DNA probe for detection of serogroup 0157 enterohemorrhagic Escherichia coli

International Journal of Food Microbiology 25 (1995) 277-287

DNA probe for detection of serogroup 0157 entero~e~orr~agic E~c~eric~i~cd Leslie G. Huck I, C. Richard Dorn *, Elisabeth J. Angrick Departmentof VeterinaryPreuentiueMedicine, llre Ohio State University,1900 Coffey Road, Columbus, OH 43210, USA Received 21 October 1993; revision received 22 April 1994; accepted 31 May 1994

Abstract To develop a probe for the detection of serogroup 0157 enterohemorrhagic Exheric&a plasmid DNA extracts from 16 E. coli strains that hybridized with the CVD419 probe were screened for restriction fragments present in plasmids of serogroup 0157 E. coli strains, but not in plasmids of non-0157 E. co& strains. Using a single 0157:H7 E. coli strain (63911, 10 serogroup 0157 E. coli specific fragments were then removed, radiolabeled and hybridized (42°C) with colony blots of both groups of strains. A 2.0 kb SmaI fragment probe WPMl) was the most specific for serogroup 0157 EHEC: Using a larger set of 41 non-E. coli and 107 E. coii strains from human, animal and meat sources, VPMl hybridized with all 49 serogroup 0157 EHEC strains. None of 8 enterotoxigenic E. coli (ETEC), including serogroup 0157 strains, nor any of the 41 non-E. co& hybridized with the VPMl probe. However, this probe hybridized with 5 of 50 non-0157 E. coli which were verotoxin (VT) or CVD419 probe-positive. Increased hybridization stringency (45°C) reduced the 5 false-positives to 2 negatives and 3 trace responses, which were easily distinguishable from positive responses. coli (EHEC),

Keywords: ~nterohemorrhagic

DNA probe; Foodborne

E~cherich~o co&; EHEC; Serotype 0157 : H7 Escherich~u co&

illness

* Corresponding author. Tel.: + 1 (614) 2921206. Fax: + 1 (614) 2924142. ’ Present address; U.S. Army 10th Medical Laboratory; Box 1423, Landstuhl, Germany, APO AE

09180-3619. Old-1605,/95/$~.50 0 1995 Elsevier Science B.V. All rights reserved S~D~Ol68-1605(94)00084-O

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I,. G. Huck et al. f Int. J. Food Microbiology 25 (1995) 277-287

1. introduction Of the several serotypes represented among enterohemorrhagic E. coil (EHEC), 0157 : H7 is the most often isolated from hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) cases, and associated with major outbreaks (Karma& 1989; Griffin and Tauxe, 1991). Non-motile strains (0157: NM) have also been isolated from outbreak and sporadic cases of HC and HUS (Scotland et al., 1988; Griffin and Tauxe, 1991). Escherichia coli 0157 : H7 was recognized as a new bacterial pathogen in 1982, following its isolation in two outbreaks from patients with HC and from ground beef linked to that served by a fast-food chain involved in both outbreaks (Riley et al., 1983). Most subsequent outbreaks have been attributed to consumption of E. coli 0157:H7 contaminated beef or unpasteurized milk (Martin et al., 1986; Borczyk et al., 1987; Bryant et al., 1989; Griffin and Tauxe, 1991, potatoes (Morgan et al., 19881, unpasteurized apple cider (Besser et al., 19931, and water (McGowan et al., 1989; Dev et al., 1991). Person-to person contact (Spika et al., 1986; Karmali et al., 1988; Ostroff et al., 1989; Belongia et al., 1993) has also been reported as a cause of infection. Several immunoassays using polyclonal and monoclonal antibodies to identify serogroup 0157: H7 E. cob have been developed; however, they are either non-specific or too time consuming for testing large numbers of samples (March and Ratnam, 1989; Okrend et al., 1990; Doyle, 1991). A dipstick immunoassay used to screen ground beef for 0157: H7 is easy to perform, but 2 of 98 negative control strains tested positive, perhaps due to denaturation or degradation of the capture antibody (Kim and DoyleJ992). Bioassays detecting the slow fermentation of sorbitol by serotype 0157 : H7 and its lack of ~-glucuronidase activity are widely used in diagnostic laboratories, but they are time consuming and individually lack specificity (Borczyk et al., 1987). DNA probes have the advantage of high sensitivi~ and specificity and probe assays can be performed on large numbers of samples in a single hybridization. However, none of the presently available probes are specific for serogroup 0157 EHEC, i.e., both 0157: H7 and 0157 : NM. Reports of two probes specific for serotype 0157: H7 have been published: a probe developed from the eae gene homologue cloned from a serotype 0157: H7 EHEC strain (Beebakhee et al., 1992) and an oligonucleotide probe based on the uidA gene sequence (Feng, 1993). The probes that hybridize with DNA sequences encoding production of the two verotoxins (VT1 and VT2; also designated SLT I and SLT II), identify a variety of E. coli serotypes that produce these toxins (Scotland et al., 1988; Newland and Neill, 1988) including serotypes that have never been associated with human illness. Another probe, CVD419, developed from a 3.4 kb Hind111 fragment of the large ca. 60 MDa plasmid of serotype 0157: H7, was shown to hybridize with 99% of serotype 0157:H7 EHEC, but also with 77-81% of non-0157 E. coli that produced VT (Levine et al., 1987). A probe derived from a chromosomal gene (cue) of an enteropathogenic E. coli identifies a broad group of attaching and effacing E. co/i, including serogroup 0157 and non-0157 EHEC

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219

{Jerse and Kaper, 1991; Willshaw et ai., 1992). This study was undertaken to develop a DNA probe that would be specific for the detection of only serogroup 0157 EHEC.

2. Materials and methods Sources ofstrains. Two sets of bacterial strains were used in this study. The first set, consisting of 16 CVD419 probe-positive E. cob, was used to search for plasmid DNA restriction fragments that were specific for serogroup 0157 EHEC. This set consisted of 9 E. coli 0157: H7 strains from bovine, human and meat (beef) sources; an 0157: NM strain from a human diarrhea case; one 026:Hll caIf strain; one 026:Hll human strain; an 0103:H2 human isolate derivative (62R703 which has a single plasmid (pDEP12) that hybridizes with the CVD419 probe; and three non-VT producing meat strains of serotypes 0159:H7, OC70/86:H49 and O?:H7 respectively. Except for the three non-VT producing strains, filtrates of all of the strains in this set were cytotoxic for Vero cells and produced either or both VT1 and VT2 when first isolated. The second set, consisting of 107 E. cob and 41 non-E. co& strains, was used to evaluate the specificity of the new DNA probe for serogroup 0157 EHEC. This set consisted of 49 serogroup 0157 EHEC and 50 VT or CVD419 probe-positive strains of 0 serogroups other than 0157. The two serotype 0103:H2 strains were derivat.ives of a human isolate: strain 62R70 contains pDEP12 which hybridizes with the CVD419 probe and strain 62R64 contains plasmid pDEP13 which does not hy~~ridize with the CVD419 probe. The following control strains were used in hybridization experiments: E. coli 026:H:ll strain E3787, VT1 positive, VT2 negative, CVD419 positive; E. coli 0157: 1~7 strain E32511, VT1 negative, VT2 positive, CVD419 positive; E. coli 0157 : 1~7 strain 6391, VT1 positive, VT2 positive, CVD419 positive; and E. coli K-12 strain 14R519, a negative control. Ptasmid DNA ~tractio~, gel electro~horesis and DNA blots. Plasmid DNA was prepared using the alkaline extraction method of Birnboim and Doly (1979). DNA was electrophoresed on 0.6% (w/v) Type II (Sigma, St. Louis) agarose gels using TBE buffer. The gels were stained with ethidium bromide and examined under ultraviolet (UV> light (Fotodyne Fotoprep I/MP4), and photographed using Poiaroid and Ektapan films. The sizes of the plasmids were determined using the eight molecular size standard plasmids of E. coli V517 and the 77.8 MDa plasmid of strain 4OR448. Digested DNA fragments were transferred (Southern blot) to Hybond-N membranes (Amersham; Amersham, UK) and baked for 2 hours at 80°C. For colony blots, broth cultures were spotted on Hybond-N grid membranes, aseptically placed on nutrient agar plates, and grown on blood agar plates. The membr,anes were prepared for hybridization as described by Maniatis et al. (19821, dried for at least 1 h and exposed to UV light (3OOnm) for 4 min. The following restriction enzymes were used to digest plasmid DNA according to the manufacturer’s instructions: HindIII, SmaI and BamHI (Gibco BRL,

Gaithersburg, MD). After digestion, the restriction fragments were electrophoresed on 0.8% agarose gel. The gel was stained with ethidium bromide and photographed, and the molecular sizes of the fragments were determined by comparison to restriction enzyme (hTindII1, SmaI, BumHI, and pstI) digests of lambda phage DNA. Hybridizations and DNA probes. Plasmid DNA fragments used as probes were cut out from low-melting point agarose gels and labeled with deoxyadenosine 5’-(r[35S]thiotriphosphate. Hybridization with the 35S-labeled probes was carried out according to the method of Maniatis et al. (1982) with the following modifications: membranes were prehybridized in a mixture of 5 x SSPE, 5 X Denhardt’s solution, 0.1% SDS, 50% formamide, 0.01 M dithiothreitol and 100 pg/ml denatured, fragmented salmon sperm DNA for 4 to 6 h at 42°C. Membranes were then hybridized with the radioactive probe (10 X lo6 dpm) and fresh prehybridization mixture overnight at 42°C except for some experiments performed with increased stringency at 45°C. Membranes were washed three times in 2 x SSC, 0.1% SDS at room temperature and washed twice in 1 X SSC, 0.1% SDS for 1 h at 68°C. The VT1 probe was a 0.75 kb IiincII fragment specific for VT1 sequences obtained from a VU-encoding phage carried by E. coli serotype 026:Hll strain H19 (Willshaw et al., 1985). The VT2 probe was a 0.85 kb AuaI-&I fragment specific for VT2 sequences cloned from a VIZ-encoding phage carried by E. coli E32511 of serotype 0157 : NM (Wilishaw et al., 1987). The CVD419 probe was a 3.4 kb Hind111 fragment that was cloned into pBR325 (Levine et al., 1987). To select restriction fragments for probe candidates, plasmid DNA of 0157 : H7 isolate 6391 was digested with either SmaI, BumHI or both endonucleases and electrophoresed on agarose gel. Ten fragments which were present in all 10 serogroup 0157 EHEC of the first set, but absent in the 7 non-0157 EHEC strains, were selected as probe candidates, radiolabeled and hybridized as previously described. Vero cell culture and cytotoxin assay. Vero cells, obtained from the American Type Culture Collection, Rockville, MD, were cultured in minimum essential medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum and glutamine (292 mg/l). A modification of the cytotoxin assay of Scotland (1985) was used. T~sin-EDTA-ha~ested Vero cells were diluted in tissue culture medium with gentami~in and amphotericin B (Fungizone) to a final concentration of 2.5 X lo5 cells per ml, and 0.2 ml portions of the suspension placed in each well of a 96-well Falcon 3072 tissue culture plate (Becton-Dickinson, Lincoln Park, NJ). Monolayers were obtained in one day using this procedure.

3. Results and discussion Among the initial set of 16 CVD419 probe-positive strains used to search for a serogroup 0157 specific restriction fragment, all 10 serogroup 0157 strains yielded filtrates that were cytotoxic for Vero cells. Of the other six CVD419 positive

LG. Hack et al. /ht.

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Table 1 Selected E. co& strains, possessing plasmids that hybridize with the CVD419 probe, used to develop the serogroup 0157 specific probe Sero~pe/strain

a

Pfasmid size (MDa1

Smaf fragments size (kb) hybrjdiz~g with probe: CVD419

0157:N’I c363-83 P673 ~622l..~S2/0 B9253aDMSl E29962 6391 10041 9321 9691 GlS7:NM 4551 OlS9:W F370-2 OC70/86: II49 b F374-3 O?:H7 F3-2 026:HlX SDss-:lS77 E3787 0103:H2 62R70

VPMl ’

57 64 58 60 59 52 53,3.0 52 53,2.0

II If 6.5 11 11 11 11 11 11.2

2 2 2 2 2 2 2 2 2

53

11

2

56

11.6

52

15

75

8.5

55, 4.6, 3.6, 1.8 s4,44,5.3,4.2

7.7 7.7,x5

44

It, 6.8

12

a Water buffalo isolate (Egypt) ~363-83 was obtained from International ~c~~~~~j~ and ~eb~~e~~~ Centre, ~~~nhagen, Denmark; bovine isolate P673 was from Pubtic Health Laboratory, Sheffield, England; bovine isolates B6221-MSZ/O, B92S3-DMSl were from Centers for Disease Control, Atlanta, GA; human isolate E29962 was from Division of Enteric Pathogens, Central Public Health Laboratory, London, England; human isolates 4S5I, 6391, 9321, 969I, and lOO4I were from Ohio Public Health Laboratory, Coiumbus, OH* beef isolates F370-2, F3?4-3, and F3-2 were from Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; calf isolates SD881577 was from South Dakota Animal Disease Research and Diagnostic Laboratory, Department of Veterinary Science, South Dakota University; human isolate E3787 was from Division of Enteric Pathogens, Central Public Health Laboratory, London, England; and 62R70, a single plasmid bearing derivative of a serotype 0103:H2 isolate (8X-l) from a human diarrhea1 case was from Division of Enteric Pathogens, Central Public Health Laboratory, London, England. b OC70/% is a provisional 0 group that has not yet been numbered. ’ Hybridization temperature was 42°C; 62R70 hybridized at this temperature, but not at 45°C.

strains, three fiitrates were positive and three were negative. Crnly the Vero cytotox:in producing strains hybridized with one or both of the two VT probes. Ali of these 16 strains had a large plasmid which hybridized with the CVD419 probe (Table 1). When plasmid DNA digests of the 16 strains were electrophoresed on agarose gel, fragments found to be specific for 0157 : H7 E. cc&

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L.G. Huck et al. /IN. J. Food Microbiology 25 (1995) 2X7-287

Table 2 Results of 10 candidate DNA probe hydridizations (42°C) with E. coli serotypes a Candidate serogroup OI.57 probe fragments (kb)

hybridized with:

SmaI

4.3 2.0 1.6 BarnHI 4.0 1.0

All serotypes tested 0157:H7,0157:NM, 0103:H2 O1S3:H7,0157: NM, 0159:H7,OC70/86:

H49,0?:H7,026:HIl

0157:H7,0157:NM+ 0103 : HZ AI1 serotypes tested

0159:H7,0(370/86:H49,0?:H7,026:R11,

OW:H7,0157:NM, 0157:H7,0157: NM, 0157:H7,0157:NM, 0157:H?, 0157:NM, 0157 : H7,01.57 : NM,

026:Hll, 0103:HZ OC70~~6:H49~ O?: Hlil, 026:Hll 026:Hl1,0103:H2 026:Hll, 0103:HZ 0159 : H7, O? : H7,026 : Hl 1

SmaI and BamNI

1.8 1.5 1.2 1.1 1.0

a All strains listed in Table 1 were tested. These probes were prepared from plasmid DNA obtained from 6391.

were 4.3, 2.0 and 1.6 kb (S’maI); 4.0 and 1.0 kb (BmaHI); and 1.8, 1.5, 1,2, 1.1 and 1.0 kb (&zaI and BumHI), Plasmid DNA fragments of these sizes from strain 6391 were excised from low melting point agarose and radiolabeled as probes for hybridization with each of the 16 E. coli strains. The SmaI 2.0 kb radiolabeled probe WMl) bybr~d~ed with all 10 of the serogroup 0157 : H7 E. CO& strains and none of the non-0157 strains, except for the serotype 0103:H2 derivative strain 62R70 (Table 2). This VPMl probe was more specific than other candidate probes. It hybridized with a corresponding 2.0 kb SmaI fragment of Southern blots of the 10 probe-positive serogroup 0157 EHEC, but also with a larger 12 kb fragment of the serotype 0103:HZ strain (Fig. 11.

Notes to table 3: a Strains that hybridized with the VPMl probe at 42”C, but did not hybridize with VPMl at 45°C. b Strains that hybridized with VPMl at 42”C, but had a trace response with the VPMl probe at 45°C. ’ The 0103: I-X2derivatives are from strain S22-1 which was VT2 positive and CVD419 probe-positive. Strain 62R64 bears pDEP13 which does not hybridize with the CVD4f9 probe> and strain 62R70 bears pDEPl2 which does hybridize with this probe. d Other bacteria1 species from various countries and sources (number of strains): Aeromomzs ~ydropb~~a (I), Arizona (l), Acinetobucter c~lcoacetic~ (l), ~~~~0~~~1~~ (l), ~c~i~~ subtile (I), Bordetella bronchjsept~c~ (l), Citrobacter fre~~dii (l), E~erobacter nerogenes (41, Ente~~c~ fecalis (l), ~e~~ll~ pneumoniae (21, Mo~ane~~ morganii (I), P~teure~~~ rn~l~~~da (Z), Proteus ~.rabii~ Of, Proteus &g&s f2)* P~e~d~~~~ aeruginosa (31, ~a~~e~a agona (2), ~~one~~a enter&&% f2), ~~~~~e~ta monte&eo (2), ~~~rn~el~~ ~p~mur~urn (21, Serratia marcescens (31, ShigeUa Group 3 , Shige~fa boydii (I}, Shigelra flexneri (I), SkigeIla sonnei (11, Staphykxoccus aweus (l), ~rr~p~~~~~ e&sin&s (l), Streptococcus fecalis (1).

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When the VPMl probe was hybridized with colony blots from the second set of 107 E. coli and 41 non-E. coli isolates, it correctly identified all 49 serogroup 0157 EHEC strains (Table 3). However, it also hybridized with 5 VT or CVD419 probe-positive strains of the following serotypes: 022,OlOl: H7, 045 : NM, Table 3 Hybridization of EHEC, ETEC and other bacterial strains with CVD419 and VPMl probes Classifical ion

Serogroup 0157 EHEC 0157 : H7 0157: NM

Countries of origin

No. of strains from: human

animals

meat

CVD419

VPMl

US, Canada. UK Egypt US, UK

31

10

3

+

+

0

0

+

+

0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

3 2 0 0 0 3 1 0 0 0 1 1 1 0 1 0 1 0 15 1 1 0 0 0 0 0 0

0 0 1 1 1 0 0 1 1 1 0 0 0 1 0 1 0 1 0 0 0 2 1 1 1 1 0

+

UK

1

0

0

us us us

0 0 0

us

0

3

0

d

d

d

d

Other VT and/or CVD419 positive E. coli O? : NM US O? : NM us O?:H7 Thailand O? : H45 Thailand 04 : H2l Thailand 05 : NM us 05 : NM us Oll:H:! Thailand 022 : HI< Thailand 022,O LO1: H7 Thailand 026 : HI 1 US, UK 045 : NM us 045 : H:!, 6, 12 us 054 : H21 Thailand 069 : NM us 076 : H’r Thailand 0103 : H2, 12 us OllO:H16 Thailand 0lll:NM us 0lll:NM us 0lll:Hll us 0117:H8 Thailand 0146:HlO Thailand 0149 : H45 Thailand 0159:H7 Thailand OC7O/SO : H49 Thailand 0103 : H2 derivative: 62R70 ’ UK 0103: H2 derivative: 62R64 ’ Serogroup 0157 ETEC 0157:hM 0157:K88ac:HlO 0157:K88ac:H13 Non-serogroup 0157 ETEC 0149: K87,88 Other species

Hybridization with probe

0 0 0

+ + +

_ _ _ _ _

+ + + + + + + + +

_a _b _ _ _=

_b _

+ + + + + + + +

_ _ _ _h

II Fig. 1. Autoradiography of Southern transfer of Smal restrictian fragments of E. coli plasmids that hybridized with the (I) CVD419 probe or with the (II) VPMl probe. Lanes: A, ~363-83; B, Pfi73; C, B6221-MS2/0; D, B9253DMSl; E, E29962; F, 6391; G. 10041; H, 4551; I, 9321; .I. 9691; K, F370-2; L, F374-3; M, FJ-2; N, SD88-1577; 0, E3787; P, 62R70. Serotypes of the lanes are: A, B, C and D (bovine) 0157:H7; E, F, G, I and J (human) 0157:H7; H (human) 0157:NM; K (bovine) 0159:H7; L (bovine) 0(370/86:H49; M (bovine) G?:H7; N (bovine) 026:Hll; 0 (human) 026:Hll; and P (human) 0103:H2.

L.G. Huck et al. /ht.

A

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B

Fig. 2. Colony hybridization using the 2 kb 35S-labeled probe (VPMl). A, 42°C hybridization; B, 45°C hybridizataon. Identification of the numbered colonies: 1,4, 5, 6, 7 and 10 are 0157: H7; 2 is 0103 :H2; 3 and 12 are 0157:NM; 8 and 11 are 026:Hll; 9 is 045:NM; and 13 is a K12 negative control.

067 : Hi’, 0103 : H2, and 0103 : H2,12. The VPMl probe did not hybridize with any of the remaining 45 non-0157 strains that were VT or CVD419 probe-positive, 5 serogroup 0157 EHEC strains, 3 non-0157 ETEC strains, nor the 41 non-E. coli strains. With the more stringent condition of overnight hybridization at 45°C (vs. 42”(Z), 2 of the 5 strains that tested false-positive gave negative results (022,OlOl: H7 and 076 : H7) and the other 3 showed only trace responses (045 : NM and the two 0103 derivative strains) (Table 3). The trace responses were easily distinguishable from a positive response (Fig. 2). Hybridization results for the other strains remained unchanged. The %zaI Southern fragments that hybridized with the CVD419 probe were all larger than the fragments that hybridized with the VPMl probe (Table 1). Thus the VPMl probe does not include any portion of the CVD419 probe. VPMl probe hybridiz~~tion with the Southern transfers of SmaI digested plasmid DNA indicated that the 2.0 kb fragment is highly conserved among serogroup 0157 EHEC strains (Fig. 1). Southern transfer hybridizations with the CVD419 kb probe revealed that the hybridizing SmaI fragment was not as highly conserved in these serogroup 0157 EHEC strains (Fig. 1). Further examination of the VPMl 2.0 kb probe fragment could determine if this region encodes for virulence properties, such as intestinal colonization, adherence and the ability to produce attaching and effacing lesions. It would also be interesting to compare the sensitivity and specificity of the VPMl probe with that of the probe developed from the cue gene homologue (Beebakhee et al., 1992) and the oligolnucleotide probe reported to be specific only for 01.57 : H7 E, coli strains (Feng, P., 1993). The 107 E. coli strains were originally isolated from humans (n = 40), animals (n = 49) and meat (n = 18) samples from 5 different countries: US, Canada, UK, Egypt and Thailand (Table 3). The 0157 EHEC strains, originating from humans, animals and meat from four of these countries (US, Canada, UK and Egypt), were correctly identified by the VPMl probe. Thus the results of the present study indicate that the VPMl derived probe has potential as a sensitive and specific method for food quality assurance testing and epidemiologic investigations. Labo-

286

L.G. Huck et al. /ht.

ratories can utilize this VPMl EHEC pathogens.

.I. Food Microbiology 25 (1995) 277-287

probe to detect both 0157: H7 and 0157 : NM

Acknowledgements

We thank G.A. Willshaw of the Division of Enteric Pathogens, Central Public Health Laboratory, Colindale, London, England, for the VT1 and VT2 probes. The CVD419 probe was kindly provided by M.M. Levine, Center for Vaccine Development, University of Maryland, School of Medicine, Baltimore. We are also indebted to the following persons for providing bacterial cultures: F. Orskov, I. Orskov, P.A. Chapman, J.G. Wells, SM. Scotland, R. Genevive, M. Bundesen, P. Escheverria, D.H. Francis, J.I. Speirs, H. Lior, P.M. Sherman, R.A. Wilson, W.C. Swoager and A.E. Lawrence. Supported in part by a grant from the U.S. Army.

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