranging from 2 to 10 4 cfu / g sample) of E. coli O157:H7 strain HF1 (lanes 1 to 5) and enriched for 250 min as described in the text. The E. coli O157:H7 cells were concentrated by immunomagnetic separation, and the Stx gene sequences were amplified by the polymerase chain reaction and analyzed by agarose (1.5%) gel electrophoresis. The appearance in the gel of the expected 215 / 212-base pair band (as indicated on the side) was taken as positive indication for contamination of the investigated food sample with E. coli O157:H7. Lane M, DNA size markers: fX 174 RF DNA HaeIII fragments (bp, from top to bottom: 1,353; 1,078; 872; 603; 310; 281 / 271; 234; 194; 118; 72); Lanes 1 to 5, samples: lane 1, unspiked control sample; lane 2, sample spiked with 2 cfu / g; lane 3, 30 cfu / g; lane 4, 400 cfu / g; lane 5, 3310 4 cfu / g; lane 6, negative control (no DNA or E. coli O157:H7 cells added); lane 7; positive control (HF1 culture, 10 ml).
Table 2 Sensitivity limits of the immuno-PCR procedure for detecting three different strains of Escherichia coli O157:H7 in spiked ground beef Inoculum size
Escherichia coli O157:H7 strain
Cfu / g Ground beef
HF14 (Stx status unknown)
HF4 (Stx 2 )
HF5 (Stx 1 )
.10 000 1000–10 000 100–1000 10–100 1–10 .0.1,1 0
1 1 1 1 1 NT 2
1 1 1 1 1 1 2
1 1 NT 1 1 2 2
Cfu, colony forming units; 1, PCR-positive; 2, PCR-negative; NT, not tested.
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Table 3 Culture and enrichment-immuno-PCR detection of Escherichia coli O157:H7 in naturally contaminated beef Code [
CMDL culture results a
Enrichmentimmuno-PCR (6 h)
Enrichmentimmuno-PCR (250 min)
Fig. 2 gel lanes
MDL 001 MDL 002 MDL 003 MDL 004 MDL 005
1 1 1 2 2
1 1 1 2 2
1 1 2 2 2
14, 15 12, 13 10, 11 8, 9 6, 7
a
CMDL samples were cultured using standard non-selective enrichment, followed by plating, colony picking and biochemical identification.
Microbial Diseases Laboratory (CMDL), Berkeley, CA. These included two hamburger patties (MDL001, MDL002), two ground beef samples (MDL003, MDL005) and one meat ball (MDL004). Three samples (MDL001, MDL002 MDL003) had been found to be positive and two (MDL004, MDL005) negative for E. coli O157:H7, using standard culture techniques (CMDL, unpublished). The samples were homogenized in the stomacher, enriched for 250-min and 6-h separately and subjected to immunocapture, followed by PCR amplification. The PCR products were analyzed by agarose gel electrophoresis, as described.
3. Results and discussion The aim of this work was to develop and optimize an immuno-PCR method for detecting E. coli O157:H7 in ground beef with reduced overall assay time, but without compromising the specificity or sensitivity. We accomplished this goal by devising a method incorporating a combination of the steps: enrichment of the pathogen (250 min), selective capture of the pathogen using IMS (30 min), amplification of the pathogen-selective DNA sequences using PCR (110 min), and detection of the amplified DNA by agarose gel electrophoresis (30 min); approximately 60 min was spent in making various preparations for the total procedure. Our hypothesis, that optimal efficiency can be achieved by a synergistic combination of these steps, was substantiated by the considerably shortened assay time of the new method (8 h).
3.1. Pre-enrichment of the culture The pre-enrichment step helps to increase the target bacterial cell number to or above a minimum detection threshold. Conventionally, the enrichment step is carried out arbitrarily for periods of up to 48 h, without paying particular attention to the number of target bacteria required. This prolongs the assay time. Further, it is also customary to use media that selectively suppress the growth of non-target microorganisms. However, selective media slow down the growth and impede the recovery of the target organisms too, especially of the stressed cells (McCleery and Rowe, 1995) and result in longer assay times. Our results showed that by using media (TSB) without any selection, E. coli 0157:H7 could be enriched from a single cfu to .800 cfu within 250 min, sufficient for the production of a detectable PCR product. The generation time for HF14 was 26 min in TSB, 43 min in mEC and 55 min in mEC with novobiocin. However, cold-stressed cells grew in TSB with a generation time of 28 min after an initial lag period of 20 min and failed to grow in mEC with novobiocin. It was also apparent from our results that co-enrichment of non-target microorganisms does not negatively impact either the growth rate of E. coli 0157:H7, its selective capture by IMS or PCR (data not shown). We surmise that the enrichment period used in our procedure was too short for the competing non-target bacteria to effectively deprive the target bacteria of nutrients. The use of a non-selective growth medium and limiting the culture enrichment step to a defined period together were the key to achieving considerable reduction in the overall assay time of E. coli
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O157:H7 detection using our method, relative to the previously published methods.
3.2. Immunomagnetic separation Immunocapture using IMS offers a considerably faster means of collecting cells than centrifugation and saves a lot of time in isolating, washing, collecting and concentrating bacterial cells from food samples (Chapman et al., 1997; Bennett et al., 1996). More importantly, it eliminates matrix effects, including potential PCR-inhibitors from food and enrichment media (Weagent et al., 1995; Lantz et al., 1994). For these advantages, we chose to optimize and use IMS in our method. A 30-min incubation of the beads with enriched culture (1 ml) yielded a PCR product of the same intensity as that obtained after 45 or 60 min. However, insufficient product was obtained from the sample with 15-min incubation (data not shown). We found that just 20 ml IMS beads could recover 80% of the total E. coli O157:H7 population from the enrichment culture containing 10 9 cfu, 95% of 10 7 cfu, over 99% of 10 4 , and 100% of 10 2 cfu / ml. It was clear from these results that 20 ml IMS beads were sufficient to capture the minimum number of E. coli O157:H7 cells required for obtaining a PCR product detectable reproducibly by agarose gel electrophoresis.
3.3. PCR conditions The specificity of the primers was ascertained by testing them against a panel of target and non-target bacterial strains (Table 1). PCR products of the expected size were only obtained with E. coli strains of the O157:H7 serotype, carrying either copy of the Stx 1 or Stx 2 genes (Table 2), but not with nonpathogenic species E. coli (free from the Stx genes) or with non-E. coli (control) bacteria tested (data not shown). Detection of non-target O157 serovars and Shigella strains carrying Stx genes can be eliminated by substituting beads coated with H7-specific antibodies (He et al., 1996) for the Dynal anti-E. coli O157 beads used in this study. The use of primers derived from the flagellar H7 antigen gene (Gannon et al., 1997), the plasmid-borne catalase-peroxidase gene (KatP) (Brunder et al., 1996) or the hemolysin
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gene of E. coli O157:H7 (Schmidt et al., 1996) in PCR can also potentially restrict detection specifically to E. coli O157:H7. Most PCR protocols include a step of lysing the bacterial cells to make the genomic DNA accessible to primers in PCR. In our method, we lysed the bacteria directly in the PCR reaction by heating at 958C for 5 min. Our results corroborate the demonstration by Begum and Jackson (1995) of Stx 2 sequence amplification directly from ground beef samples inoculated with verotoxigenic E. coli, eliminating the need to isolate E. coli from the food samples or purify whole cell DNA for the PCR. Matrix effects are a serious problem in direct application of PCR to the analysis of food samples (Weagent et al., 1995; Lantz et al., 1994). One potential problem is chelation of free magnesium ions required for PCR. Therefore, we investigated the influence of magnesium (MgCl 2 ) concentration, in the range of 0.5 to 2.5 mM, on PCR in a titration experiment, with 1 to 100 target bacterial cells as template source. An amplicon of the expected size (215 / 212 bp) and intensity was obtained at all the concentrations of MgCl 2 above 1 mM, with the band intensity being highest in the 1.5 to 2.5 mM range. Hence, we used 1.5 mM MgCl 2 in all the subsequent assays. In efforts to establish the lower limits of the sensitivity of PCR we found a 30-cycle reaction to need a minimum threshold of 18 cfu and the 40cycle reaction 2 cfu. After 50 cycles, however, a second smaller band appeared, possibly of primer dimers. Therefore, it appeared desirable to keep the number of cycles to a minimum. Evidence from a different set of experiments that enabled detection of 1 cfu of E. coli O157:H7 also suggested the optimal number of PCR cycles to be 35 under the standard reaction conditions used in our method (data not shown).
3.4. Sensitivity of the method In determining the detection limits, the method was found to be sensitive to a pre-enrichment titer of 1 cfu of the HF4, HF5 and HF14 strains E. coli O157:H7 per gram meat, regardless of the strain variations (Table 2). Fig. 1 shows in all the positive test samples a direct correlation between the intensity
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of the signal of the PCR product and the preenrichment inoculum size (2 to 10 4 cfu / g sample). The 215 / 212-bp band in the agarose gel (Fig. 1) clearly shows a proportionate increase in signal strength from the inoculum size of 2 cfu / g sample (Fig. 1, lane 2) to 10 4 cfu / g sample (Fig. 1, lane 5). The negative control, with no E. coli O157:H7 cells or DNA, yielded no PCR product (Fig. 1, lane 6). These results demonstrate the sensitivity and specificity of the method and its reproducibility at different contamination levels.
3.5. Detection of E. coli O157: H7 in naturally contaminated beef Finally, the suitability of the method for field applications was demonstrated by its ability to accurately identify coded real-life samples of hamburgers and beef patties, collected from previous E. coli O157:H7 outbreaks. Using the standard 250-min enrichment the method could correctly identify all samples (Table 3) but MDL 003 (Fig. 2, lane 11), which needed a 6-h enrichment (Fig. 2, lane 10). We suspect that severe stress of the low original inoculum during collection or transit (including multiple freezings and thawings after collection) could be one reason (McCleery and Rowe, 1995; Clavero and Beuchat, 1996; Sutherland et al., 1995). The experiment could not be repeated for lack of a duplicate sample. However, in special situations such as this
where the sample is suspected to be extraordinarily stressed, false negative results could be avoided by increasing the enrichment time to 6 h.
3.6. Conclusion In conclusion, by combining the steps of preenrichment, IMS and PCR, we have created a rapid method for detection of E. coli O157:H7 in both artificially and naturally contaminated beef samples. Using this method, we project detection to a preenrichment inoculum of 1 cfu of E. coli O157:H7 in 1 g of spiked ground beef in 8 h. The demonstrated rapidity, sensitivity, specificity and reproducibility of the method should prove valuable for the public health laboratories, regulatory agencies and beef industry in using it as a high-throughput early warning screening system and in implementing the HACCP plans.
Acknowledgements We thank Rita Brenden, Ray Bryant and Greg Inami of the California Department of Health Services Microbial Diseases Laboratory for providing the naturally contaminated beef samples, data from standard culture techniques and constructive comments. Thanks are also due to Jennie Hunter-Cevera
Fig. 2. Gel electrophoretic analysis of the Stx DNA sequences amplified by immuno-PCR of ground beef samples, naturally-contaminated with Escherichia coli O157:H7 (samples received from CA Department of Health Services Microbial Diseases Laboratory). The experimental details were as in Fig. 1 legend. Lanes 1,16: DNA Mr markers; lanes 2,3: positive controls; lanes 4,5: negative controls; lane 6: sample MDL005 enriched for 6 h; lane 7: MDL005 enriched for 250 min; lane 8: MDL004 enriched for 6 h; lane 9: MDL004 enriched for 250 min; lane 10: MDL003 enriched for 6 h; lane 11: MDL003 enriched for 250 min; lane 12: MDL002 enriched for 6 h; lane 13: MDL002 enriched for 250 min; lane 14: MDL001 enriched for 6 h; lane 15: MDL001 enriched for 250 min.
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and Sharon Shoemaker for helpful discussions. This research was supported by a grant (CHOUD941900) from the California Department of Justice and the Office of the Alameda County District Attorney. The contents of this paper are subject to a UC patent application (UC 97-019).
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Flint, S.H., Hartley, N.J., 1995. Evaluation of the TECRA Escherichia coli O157 visual immunoassay for tests on dairy products. Lett. Appl. Microbiol. 21, 79–82. Gannon, V.P.L., King, R.K., King, J.Y., Goldstein-Thomas, E.J., 1992. Rapid and sensitive method for the detection of Shigalike toxin producing Escherichia coli in ground beef using the polymerase chain reaction. Appl. Environ. Microbiol. 58, 3809– 3815. Gannon, V.P., D’Souza, S., Graham, T., King, R.K., Rahn, K., Read, S., 1997. Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains. J. Clin. Microbiol. 35, 656–662. Gooding, C.M., Choudary, P.V., 1997. A rapid and sensitive immunomagnetic separation-polymerase chain reaction method for the detection of Escherichia coli O157:H7 in raw milk and ice cream. J. Dairy Res. 64, 87–93. He, Y., Keen, J.E., Westerman, R.B., Littledike, E.T., Kwang, J., 1996. Monoclonal antibodies for detection of the H7 antigen of Escherichia coli. Appl. Environ. Microbiol. 62, 3325–3332. Izumiya, H., Terajima, J., Wada, A., Inagaki, Y., Itoh, K.I., Tamura, K., Watanabe, H., 1997. Molecular typing of enterohemorrhagic Escherichia coli O157:H7 isolates in Japan by using pulsed-field gel electrophoresis. J. Clin. Microbiol. 35, 1675–1680. Johnson, R.P., Durham, R.J., Johnson, S.T., MacDonald, L.A., Jeffrey, S.R., Butman, B.T., 1995. Detection of Escherichia coli O157:H7 in meat by an enzyme-linked immunosorbent assay EHEC-Tek. Appl. Environ. Microbiol. 61, 386–388. Keene, W.E., Hedberg, K., Herriott, D.E., Hancock, D.D., McKay, R.W., Barrett, T.J., Fleming, D.W., 1997. A prolonged outbreak of Escherichia coli O157:H7 infections caused by commercially distributed raw milk. J. Inf. Dis. 176, 815–818. Lantz, P.-G., Hahn-Hagerdal, B., Radstrom, P., 1994. Sample preparation methods in PCR-based detection of food pathogens. Trends Food Sci. Technol. 5, 384–389. March, S.B., Ratnam, S., 1986. Sorbitol-MacConkey medium for the detection of Escherichia coli O157:H7 associated with haemorrhagic colitis. J. Clin. Microbiol. 23, 869–872. McCleery, D.R., Rowe, M.T., 1995. Development of a selective plating technique for the recovery of Escherichia coli O157:H7 after heat stress. Lett. Appl. Microbiol. 21, 252–256. Meng, J., Zhao, S., Doyle, M.P., Mitchell, S.E., Kresovich, S., 1997. A multiplex PCR for identifying Shiga-like toxin-producing Escherichia coli O157:H7. Lett. Appl. Microbiol. 24, 172– 176. Paton, A.W., Paton, J.C., Goldwater, P.N., Manning, P.A., 1993. Direct detection of Escherichia coli Shiga-like toxin genes in primary faecal cultures by polymerase chain reaction. J. Clin. Microbiol. 31, 3063–3067. Qadri, S.M., Kayali, S., 1998. Enterohemorrhagic Escherichia coli: a dangerous food-borne pathogen, Postgrad. Med. 103, 179–180; 185–187. Riley, L.W., Remis, R.S., Helgerson, S.D., McGee, H.H., Wells, G.J., Davis, B.R., Hebert, R.J., Olcott, E.S., Johnson, L.M., Hargrett, N.T., Blake, P.A., Cohen, M.L., 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype. New Engl. J. Med. 208, 681–685.
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Sambrook, J., Fritch, E.F., Maniatis, T. (Eds.), 1989. Agarose gel electrophoresis. In: Molecular Cloning: a Laboratory Manual, 2nd ed., Vol. 1, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 6.3–6.19. Scheu, P.M., Berghof, K., Stahl, U., 1998. Detection of pathogenic and spoilage microorganisms in food with the polymerase chain reaction. Food Microbiol. 15, 13–31. Schmidt, H., Kernbach, C., Karch, H., 1996. Analysis of the EHEC hly operon and its location in the physical map of the large plasmid of enterohaemorrhagic Escherichia coli O157:H7. Microbiol. 142, 907–914. Sutherland, J.P., Bayliss, A.J., Braxton, D.S., 1995. Predictive modelling of growth of Escherichia coli O157:H7: the effects of temperature, pH and sodium chloride. Int. J. Food Microbiol. 25, 29–49.
Swinbanks, D., 1996. Outbreak of E. coli infection in Japan renews concerns. Nature 382, 290. Watanabe, Y., Ozasa, K., 1997. An epidemiological study on an outbreak of Escherichia coli O157:H7 infection. Rinsho Byori Jap. J. Clin. Pathol. 45, 869–874. Weagent, S.D., Bryant, J.L., Jinneman, K.G., 1995. An improved rapid technique for isolation of Escherichia coli O157 from foods. J. Food Protect. 58, 7–12. Wells, J.G., Davis, B.R., Wachsmuth, I.K., Riley, L.W., Remis, R.S., Sokolow, R., Morris, G.K., 1983. Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J. Clin. Microbiol. 18, 512–520. Zadik, P.M., Chapman, P.A., Siddons, C.A., 1993. Use of tellurite for the selection of verotoxic Escherichia coli O157. J. Med. Microbiol. 39, 155–158.
Journal of Microbiological Methods
Detection of Escherichia coli O157:H7 in ground beef in eight hours Christopher M. Gooding, Prabhakara V. Choudary* Antibody Engineering Laboratory and the Department of Entomology, University of California, Davis, CA 95616 -8584, USA Received 28 April 1998; received in revised form 10 July 1998; accepted 19 July 1998
Abstract A new method involving enrichment and immuno-polymerase chain reaction is presented for rapid and sensitive detection of Escherichia coli O157:H7 in ground beef. The bacteria in the spiked beef sample were enriched in a non-selective medium for 250 min and the E. coli O157:H7 cells were captured rapidly from the culture medium, using magnetic beads coated with E. coli O157-specific antibody. Partial stretches of sequences encoding Shiga toxins (Stx1 and Stx2) were amplified by the polymerase chain reaction and detected by agarose gel electrophoresis. Detection of a 215 / 212-base pair amplicon indicated contamination of the test sample with E. coli O157:H7. The method enabled detection of a single colony-forming unit of E. coli O157:H7 conclusively in 8 h. The suitability of the method to real life pathogen monitoring applications was demonstrated by accurate confirmation of E. coli O157:H7 in coded culture-positive samples of naturally contaminated ground beef and hamburger patties. 1998 Elsevier Science B.V. All rights reserved. Keywords: EHEC; Food safety; Hamburger; HACCP; Immuno detection; Infectious disease; PCR
1. Introduction Escherichia coli O157:H7 has been identified conclusively as a food pathogen in 1982 with the demonstration of its causal association with two outbreaks of hemorrhagic colitis, following the consumption of hamburgers (Riley et al., 1983; Wells et al., 1983). Escherichia coli O157:H7 has since been linked with many a foodborne bacterial outbreak in the USA, Canada and the UK and is now the most frequently isolated diarrheagenic type of E. coli in North America (Qadri and Kayali, 1998). An epidemic of E. coli O157:H7 with over 6000 cases has *Corresponding author. Tel.: 11-530-752-5563; fax: 11-530752-1537; e-mail: [email protected]
occurred recently in Japan (Izumiya et al., 1997; Swinbanks, 1996; Watanabe and Ozasa, 1997). In 1996, an E. coli O157:H7 outbreak associated with cooked beef pies has caused the death of 16 people in Scotland (Coia et al., 1998). Most E. coli O157:H7 outbreaks have been associated with the consumption of undercooked beef or raw milk (Centers for Disease Control, 1997; Cieslak et al., 1997; Keene et al., 1997). To address epidemiological issues and develop effective management strategies such as HACCP (hazard analysis critical control points) system for the prevention and control of this pathogen, a rapid and sensitive method of identification is required (Chapman et al., 1997). Traditionally, selective microbiological culture
0167-7012 / 98 / $ – see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S0167-7012( 98 )00070-0
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techniques are the primary methods of isolation and identification of E. coli O157:H7, with confirmation obtained from biochemical and serological tests (Johnson et al., 1995). Recently, however, there has been a surge of interest in more rapid and sensitive techniques, especially the polymerase chain reaction (PCR). Using a thermostable DNA polymerase and specific oligonucleotide primers in an iterative cycle of DNA synthesis, PCR amplifies a target stretch of DNA sequence exponentially. PCR procedures of multiple formats and combinations have been developed and used by several investigators for the detection of E. coli O157:H7 (Fields et al., 1997; Meng et al., 1997; Scheu et al., 1998). Gannon et al. (1992) had used Stx 1 and Stx 2 genes (previously known as Shiga-like toxin, SLT-I or verotoxin, VT1 and SLT-II or VT2, respectively) as templates in PCR to detect 1 cfu of E. coli O157:H7 in 1 g of ground beef in 15 h. It involved a 6-h culture enrichment step, followed by a 9-h step of isolation of DNA, PCR and agarose gel electrophoresis. Others have used immunomagnetic separation (IMS) for rapid isolation of E. coli O157:H7 from enrichment broth (Blanco et al., 1996; Weagent et al., 1995). Commercial tests with comparable sensitivities have also become available. Each of these methods has individual merits over the culture methods, but they are still too time-consuming for wide application in effective prevention and control strategies. For instance, the Tecra E. coli O157:H7 kit with 2-h assay time requires 18-h pre-enrichment (Flint and Hartley, 1995). The Ampcor Diagnostics MicroScreen test kit with 15-min detection time requires a minimum enrichment of 20 h (Czajka and Batt, 1996). Similarly, several PCR-based methods are also making their debut in the market. The Bax system from Qualicon is one such method without IMS, but requires enrichment overnight. Thus, there is a strong need for a sensitive and specific method that can be completed within a working day (Gooding and Choudary, 1997). To address this gap, we devised and optimized an approach using a combination of the above-described steps as modules of an integrated procedure. It can be readily assembled in a desired format to fit a specific contamination situation, by combining only the required modules. The suitability of the method
for contamination-monitoring purposes was demonstrated by accurate detection of E. coli O157:H7 in coded, culture-positive beef samples from previous outbreaks. This paper reports the method development, optimization and evaluation.
2. Materials and methods
2.1. Strains, media and growth conditions Twenty-four strains of bacteria were tested in this study as listed in Table 1. Ten strains were E. coli O157:H7, one was E. coli O111, and the rest were non-E. coli control bacteria. Only E. coli O157:H7 carried the Stx genes. The bacterial stocks were stored frozen at 2 708C in broth containing 15% glycerol and resuscitated by plating on agar (1.5%, w / v) plates and incubating overnight at optimal temperatures. E. coli O157:H7 stocks were streaked on sorbitol MacConkey (SMAC) (March and Ratnam, 1986) agar plates and incubated overnight at 378C, and the non-E. coli control strains on TYS (tryptone, 1%; yeast extract, 1%; sodium chloride, 0.5%) agar plates and incubated overnight on a lab bench (25–288C). Different strains of bacteria were each cultured in 20 ml of broth in a 250-ml Erlenmeyer flask inoculated with a single colony from the agar plates and shaking at 378C at 120 rpm on a gyrotory shaker (Model G24; New Brunswick Scientific, Edison, NJ, USA). Tryptose soy broth (TSB; DIFCO, Detroit, MI, USA) or modified EC broth (mEC; DIFCO, Detroit, MI, USA) with or without novobiocin (20 mg l 21 ) (Sigma Chemical, St. Louis, MO, USA) was used for growing E. coli strains and TYS broth for the rest. The identity of E. coli O157:H7 was confirmed by spread-plating on sorbitol MacConkey agar, containing cefimide (0.05 mg / l) and potassium tellurite (2.5 mg / l) (SMACCT) (Zadik et al., 1993). The non-E. coli control strains, CG1, CG2 and CG4, were grown at 308C, and CG5 at 378C in Brain Heart Infusion broth (DIFCO, Detroit, MI, USA). Serial dilutions of these cultures prepared in broth were used as inoculum to spike the ground beef samples. The inoculum size (cfu / ml) was determined by counting the number of colonies obtained from plating a portion (100 ml) of each dilution on SMAC
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91
Table 1 List of the bacterial strains used in the study, their Stx gene status and sources UCD number
Strain
Stx genotype
Original source
HF 1 HF 2 HF 3 HF 4 HF 5 HF 13 HF 14 HF 15 HF 51 HF 52 J5 CG 1 CG 2 CG 3 CG 4 CG 5 CG 6 CG 7 CG 8 CG 9 S 36 CG 10 CG 11
Escherichia coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O157: H7 E. coli O111 Aeromonas hydrophila Bacillus cereus Citrobacter freundii Enterobacter cloacae Enterococcus faecalis Hafnia alvei Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa Salmonella enteritidis Shigella sonnei Staphylococcus aureus
1&2 1&2 0e 2 1 NK NK NK 2 1&2 0 0 0 0 0 0 0 0 0 0 0 0 0
ATCC a 42895 ATCC 43894 ATCC 43888 ATCC 43889 ATCC 43890 CMDL b (Beef) CMDL (Beef) CMDL (Beef) USUHS c USUHS NK d ATCC 7965 ATCC 14579 ATCC 8090 ATCC 13047 ATCC 19433 ATCC 29926 ATCC 13883 ATCC 13315 ATCC 10145 ATCC 13076 ATCC 29930 ATCC 12600
a
ATCC, American Type Culture Collection, Rockville, MD. CMDL, California Department of Health Services Microbial Diseases Laboratory, Berkeley, CA. c USUHS, Uniformed Services University of Health Sciences, Bethesda, MD. d NK, not known. e 0, Stx gene(s) absent. b
agar plates and incubating at 378C. Unspiked beef samples were included as negative controls. To examine the effect of cold-stress (48C) on the viability and recovery of E. coli O157:H7, cultures with different inoculum sizes were each grown in TSB / mEC in Erlenmeyer flasks, shaking at 120 rpm for 7 days at 48C, followed by overnight at 378C and were tested using the new assay. The growth rates of the test cultures were monitored as a function of the A 600 values of the cultures at various time points.
a decimal serial dilution (in the range of 10 6 to 10 9 ) of each test culture in TSB (100 ml), using a pipetman (P-200) tip. The inoculated meatballs, in triplicates, were held at room temperature for 1 h to allow the inoculum to diffuse and were then frozen at 2 208C for 24 h or until further processing.
2.2. Experimental contamination of ground beef samples with E. coli 0157: H7
The frozen meatballs were each transferred aseptically to the mesh insert of a stomacher bag (Applied Biosolutions, New York, NY, USA) and thawed at room temperature for 30 min. Then, TSB (225 ml) was added to each bag, and the meatballs were homogenized for 1 min at room temperature in a stomacher (Tekmar, Cincinnati, OH, USA). The beef homogenate was incubated in the bag at 378C for
Ground beef (20% fat), purchased from a local grocery, was divided aseptically into 25-g portions, individually patted into balls and placed in an ethanol-sterilized plastic box. The meatballs were each inoculated in the center (ca. 1.5-cm deep) with
2.3. Pre-enrichment of the bacteria present in the spiked ground beef
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250 min, without shaking. The controls were processed without the 250-min incubation step.
2.4. Immunocapture of E. coli O157: H7 cells After the 250-min enrichment step, 1 ml-samples of each culture were removed from the stomacher bag and mixed with 20 ml of Dynabeads anti-E. coli O157 (Dynal, Lake Success, NY, USA) in a 1.8-ml microfuge tube. The tubes were shaken gently (150 rpm, Vortex Genie II, Fisher Scientific, Pittsburgh, PA, USA) for 30 min at room temperature, and the culture medium was aspirated, holding the beads in place using a magnetic particle concentrator (Promega, Madison, WI, USA). The beads were washed 3 3 with phosphate-buffered saline (PBS; 0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.4) containing 0.05% Tween 20. As a negative control, a 1-ml portion of the enriched culture was centrifuged in a microfuge at 14 000 3 g for 2 min, the supernatant was removed, and the PCR reaction mixture added directly to the pellet. The immunocapture step was optimized with respect to the incubation time, capacity and saturation. For optimization, the parameter under test was examined in a selected range, maintaining the rest constant. The optimal bead concentration was determined by incubating 1 ml-samples of each enriched culture for 30 min with 10, 20 or 50 ml of Dynabeads Anti-E. coli O157, washing and subjecting to PCR, followed by gel electrophoresis. To determine the optimal length of incubation time, the tubes containing immunobeads and the meat homogenate were shaken gently at room temperature for 15, 30, 45 or 60 min, washed and subjected to PCR, followed by gel electrophoresis. To estimate the capacity of the beads to capture E. coli O157:H7 cells, different dilutions of E. coli O157:H7 in TSB were treated with the beads as described above, and plate counts of the residual bacteria in the supernatant were determined.
2.5. PCR The PCR amplifications were carried out as described earlier (Gooding and Choudary, 1997), using a primer set derived from the Shiga toxin genes, Stx 1
and Stx 2 (Paton et al., 1993). This primer pair amplifies a 215-bp from the 586–800-bp stretch of the Stx 1 coding sequence and / or a 212-bp segment off the 583–794 bp portion of the Stx 2 coding sequence, respectively. Paton et al. (1993) designed these primer sequences originally and demonstrated their E. coli O157:H7-specificity in the PCR examination of human fecal cultures. The primers, with a GC content of 34% and a T m of 568C, were as follows: Forward: 59 . ATACAGAG(GA)G(GA)ATTTCGT , 39 Reverse: 59 . TGATGATG(AG)CAATTCAGTAT , 39 Amplification of the target bacterial DNA (Stx 1 and / or Stx 2 sequences) was performed in 0.5-ml PCR tubes (Fisher Scientific, Pittsburgh, PA, USA) using 100-ml total reaction volumes, containing 20 ml of the Dynabeads-bound bacteria as the source of template DNA. The reaction mixture consisted of (final concentrations): 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl 2 , 1 ml / l Triton X-100, 200 mM each of the deoxynucleoside triphosphates (Pharmacia Biotech, Piscataway, NJ, USA), 1.5 U of Taq polymerase (Promega, Madison, WI, USA) and 0.5 mM each of the primers, overlaid with 70 ml of sterile mineral oil (Sigma Chemical, St. Louis, MO, USA). The PCR was carried out in a programmable thermocycler (Model PTC150; M.J. Research, Watertown, MA, USA) using the following conditions: an initial cell lysis-cum-denaturation step at 958C for 5 min, followed by 35 cycles of denaturation at 928C for 1 min, primer annealing at 508C for 1 min and primer extension at 728C for 1 min. Experimental parameters of the PCR were each optimized in a separate set of experiments. For optimization of Mg 21 concentration, MgCl 2 was varied between 0.5 and 2.5 mM, with 1 to 100 target bacterial cells as template source, and the size and intensity of the amplicons were monitored and compared for all the concentrations of MgCl 2 tested. Using 1.5 mM MgCl 2 in the assay, the sensitivity of PCR was analyzed in terms of the maximum number of cycles required to yield a detectable PCR
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product from a minimal number of target bacterial cells. A target range of 2 to 165 cells was evaluated in 30, 40 or 50 cycles. The specificity of the primers was ascertained by testing a panel of target and non-target bacterial strains for PCR products of the expected size (215 / 212 bp). The products of PCR amplification from each reaction were analyzed by agarose gel electrophoresis (Sambrook et al., 1989). Typically, 10 ml of the amplicons were mixed with 2 ml of the loading solution with marker dyes (Sigma Chemical, St. Louis, MO, USA) and resolved by electrophoresis for 30 min at a constant power of 100 V in a 1.5% horizontal slab agarose (FMC Bioproducts, Rockland, ME, USA) gel in Tris–acetate–EDTA buffer (Sambrook et al., 1989), containing 0.5 mg / ml ethidium bromide (Sigma Chemical, St. Louis, MO, USA). HaeIII-digested fX 174 RF DNA (Life Technologies, Gaithersburg, MD, USA) was used as markers for DNA size. The DNA bands in the gels were visualized by UV transillumination and were photographed using a Polaroid camera system (Photo / PrepI, Fotodyne, Hartland, WI, USA). Each experiment was repeated twice with two replicates of each sample.
2.6. Determination of sensitivity limits of the method The detection limits of the method were established by assaying for the target-specific PCR amplification products of the Stx sequences (215 / 212 bp) from a range of inoculum sizes of the E. coli O157:H7 strains. The strain HF1 was tested in the range of 2 to 10 4 cfu / g of sample (Fig. 1), and HF4, HF5 and HF14 were tested in the range of 0.1 to .10 4 cfu / g sample (Table 2).
2.7. Detection of E. coli O157: H7 in naturally contaminated beef Five coded frozen samples (MDL001-MDL005) of naturally-contaminated beef from previous outbreaks of E. coli O157:H7 (Table 3) were obtained from the California Department of Health Services
93
Fig. 1. Agarose gel electrophoresis of the immuno-PCR products of Stx 1 and Stx 2 genes from ground beef samples, spiked with Escherichia coli O157:H7. Ground beef samples (25 g) were each inoculated with 100 ml of serial dilutions, each comprising a different inoculum size (ranging from 2 to 10 4 cfu / g sample) of E. coli O157:H7 strain HF1 (lanes 1 to 5) and enriched for 250 min as described in the text. The E. coli O157:H7 cells were concentrated by immunomagnetic separation, and the Stx gene sequences were amplified by the polymerase chain reaction and analyzed by agarose (1.5%) gel electrophoresis. The appearance in the gel of the expected 215 / 212-base pair band (as indicated on the side) was taken as positive indication for contamination of the investigated food sample with E. coli O157:H7. Lane M, DNA size markers: fX 174 RF DNA HaeIII fragments (bp, from top to bottom: 1,353; 1,078; 872; 603; 310; 281 / 271; 234; 194; 118; 72); Lanes 1 to 5, samples: lane 1, unspiked control sample; lane 2, sample spiked with 2 cfu / g; lane 3, 30 cfu / g; lane 4, 400 cfu / g; lane 5, 3310 4 cfu / g; lane 6, negative control (no DNA or E. coli O157:H7 cells added); lane 7; positive control (HF1 culture, 10 ml).
Table 2 Sensitivity limits of the immuno-PCR procedure for detecting three different strains of Escherichia coli O157:H7 in spiked ground beef Inoculum size
Escherichia coli O157:H7 strain
Cfu / g Ground beef
HF14 (Stx status unknown)
HF4 (Stx 2 )
HF5 (Stx 1 )
.10 000 1000–10 000 100–1000 10–100 1–10 .0.1,1 0
1 1 1 1 1 NT 2
1 1 1 1 1 1 2
1 1 NT 1 1 2 2
Cfu, colony forming units; 1, PCR-positive; 2, PCR-negative; NT, not tested.
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Table 3 Culture and enrichment-immuno-PCR detection of Escherichia coli O157:H7 in naturally contaminated beef Code [
CMDL culture results a
Enrichmentimmuno-PCR (6 h)
Enrichmentimmuno-PCR (250 min)
Fig. 2 gel lanes
MDL 001 MDL 002 MDL 003 MDL 004 MDL 005
1 1 1 2 2
1 1 1 2 2
1 1 2 2 2
14, 15 12, 13 10, 11 8, 9 6, 7
a
CMDL samples were cultured using standard non-selective enrichment, followed by plating, colony picking and biochemical identification.
Microbial Diseases Laboratory (CMDL), Berkeley, CA. These included two hamburger patties (MDL001, MDL002), two ground beef samples (MDL003, MDL005) and one meat ball (MDL004). Three samples (MDL001, MDL002 MDL003) had been found to be positive and two (MDL004, MDL005) negative for E. coli O157:H7, using standard culture techniques (CMDL, unpublished). The samples were homogenized in the stomacher, enriched for 250-min and 6-h separately and subjected to immunocapture, followed by PCR amplification. The PCR products were analyzed by agarose gel electrophoresis, as described.
3. Results and discussion The aim of this work was to develop and optimize an immuno-PCR method for detecting E. coli O157:H7 in ground beef with reduced overall assay time, but without compromising the specificity or sensitivity. We accomplished this goal by devising a method incorporating a combination of the steps: enrichment of the pathogen (250 min), selective capture of the pathogen using IMS (30 min), amplification of the pathogen-selective DNA sequences using PCR (110 min), and detection of the amplified DNA by agarose gel electrophoresis (30 min); approximately 60 min was spent in making various preparations for the total procedure. Our hypothesis, that optimal efficiency can be achieved by a synergistic combination of these steps, was substantiated by the considerably shortened assay time of the new method (8 h).
3.1. Pre-enrichment of the culture The pre-enrichment step helps to increase the target bacterial cell number to or above a minimum detection threshold. Conventionally, the enrichment step is carried out arbitrarily for periods of up to 48 h, without paying particular attention to the number of target bacteria required. This prolongs the assay time. Further, it is also customary to use media that selectively suppress the growth of non-target microorganisms. However, selective media slow down the growth and impede the recovery of the target organisms too, especially of the stressed cells (McCleery and Rowe, 1995) and result in longer assay times. Our results showed that by using media (TSB) without any selection, E. coli 0157:H7 could be enriched from a single cfu to .800 cfu within 250 min, sufficient for the production of a detectable PCR product. The generation time for HF14 was 26 min in TSB, 43 min in mEC and 55 min in mEC with novobiocin. However, cold-stressed cells grew in TSB with a generation time of 28 min after an initial lag period of 20 min and failed to grow in mEC with novobiocin. It was also apparent from our results that co-enrichment of non-target microorganisms does not negatively impact either the growth rate of E. coli 0157:H7, its selective capture by IMS or PCR (data not shown). We surmise that the enrichment period used in our procedure was too short for the competing non-target bacteria to effectively deprive the target bacteria of nutrients. The use of a non-selective growth medium and limiting the culture enrichment step to a defined period together were the key to achieving considerable reduction in the overall assay time of E. coli
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O157:H7 detection using our method, relative to the previously published methods.
3.2. Immunomagnetic separation Immunocapture using IMS offers a considerably faster means of collecting cells than centrifugation and saves a lot of time in isolating, washing, collecting and concentrating bacterial cells from food samples (Chapman et al., 1997; Bennett et al., 1996). More importantly, it eliminates matrix effects, including potential PCR-inhibitors from food and enrichment media (Weagent et al., 1995; Lantz et al., 1994). For these advantages, we chose to optimize and use IMS in our method. A 30-min incubation of the beads with enriched culture (1 ml) yielded a PCR product of the same intensity as that obtained after 45 or 60 min. However, insufficient product was obtained from the sample with 15-min incubation (data not shown). We found that just 20 ml IMS beads could recover 80% of the total E. coli O157:H7 population from the enrichment culture containing 10 9 cfu, 95% of 10 7 cfu, over 99% of 10 4 , and 100% of 10 2 cfu / ml. It was clear from these results that 20 ml IMS beads were sufficient to capture the minimum number of E. coli O157:H7 cells required for obtaining a PCR product detectable reproducibly by agarose gel electrophoresis.
3.3. PCR conditions The specificity of the primers was ascertained by testing them against a panel of target and non-target bacterial strains (Table 1). PCR products of the expected size were only obtained with E. coli strains of the O157:H7 serotype, carrying either copy of the Stx 1 or Stx 2 genes (Table 2), but not with nonpathogenic species E. coli (free from the Stx genes) or with non-E. coli (control) bacteria tested (data not shown). Detection of non-target O157 serovars and Shigella strains carrying Stx genes can be eliminated by substituting beads coated with H7-specific antibodies (He et al., 1996) for the Dynal anti-E. coli O157 beads used in this study. The use of primers derived from the flagellar H7 antigen gene (Gannon et al., 1997), the plasmid-borne catalase-peroxidase gene (KatP) (Brunder et al., 1996) or the hemolysin
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gene of E. coli O157:H7 (Schmidt et al., 1996) in PCR can also potentially restrict detection specifically to E. coli O157:H7. Most PCR protocols include a step of lysing the bacterial cells to make the genomic DNA accessible to primers in PCR. In our method, we lysed the bacteria directly in the PCR reaction by heating at 958C for 5 min. Our results corroborate the demonstration by Begum and Jackson (1995) of Stx 2 sequence amplification directly from ground beef samples inoculated with verotoxigenic E. coli, eliminating the need to isolate E. coli from the food samples or purify whole cell DNA for the PCR. Matrix effects are a serious problem in direct application of PCR to the analysis of food samples (Weagent et al., 1995; Lantz et al., 1994). One potential problem is chelation of free magnesium ions required for PCR. Therefore, we investigated the influence of magnesium (MgCl 2 ) concentration, in the range of 0.5 to 2.5 mM, on PCR in a titration experiment, with 1 to 100 target bacterial cells as template source. An amplicon of the expected size (215 / 212 bp) and intensity was obtained at all the concentrations of MgCl 2 above 1 mM, with the band intensity being highest in the 1.5 to 2.5 mM range. Hence, we used 1.5 mM MgCl 2 in all the subsequent assays. In efforts to establish the lower limits of the sensitivity of PCR we found a 30-cycle reaction to need a minimum threshold of 18 cfu and the 40cycle reaction 2 cfu. After 50 cycles, however, a second smaller band appeared, possibly of primer dimers. Therefore, it appeared desirable to keep the number of cycles to a minimum. Evidence from a different set of experiments that enabled detection of 1 cfu of E. coli O157:H7 also suggested the optimal number of PCR cycles to be 35 under the standard reaction conditions used in our method (data not shown).
3.4. Sensitivity of the method In determining the detection limits, the method was found to be sensitive to a pre-enrichment titer of 1 cfu of the HF4, HF5 and HF14 strains E. coli O157:H7 per gram meat, regardless of the strain variations (Table 2). Fig. 1 shows in all the positive test samples a direct correlation between the intensity
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of the signal of the PCR product and the preenrichment inoculum size (2 to 10 4 cfu / g sample). The 215 / 212-bp band in the agarose gel (Fig. 1) clearly shows a proportionate increase in signal strength from the inoculum size of 2 cfu / g sample (Fig. 1, lane 2) to 10 4 cfu / g sample (Fig. 1, lane 5). The negative control, with no E. coli O157:H7 cells or DNA, yielded no PCR product (Fig. 1, lane 6). These results demonstrate the sensitivity and specificity of the method and its reproducibility at different contamination levels.
3.5. Detection of E. coli O157: H7 in naturally contaminated beef Finally, the suitability of the method for field applications was demonstrated by its ability to accurately identify coded real-life samples of hamburgers and beef patties, collected from previous E. coli O157:H7 outbreaks. Using the standard 250-min enrichment the method could correctly identify all samples (Table 3) but MDL 003 (Fig. 2, lane 11), which needed a 6-h enrichment (Fig. 2, lane 10). We suspect that severe stress of the low original inoculum during collection or transit (including multiple freezings and thawings after collection) could be one reason (McCleery and Rowe, 1995; Clavero and Beuchat, 1996; Sutherland et al., 1995). The experiment could not be repeated for lack of a duplicate sample. However, in special situations such as this
where the sample is suspected to be extraordinarily stressed, false negative results could be avoided by increasing the enrichment time to 6 h.
3.6. Conclusion In conclusion, by combining the steps of preenrichment, IMS and PCR, we have created a rapid method for detection of E. coli O157:H7 in both artificially and naturally contaminated beef samples. Using this method, we project detection to a preenrichment inoculum of 1 cfu of E. coli O157:H7 in 1 g of spiked ground beef in 8 h. The demonstrated rapidity, sensitivity, specificity and reproducibility of the method should prove valuable for the public health laboratories, regulatory agencies and beef industry in using it as a high-throughput early warning screening system and in implementing the HACCP plans.
Acknowledgements We thank Rita Brenden, Ray Bryant and Greg Inami of the California Department of Health Services Microbial Diseases Laboratory for providing the naturally contaminated beef samples, data from standard culture techniques and constructive comments. Thanks are also due to Jennie Hunter-Cevera
Fig. 2. Gel electrophoretic analysis of the Stx DNA sequences amplified by immuno-PCR of ground beef samples, naturally-contaminated with Escherichia coli O157:H7 (samples received from CA Department of Health Services Microbial Diseases Laboratory). The experimental details were as in Fig. 1 legend. Lanes 1,16: DNA Mr markers; lanes 2,3: positive controls; lanes 4,5: negative controls; lane 6: sample MDL005 enriched for 6 h; lane 7: MDL005 enriched for 250 min; lane 8: MDL004 enriched for 6 h; lane 9: MDL004 enriched for 250 min; lane 10: MDL003 enriched for 6 h; lane 11: MDL003 enriched for 250 min; lane 12: MDL002 enriched for 6 h; lane 13: MDL002 enriched for 250 min; lane 14: MDL001 enriched for 6 h; lane 15: MDL001 enriched for 250 min.
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and Sharon Shoemaker for helpful discussions. This research was supported by a grant (CHOUD941900) from the California Department of Justice and the Office of the Alameda County District Attorney. The contents of this paper are subject to a UC patent application (UC 97-019).
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