Direct detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction

Molecular and Cellular Probes(1995) 9, 259-264

Direct detection of Shiga-like toxin-producing Escherichia coil in ground beef using the polymerase chain reaction D. Begum and M. P. Jackson*

Department of Immunology and Microbiology, Wayne State University Medical School, Detroit, MI 48201, USA (Received 28 March 1995, Accepted 17 April 1995) We recently reported the development and assessment of a technique for the detection of Shigalike toxin-producing Escherichia coil (SLTEC) using the polymerase chain reaction (PCR) and a digoxigenin-11-dUTP-labelled DNA probe. This technique has now been adapted for the direct identification of SLTEC in ground beef. Ground beef homogenates were diluted 1000-fold to reduce the concentration of components which inhibit the thermostable polymerase. Assessment of four different ground beef samples using the PCR detection technique revealed that fat content was a major inhibitory component. As few as 30 SLTEC ml -~ of a ground beef homogenate were detected using the PCR technique, although it was necessary to enrich six of the samples for positive detection. These findings indicate that the PCR detection technique is suitable for the identification of SLTEC directly from contaminated ground beef without isolation of the bacterium or purification of its DNA. © 1995 Academic Press Limited

KEYWORDS: Shiga-like toxin, PCR, ground beef. INTRODUCTION Outbreaks of haemorrhagic colitis and the haemolytic uremic syndrome (HUS) caused by Shiga-like toxinproducing Escherichia coil (SLTEC; also designated Verotoxin-producing E. coli or VTEC) are usually linked to the consumption of contaminated beef implicating cattle as a reservoir for the organismJ -3 Consequently, there is interest in developing methods for the identification of SLTEC in contaminated beef. Because strains which produce SLT type II (SLT-II)are more frequently identified as the causative agents of HUS4-6 any method designed for the reliable identification of SLTEC in food should have the capacity to detect the production of SLT-II. There have been reports that SLTECcan be detected using the polymerase chain reaction (PCR),7-" a technique which is relatively rapid, highly specific, and very sensitive. 12This detection technique is designed

to selectively identify only toxin-producing strains using PCR under highly sensitive and specific conditions, eliminating the false identification of nontoxinogenic 0157 strains or the omission of non0157 SLT-producers. This is particularly important considering the recent demonstration by Karch etal. 1~ that the sit genes can be unstable in SLTEC. A technique for the identification of SLTEC using PCR and a digoxigenin-dUTP (dig)-Iabelled DNA probe has been developed. 14'~sSensitivity and specificity levels of 99% were established for this technique using a collection of 100 toxin-producing clinical isolates (16). In this report, we demonstrated that the slt-II gene sequences may be amplified directly from several ground beef samples which have been inoculated with SLTEC. Although the ground beef homogenates

* Author to whom correspondenceshouldbe addressed. 0890-8508/95/040259 + 06 $12.00/13

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© 1995 Academic PressLimited'

260

D. Begum and M. P. Jackson

Table 1. SLTECused in this study

PCR primers

Strain

Serotype

SLT type

933 3481 86-24 3235 B9560

O157:H7 O157:NM* O157:H7 O157:H7 O165:H25

I+11 I + II II II II

The sequences of the forward and reverse PCR primers which were used for the detection of SLT-il-producing E. coli are given in Table 2.

PCR

* NM: non-motile.

were diluted to eliminate components which inhibited the thermostable polymerase, there was no need to isolate SLTEC from the samples or purify whole cell DNA for the PCR.

MATERIALS AND METHODS Bacterial strains

SLTEC which were used in this study are shown in Table 1. All of the strains used in this study were obtained from the Centers for Disease Control (Atlanta, GA, USA). The toxin phenotypes of these strains were previously evaluated using the PCR detection technique and cytotoxicity assays) 6All bacteria were cultivated overnight at 37°C in LB broth) 7

One microlitre of the ground beef slurries containing serial dilutions of SLTEC was introduced into a 20-i.d PCR mixture composed of 10 mM Tris-HCI (pH 8.8), 50ram KCI, 1-5 mM MgCI2, 0-001% (w/v) gelatin, 20pM each of the 5' and 3' primers, and 125 F.M each of dATP, dGTP, dCTP and TTP. One unit of thermostable DNA polymerase was added and the samples were incubated for 30 cycles as described previously. TM Amplification products were visualized by agarose gel electrophoresis and ethidium bromide 17or by membrane hybridization and a dig-labelled DNA probe. A dig-labelled DNA probe for the detection of the sequences was prepared by PCR as previously described [14]. Plasmid pMJ100, which contains the slt-II operon [18], was used as a template. The SLT-II probe was labelled by incorporating the nucleotide analogue dig-dUTP (17.5 pM) (Boehringer Mannheim Biochemicals, Indianapolis, IN, USA) into a TTPdeficient PCR mixture containing 501~ each of dATP, dGTP and dCTP, and 32.5 ~M TTP.

Hybridization with a dig-labelled DNA probe Ground beef

Ground beef samples inoculated with SLTEC which had been cultured overnight in LB broth were diluted 10-fold (w/v) in saline. Diluted ground beef samples containing SLTEC were introduced directly into the PCR mixture. In separate experiments, the contaminated ground beef samples were enriched at 37°C for 4 h prior to PCR analysis. Ground beef sources were as follows: (i) 'Retail' was purchased from a local supermarket; (ii) 'Restaurant' was obtained from a supplier and is composed of a mixture of ground beef and vegetable matter; (iii) 'RestaurantJ20' was obtained from a supplier and is composed of ground beef with a reduced (20%) fat content; and (iv) 'Exsanguinated' was purchased from a local supermarket and was prepared from an exsanguinated animal. Ground beef samples contained approximately 100 colony forming units per millilitre (cfu m1-1) of aerobic bacteria and no detectable SLTEC prior to inoculation with the strains shown in Table 1.

Halfa microlitre of the PCR products was immobilized on a nitrocellulose membrane and hybridized with 5 p.I of the dig-labelled DNA probe. 's

RESULTS Detection limits of SLTEC in ground beef

Ground beef samples from four separate sources containing various dilutions of SLT-ll-producing and SLTI, SLT-ll-producing E. coli (Table I) were inoculated directly into the PCR mixtures. Incubation of the samples at 95°C for 10rain induced lysis of the bacteria, liberating whole cell DNA for amplification using primers specific for the slt-ll gene sequence (Table 2). Amplification products were visualized by agarose gel electrophoresis and by hybridization using a dig-labelled DNA probe in a spot-blot assay. Nontoxinogenic strains of E. coli were not detected using the PCR technique while pure cultures of the SLTEC

Detection of Shiga-like toxin producers

Table 2.

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PCR primers* % Homology to:

Designation SLT-IIF 5LT-IIR

Sequence

Position

slt-I

slt-II

Tm(°C)

CAGAAGCCTTACGCTT CCGGAGCCTGATTCACAGG

798-813 1427-1445

81 53

100 100

48 61

* Sequencesof the primers were taken from reference14. SLT-IIRis the reversecomplement of the slt-II gene sequence.

shown in Table 1 always gave positive results. None of the four ground beef samples were naturally contaminated with SLTEC as assessed using the PCR detection technique which has sensitivity and specificity values of 99%. 1~ Various concentrations of the SLTEC cultures were inoculated into diluted ground beef samples. Bacterial concentrations and the ground beef dilution required for detection of the PCR product varied with individual samples (Table 3). As few as 30 cfu ml-1 of the SLTEC strain 86-24 were detected in a contaminated ground beef sample from a restaurant supplier. This is equivalent to 8 cfu ml -~ of the 20-1~1 PCR mixture. Conversely, the limits for detection of strain 933 in a retail ground beef homogenate were significantly higher: 7 . 8 x l 0 S c f u m l -~ which is equivalent to 2 x 10Scfu m1-1 of the total amplification reaction. Optimum detection limits were observed using the ground beef sample which had a reduced fat content

Table 3.

(Table 3). The average number of cfu detectable per millilitre for the Restaurant/20 ground beef sample was 6744 in comparison to averages of approximately 5 x l 0 4 c f u m l -I for the Restaurant, Retail and Exsanguinated samples. It was necessary to dilute the contaminated ground beef samples in saline to reduce the concentration of components which may inhibit the thermostable polymerase used in the amplification reaction. Dilution factors ranged from 10- to 10 000-fold with a median of 100 (Table 3). As with the evaluation of detection limits, the best ground beef sample was Restaurant/20 with an average dilution factor of 600 in comparison to the Retail and Exsanguinated samples which required average dilutions of 2300- and 2800fold, respectively. Significant differences in the detection limits or dilution factors were not observed upon comparison of the five different SLTEC strains (Table 3).

Detection of SLTEC in ground beef

SLTEC

Ground beef source

933 933 933 933 3481 3481 3481 3481 B9560 B9560 B9560 B9560 86-24 86-24 86-24 86-24 3235 3235 3235 3235

Exsanguinated RestaurantJ20 Retail Restaurant Exsanguinated Restaurant/20 Retail Restaurant Exsanguinated Restaurant/20 Retail Restaurant Exsanguinated RestaurantJ20 Retail Restaurant Exsanguinated RestauranlJ20 Retail Restaurant

* ND: not detected.

Detection limits (cfu ml-1)

Ground beef dilution

43 11 200 780 000 8200 ND* 10 000 210 000 2300 1900 120 1800 1200 4000 400 1120 30 2600 12 000 220 000 1400

1000 1000 100 100 1000 1000 100 10 000 100 10 000 1000 100 1000 1000 100 100 10 100 10

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D. Begum and M. P. Jackson Table 4. Enrichmentof contaminated ground beef samples Detection limits (cfu m1-1) Sample

Time 0

3481/Retail 3481/Restaurant 3235/Exsanguinated 3235/Retail B9560/Restaurant 86-24/Retail B9560/Restaurant 86-24/Retail

4500 45 12 1200 38 30 3400 560

Time 4 h* 210 000 2300 2600 220 000 1200 1120 73 000 10 000

Dilution factor

ResuIt-I-

1000 1O0 1O0 100 1000 1000 1O0 1O0

+ + + + + + -

* Enrichmentfor 4 h at 37°C. t PCRproductsvisualizedusingagarosegel and the spot-blotassay.

Hybridization detection using a dig-labelled DNA probe The success of the amplification reaction was initially assessed by visualizing the PCR products using agarose gel electrophoresis. The 20 amplification products were also immobilized on nitrocellulose membranes and hybridized with a dig-labelled DNA probe which was prepared using PCR and the same slt-II gene-specific primers which were used in the detection assay. Identification of positive amplification products using an alkaline phosphatase-conjugated antibody to dig was performed as described previously.TM Fifteen of the 19 PCR samples which were detected using agarose gel electrophoresis were visualized using the spot-blot assay (data not shown). Retail and Restaurant/20 ground beef samples contaminated with SLTEC strain 86-24, a Restaurant sample contaminated with strain B9560, and a Restaurant/20 sample containing 3481 which were positive using the PCR detection technique and visualization by agarose gel electrophoresis (Table 3) were negative using the spot-blot assay. Ground beef from an exsanguinated animal contaminated with strain 3481 was not identified using either the agarose gel or spotblot visualization methods.

Enrichment of contaminated ground beef samples Because the detection limits for some samples exceeded 10 s cfu ml-1 (Table 3), we evaluated the effect of a 4-h, 37°C enrichment on ground beef contaminated with less than detectable levels of SLTEC (Table 4). Enrichment of SLTEC strain 3235 in ground beef from an exsanguinated animal from 12 cfu ml -I to 2600cfu m1-1 (a 217-fold enrichment) and 3235

in ground beef from a retail source from 1200 cfu ml-1 to 220 000 cfu ml-1 (a 183-fold enrichment) permitted detection of the amplification products using agarose gel electrophoresis and the spot-blot assay. SLTEC strain 3481 in ground beef from restaurant and retail sources was detected using the PCR detection method following enrichment from 45 cfu m1-1 to 2300cfu m1-1 (52-fold) and from 4500cfu m1-1 tO 210 000 cfu m1-1 (46-fold), respectively (Table 4). Enrichment of samples composed of SLTEC strains 86-24 in retail ground beef and B9560 in restaurant ground beef from 38 to 1200 cfu ml -I (32-fold) and 30cfu m1-1 to 1120cfu m1-1 (37-fold), respectively, was sufficient to permit detection using PCR (Table 4). In contrast, enrichment of the same samples an average of 20-fold (560 cfu ml-1 to 10 000 cfu ml-1 for 86-24/retail and 3400 cfu ml -I to 73 000 cfu m1-1 for B9560/restaurant) was not sufficient to permit detection using PCR (Table 4). Therefore, while the final detection limits following the enrichment procedure ranged from 1120 cfu ml-1 to 220 000 cfu m1-1, an average 35-fold enrichment of the contaminated ground beef samples was sufficient to permit identification of the amplification products by agarose gel electrophoresis and spot-blot hybridization.

DISCUSSION In previous studies, we developed and evaluated a PCR detection technique for the identification of SLTEC.14-16In this study, we showed that this technique may be used for the direct identification of SLTEC in ground beef samples without prior isolation of the strain or its DNA. Ground beef homogenates inoculated with toxinogenic strains were directly subjected to PCR and the amplification products were

Detection of Shiga-like toxin producers

visualized using agarose gel electrophoresis or a spotblot hybridization assay with a dig-labelled DNA probe. While detection limits using agarose gels were superior to the spot-blot, the spot-blot is technically simpler and has the capacity for screening large number of samples simultaneously. Optimum results with most ground beef homogenates were obtained using a 100-fold dilution of the sample, presumably because this reduced the concentration of factors which inhibited the thermostable polymerase or other components of the reaction system. Evaluation of the detection limits indicated that fat content in the ground beef sample was a major inhibitory factor. The lowest detection limits and dilution factors were obtained using ground beef with a reduced (20%) fat content (Table 3). Dilution factors of ground beef from an exsanguinated animal were not superior to samples from retail or restaurant sources. This PCR technique had the capacity to detect as few as 30 SLTEC per millilitre of a ground beef sample, although the detection limits for some samples were in excess of 1 x 10 s cfu mI-L Although the detection limits and dilution factors required for the different SLTEC strain and hamburger combinations varied widely, an enrichment step permitted confirmation of all SLTEC-contaminated samples with no falsepositive identifications. Incubation of contaminated ground beef samples for 4 h at 37°C elevated cell counts of SLTEC strains with poor detection limits to levels which permitted identification using the PCR technique. Although detection limits were as variable as with the pre-enrichment samples, a 35-fold increase in the number ofcfu per millilitre was sufficient for positive detection results for most samples. Previously reported PCR detection methods 7-u may require lengthy enrichment of the specimen, purification of whole cell DNA, and/or a radioactive DNA probe for identification of the amplification product. These features render the detection methods impractical for the rapid screening of large numbers of samples. The purpose of this study was to assess the capacity of the PCR technique developed and evaluated in our laboratory 1~-16for the direct detection of SLTEC in ground beef samples. Our results showed that naturally-contaminated ground beef which contained too few organisms for direct detection may be amplified and diluted 100-fold to reduce the fat content prior to PCR analysis.

ACKNOWLEDGEMENT

This work was supported by Public Health Service grant AI29929 from the National Institutes of Health.

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REFERENCES

1. Karmali, M. (1989). Infection by Verocytotoxin-producing Escherichia coil Clinical Microbiology Reviews 2, 15-38. 2. MMWR, CDC. (1993). Preliminary report: foodborne outbreak of Escherichia coli O157:H7 infections from hamburgers--Western United States. MMWR 42, 85. 3. O'Brien, A. D. & Holmes, R. K. (1987). Shiga and Shiga-like toxins. Microbiology Review 51,206-20. 4. Kleanthous, H., Smith, H. R., Scotland, S. M., Gross, R. J., Rowe, B., Taylor, C. M. & Milford, D. V. (1990). Hemolytic uremic syndromes in the British Isles, 1985-8: association with Verocytotoxin producing Escherichia coil Part 2: microbiological aspects. Archives of Diseases in Childhood 65, 722-7. 5. Ostroff, S. M., Tarr, P. I., Neill, M. A., Lewis, J. H., Bean, N. H. & Kobayashi, J. M. (1989). Toxin genotypes and plasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections. Journal of Infectious Diseases 160, 994-8. 6. Wadolkowski, E. A., Sung, L. M., Burris, J. A., Samuel, J. E. & O'Brien, A. D. (1990). Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin type 11. Infection and Immunity 58, 3959-65. 7. Gannon, V. R, King, R. K., Kim, J. Y. & Thomas, E. J. (1992). Rapid and sensitive method for the detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Applied Environmental Microbiology 58, 3809-15. 8. Karch, H. & Meyer, T. (1989). A single primer pair for amplifying segments of distinct Shiga-like toxin genes by polymerase chain reaction. Journal of Clinical Microbiology 27, 2751-7. 9. Paton, A. W., Paton, J. C., Goldwater, P. N. & Manning, P. A. (1993). Direct detection of Escherichia coliShigalike toxin genes in primary fecal cultures by polymerase chain reaction. Journal of Clinical Microbiology 31, 3063-7. 10. Pollard, D. R., Johnson, W. M., Lior, H., Tyler, S. D. & Rozee, K. R. (1990). Rapid and specific detection of Verotoxin genes in Escherichia coli by the polymerase chain reaction (PCR).Journal of Clinical Microbiology 28, 540-5. 11. Read, S. C., Clarke, R. C., Martin, A. et al. (1992). Polymerase chain reaction for the detection of verocytotoxigenic Escherichia coli isolated from animal and food sources. Molecular and Cellular Probes 6,

153-61. 12. Saiki, R. K., Gelfand, D. H., Stoffel, S. et al. (1988). Primer-directed enzymatic amplification of DNA with thermostable DNA polymerase. Science 239, 487-91. 13. Karch, H., Meyer, T., Russmann, H. & Heesemann, J. (1992). Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coil upon subcultivation. Infection and Immunity 60, 3464-7. 14. Jackson, M. P. (1991). Detection of Shiga toxin producing Shigella dysenteriae type 1 and Escherichia coli by using the polymerase chain reaction with incorporation of digoxigenin-11 -d UTP.Journal of Clinical Microbiology 29, 1910-14. 15. Jackson, M. P. (1992). Identification of Shiga-like toxin type II producing Escherichia coliusing the polymerase

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chain reaction and a digoxigenin labelled DNA probe. Molecular and Cellular Probes 6, 209-14. 16. Begum, D., Strockbine, N. A., Sowers, E. G. & Jackson, M. P. (1993). Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli using PCR and digoxigenin-labelled probes. Journal of Clinical Microbiology 31, 3153-6.

17. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. 2nd ed. New York. Cold Spring Harbor Laboratory Press. 18. Jackson, M. P., Deresiewicz, R. L. & Calderwood, S. B. (I 989). Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits. Journal of Bacteriology 172, 3346-50.