Prevalence and characteristics of Escherichia coli O157 from major food animals in Korea

International Journal of Food Microbiology 95 (2004) 41 – 49 www.elsevier.com/locate/ijfoodmicro

Prevalence and characteristics of Escherichia coli O157 from major food animals in Korea Mi-Yeong Jo a, Ji-Hyun Kim b, Jae-Hyang Lim a, Mi-Young Kang a, Hong-Bum Koh a, Yong-Ho Park b, Do-Young Yoon c, Joon-Seok Chae d, Seong-Kug Eo d, John Hwa Lee d,* a

College of Veterinary Medicine, Chonnam National University, Kwangju 500-757, South Korea b College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea c Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, South Korea d College of Veterinary Medicine, Chonbuk National University, Chonju 561-756, South Korea

Received 23 September 2003; received in revised form 22 December 2003; accepted 5 January 2004

Abstract Escherichia coli O157:H7/NM (E. coli O157) is now recognized as an important cause of diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome worldwide. There have been several cases of human E. coli O157 infection in Korea since it was first isolated from a patient with hemolytic-uremic syndrome in 1998. Meat, other foods, and recreational and drinking water contaminated with animal feces are probably the major sources of the E. coli O157 infection. In this study, we investigated the prevalence of E. coli O157 in fecal and meat samples of cattle, pigs and chicken in Korea from April 2000 to July 2002. Eightysix (3.03%) of 2843 samples were positive for E. coli O157. Most of the E. coli O157 strains were isolated from fecal samples of beef and dairy cattle from May to October of each year. Of 86 E. coli O157 isolates, 73 were serotype O157:H7 and 13 were serotype O157:NM. Polymerase chain reaction (PCR) analysis of E. coli O157 virulence markers revealed that all O157:H7/ NM isolates were positive for EhlyA, eaeA and rfbO157, and 77 isolates were positive for stx1 and/or stx2. Cytotoxicity analysis revealed that many of the E. coli O157 isolates showed high cytotoxicity on Vero cells. Our data suggest that the majority of Korean E. coli O157 isolates from food animals can cause serious diseases in humans. D 2004 Elsevier B.V. All rights reserved. Keywords: E. coli O157; Prevalence; Genetic and phenotypic characteristic; Food animal

1. Introduction Escherichia coli O157:H7/NM (E. coli O157) has been globally recognized as an important food-borne * Corresponding author. Tel.: +82-63-270-2553; fax: +82-63270-3780. E-mail address: [email protected] (J.H. Lee). 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.01.016

pathogen since the outbreak was first reported in the United States in 1982 (Allerberger et al., 1996; Cordovez et al., 1992; Griffin, 1995; Kim et al., 1998; Riley et al., 1983; Tamura et al., 1996). More than 30 countries have reported E. coli O157 outbreaks in humans. In Japan, 29 outbreaks of E. coli O157 infections were reported between 1991 and 1995 (NIH, Japan, 1996). In 1996, multiple outbreaks of

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E. coli O157 infection occurred in Japan and involved 11,826 cases and 12 deaths (Watanabe et al., 1999). Hundreds of cases have been reported yearly since then. There have been several cases of the human infections in Korea with a gradual increase of incidence since the first isolation of E. coli O157 from a patient with hemolytic-uremic syndrome in 1998 (Kim et al., 1998; NIH, Korea, 2001 –2003). E. coli O157 is a major cause of bloody diarrhea and is also associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans (Griffin, 1995; Karmali, 1989). The high morbidity and mortality of these diseases have rendered E. coli O157 as one of major threats to public health (Minami, 1997). E. coli O157 outbreaks are associated with the consumption of raw or undercooked meat of food animals and other foods contaminated with animal feces (Su and Brandt, 1995). Cattle have been implicated in the majority of food-borne outbreaks of E. coli O157 (Laegreid et al., 1999). Other food animals such as pigs and chickens also appear to be reservoirs of these organisms (Beutin et al., 1993). The pathogenicity of E. coli O157 is associated with a number of virulence factors, including Shiga toxin 1 and 2 (encoded by the genes stx1 and stx2), intimin (encoded by the gene eae), and the plasmid-encoded enterohemolysin (encoded by the gene hly) (Barrett et al., 1992; Beutin et al., 1993, 1995; Schmidt et al., 1995). Shiga toxins appear to play a major role in the pathogenesis of hemorrhagic colitis and hemolytic-uremic syndrome. Intimin facilitates adherence to intestinal villi and effacement. Several methods have been developed for the detection of E. coli O157. Detection of these pathogens can be accomplished either by testing broth culture of suspected foods or feces with Vero cell cytotoxicity assays or by enzyme-linked immunosorbent assay (Konowalchuk et al., 1977; Smith and Scotland, 1993). DNA colony blot hybridization can also be used to detect these pathogens by identifying genes encoding Shiga toxins and intimin (Samadpour et al., 1990). Special biochemical media and diagnostic kits containing latex reagents directed against O157 and H7 antigens have been developed for selective isolation and specific detection of E. coli O157 (March and Ratnam, 1986; Ojeda et al., 1995; Sowers et al., 1996). To isolate E. coli O157, specimens can be directly plated onto selective and/or differential agars or can be selectively enriched in

broth followed by plating onto selective and/or differential agars. This selective enrichment step is made more effective by immunomagnetic separation using beads coated with O157-specific antibody before plating onto agar (Hoyle, 2000). Polymerase chain reaction (PCR) has become a useful diagnostic tool, and various studies have also used PCR techniques to screen broth enrichment cultures for the presence of E. coli O157 (Gannon et al., 1997a; Gannon et al., 1997b; Karch and Meyer, 1989). Because meat and other foods contaminated with animal feces are probably the major sources of the E. coli O157 infection, in this study we investigated the prevalence of E. coli O157 in fecal and meat samples of cattle, pigs and chicken in Korea. To increase sensitivity, specimens were cultivated in selective enrichment broths followed by immuno-magnetic bead separation before plating onto SMAC agar (Elder et al., 2000; McDonough et al., 2000). Subsequently, suspect E. coli O157 isolates were characterized by biochemical, genetic, and serological assays as well as Vero cell cytotoxicity assay to determine the actual virulence potential. Our study was the first systematic investigation on E. coli O157 in Korea and the data add to information of global epidemiology of E. coli O157.

2. Materials and methods 2.1. Collection of specimens Fecal and meat samples of beef cattle, dairy cattle, pigs, and chicken were collected at monthly intervals from 15 slaughterhouses, 7 meat processing facilities, 60 farms, and 11 food stores, which were located through out Korea, including the provinces of Gyeonggi, Chungcheong, Gyeongsang, Gangwon, and Jeolla, from April 2000 to July 2002. A total 2843 samples were collected from fecal samples of beef cattle (864), dairy cattle (990), pigs (345) and chicken (418), and from samples of retail beef (94), pork (80) and chicken meat (52). At each of the sampling locations, 2 to 5 or 5 to 15 samples were collected per farm and food store or slaughterhouse and meat processing facility, respectively. For chicken fecal samples, one pooled fecal sample was taken per flock from 98 different places. All samples were immediately transported to the laboratory in ice-cooled containers.

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2.2. Isolation of presumptive E. coli O157 The microbiological examination was started within 6 h after collection of the samples. A 10% suspension (5– 10 g of sample/50– 100 ml of broth) was prepared by homogenizing feces and meat in GN broth (Difco, Detroit, MI, USA) containing 8 mg/ l vancomycin (Sigma, St. Louis, MO, USA), 0.05 mg/l cefixime (Dynal, Oslo, Norway), and 10 mg/ l cefsuludin (Sigma), or in modified E. coli broth containing 20 mg/l novobiocin (Sigma). The suspension was incubated at 37 jC for 6 to 12 h followed by immunomagnetic bead separation as described previously (Elder et al., 2000). In brief, 1 ml of enrichment broth culture was incubated with 20 Al of anti-O157 immunomagnetic beads (Dynal) on a rocker (60 cycles/min) at room temperature for 30 min. The immunomagnetic bead suspensions were washed three times in 1 ml of PBS containing 0.05% Tween 20 and resuspended in 100 Al of PBS. Fifty microliter of the bead suspension was spread onto sorbitol MacConkey (Difco: SMAC) or choromogenic SMAC agar plates (Biomerieux, Lyon, France) containing 0.05 mg/l cefixime and 2.5 mg/l potassium tellurite (Dynal). Sorbitol-negative colonies exhibiting colony morphology typical of E. coli O157 were selected and tested by using O157 latex reagents (Oxoid, Basingstoke, UK), or ImmunoCard Stat E. coli O157:H7 (Meridian Diagnostics, Cincinnati, OH, USA). Positive isolates were considered as presumptive E. coli O157 and were confirmed by biochemical test, motility assay, serotyping, and PCR. 2.3. Biochemical and motility assays Biochemical assays of isolates was completed with triple sugar iron, phenylalanine deaminase, maltose, mannitol, Indole, methyl red, Voges-Proskauer, urease, citrate and h-glucuronidase (MUG) or with API 32E (Biomerieux). Motility was examined by agar stab method using motility GI medium (Difco). 2.4. O157 and H7 serotyping Serotypes of the suspect E. coli O157 isolates were examined by using O157 and H7 antisera (Difco). For O157 serotyping, the suspect isolate was cultured in

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TSB (Difco) at 37 jC for 18 h and centrifuged at 5000 rpm for 5 min. After removal of the supernatant, the pellet was adjusted in PBS to 1  109 cfu/ml. The suspension was boiled at 95 jC for 30 min and diluted 1:1 with 1% formalin in physiological saline solution. Other conditions were according to the manufacturer’s instructions. For H7 serotyping the isolate was grown overnight in heart infusion broth (HIB) (Difco) at 37 jC and was then serially passaged in motility GI medium at least three times to ensure that the isolate was highly motile. The final passaged culture was grown overnight in HIB or Veal infusion broth (Difco). One milliliter aliquot of the overnight broth culture was mixed with 1 ml of 1% formalin/saline solution. The diluted H7 antiserum (1:500 in saline) was mixed with the same volume of the formalized broth culture and incubated in a 50 jC water bath for 1 h and observed for agglutination. 2.5. PCR amplification E. coli O157 isolates were examined by polymerase chain reaction (PCR) assay to determine the presence of Shiga toxin 1 and 2 genes (stx1 and stx2) (Jackson et al., 1987a,b), E. coli attaching-andeffacing (eaeA) (Yu and Kaper, 1992), enterohemolysin (EhlyA) (Schmidt et al., 1995), and the E. coli O157 specific gene rfbEO157 (Schmidt et al., 1995). Template DNA was prepared from pure culture of isolates, grown in mEC or TSB for 18 h at 37 jC. One and a half milliliters of culture was centrifuged, and the pellet was resuspended in 0.1 ml of InstaMatrix (Bio-Rad, Richmond, CA, USA). The suspension was heated at 100 jC for 10 min and then centrifuged at 12,000  g for 5 min. The supernatant was used for the PCR template. Multiplex PCR or non-multiplex PCR was performed in a 50 Al final reaction volume containing 0.2 AM primers, 200 AM dNTPs, 10 mM Tris – HCl [pH 8.3], 50 mM KCl, 2.5 mM MgCl2, 1 U of Taq polymerase, and 4 Al of template DNA. Cycling condition in a GeneAmp 2400 Thermocycler (Perkin-Elmer Cetus, Norwalk, CT, USA) was as follows: initial denaturation at 94 jC for 3 min; 35 cycles of 94 jC for 20 s, 58 jC for 40 s, and 72 jC for 90 s; and final extension at 72 jC for 5 min. PCR amplicons were run on a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV illumination.

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2.6. Vero cell cytotoxicity assay The cytotoxicities of the E. coli O157 isolates were determined using Vero cells and compared with the cytotoxicities of the E. coli O157 strains of ATCC43889 and ATCC43894. For toxin preparation, bacterial cultures were grown in 15 ml of brain heart infusion (BHI) broth at 37 jC for 24 h with constant agitation. A 1.5-ml aliquot of bacterial cultures was centrifuged (8160  g, 3 min) and cell-free supernatants were stored in sterile tubes. Cell pellets were resuspended in 75 Al of polymyxin B sulfate (Sigma) solution (2 mg/ml in PBS) and incubated in a shaker-incubator for 30 min to release cell bound toxins. After centrifugation (8160  g, 5 min),

the supernatants were collected and combined with the original cell-free supernatants and filtered through 0.2Am disc filters (Corning, NY, USA). The filtrates were either used immediately or held at 20 jC. African green monkey kidney (Vero) cells were maintained in Dulbecco’s modified Eagles medium (DMEM) (Sigma) with 5% fetal bovine serum. The cells were grown at 37 jC with 5% CO2 under humidity. The cells were adjusted to 105 cells per milliliter in serum-free medium. Half milliliter aliquots of the adjusted cells were seeded into 24-well plates and were incubated for 1 h at 37 jC in 5% CO2. Half milliliter DMEM medium with 5% fetal bovine serum was added to each well and re-incubated for 24 h. The cell monolayers were inoculated with 0.5 ml of

Table 1 Isolation of E. coli O157 from major food animals in Korea, 2000 to 2002 Date

2000, Apr May Jun Jul Aug Sep Oct Nov Dec 2001, Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2002, Jan Feb Mar Apr May Jun Jul Total

Cattle

Pig

Chicken

Samp No (fecal/meat)

Posi No (fecal/meat)

Samp No (fecal/meat)

Posi No (fecal/meat)

Samp. No (fecal/meat)

Posi No (fecal/meat)

64/0 44/10 61/7 30/3 79/15 170/10 194/10 60/0 59/0 73/0 71/0 160/16 70/0 87/0 120/0 141/13 32/0 42/0 96/10 54/0 0/0 26/0 0/0 0/0 51/0 12/0 25/0 18/0 1854/94

0/0 14/0 9/1 2/0 8/0 8/0 6/0 0/0 0/0 0/0 0/0 0/0 0/0 9/0 4/0 9/1 0/0 0/0 2/0 1/0 0/0 0/0 0/0 0/0 0/0 2/0 3/0 4/0 81/2

21/0 10/5 0/0 20/16 10/5 0/0 0/0 0/0 20/7 20/7 15/12 7/0 0/0 12/0 52/0 84/7 31/0 0/0 0/0 0/0 20/16 15/5 0/0 0/0 0/0 0/0 0/0 8/0 345/80

0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/1 1/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/2

0/0 0/0 74/7 27/0 70/8 28/0 0/0 29/0 27/0 27/0 72/9 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 35/13 29/15 0/0 0/0 0/0 0/0 418/52

0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

Samp No: Numbers of animals or flocks (chicken) sampled. Posi No: Numbers of presence of E. coli O157 from the samples.

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the toxin preparations and were incubated for additional 24 h. Half milliliter BHI broth was used as negative control. To determine the cytopathic effects on Vero cells, the cell monolayers were evaluated for the degree of cell death by staining with Trypan blue (0.4%) and examining under a Nikon phase contrast microscopy (400 ), and were scaled arbitrarily to values of 0 to 10. Higher degree indicated that many Vero cells were damaged.

3. Results 3.1. Overall prevalence of E. coli O157-positive samples Of 2843 samples, 86 (3.03%) were positive (Table 1). The numbers of individual positive samples from fecal samples of beef cattle, dairy cattle, pigs, and chicken were 15 (1.7%), 66 (6.7%), 1 (0.3%), and 0,

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Table 2 Characteristics of E. coli O157 isolates from major food animals in Korea Origin Serotype No. of Presence of the following genes isolates rfbE hlyA eaeA stx1

stx2

Cattle Cattle Cattle Cattle Cattle Cattle Cattle Pork Pork Pig

+ – + – + – + + – –

H7 H7 H7 H7 NM NM NM H7 H7 NM

8 7 48 8 10 1 1 1 1 1

+ + + + + + + + + +

+ + + + + + + + + +

+ + + + + + + + + +

+ + – – + + – + + –

respectively. The numbers of positive samples from beef meat, pork, and chicken meat were 2 (2.1%), 2 (2.5%), and 0, respectively. Most of the E. coli O157 isolates were detected from May to October of each year. The E. coli O157 isolates were isolated from five different provinces in Korea without any significant regional difference in prevalence (Fig. 1). 3.2. Phenotypic characteristics of isolates Of 86 E. coli O157 isolates, 73 were serotype O157:H7, and 13 were serotype O157:NM (Table 2). Eleven of the O157:H7 were isolated from fecal samples of beef cattle, 58 were from fecal samples of dairy cattle, two were from beef meat samples, and two were from pork samples. Four of the O157:NM were isolated from fecal samples of beef cattle, eight were fecal samples of dairy cattle, and one was from pig fecal sample. All 86 E. coli O157 isolates found in this study were negative for sorbitol fermentation. 3.3. Presence of virulence determinants

Fig. 1. Provinces in Korea and prevalence of E. coli O157 in each region. Numbers in parentheses represent E. coli O157 positive samples per total collected specimens from indicated provinces.

Genetic profiling for E. coli O157 virulence markers such as stx1, stx2, eaeA, EhlyA, and rfbO157 was performed by PCR. All the E. coli O157:H7/NM isolates were positive for EhlyA, eaeA, rfbO157, and stx1 and/or stx2, except nine isolates that were stx negative (Table 2). Of 77 stx positive isolates, 9, 49, and 19 isolates have stx1, stx2, and both stx1 and stx2, respectively.

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Table 3 Vero cell toxicity of E. coli O157 isolates Toxicitya

9.1 – 10.0 8.1 – 9.0 7.1 – 8.0 6.1 – 7.0 5.1 – 6.0 4.1 – 5.0 3.1 – 4.0 2.1 – 3.0 1.1 – 2.0 0.0 – 1.0

Number of isolates with presence of stx1, stx2

stx1

stx2

None of stx

3 3 6 1 0 0 2 2 2 0

0 0 0 1 1 2 0 1 3 1

0 2 5 2 6 7 8 8 5 6

0 0 0 1 2 3 1 1 1 0

Toxicities of reference strains ATCC43889 (E. coli O157:H7/stx2) and ATCC43894 (E. coli O157:H7/stx1, stx2) were shown 7.4 and 9.6, respectively, while negative control (medium) was 0.0. a Vero cell toxicities for the isolates were arbitrarily scaled to values from 0 to 10 (0, non-toxic to Vero cells; 10, toxic to 100% Vero cells).

3.4. Vero cell cytotoxicity The cytotoxicity of toxin preparations from the positive control strains ATCC43889 (E. coli O157:H7/ stx2) and ATCC43894 (E. coli O157:H7/stx1, stx2) were scaled at 7.4 and 9.6, respectively. Toxin preparations from all 86 E. coli O157 isolates were screened for cytotoxicities, and data were presented in Table 3. The degrees of toxicity of the isolates varied. Many of the isolates positive for stx2 or both stx1 and stx2 induced higher than degree 7 of cytotoxicity while all the isolates positive for only stx1 induced lower than degree 7. In addition, six isolates positive for stx2 induced very weak cytotoxicity (lower than degree 1). Interestingly, the isolates negative for stx still showed some degrees of cytotoxic effects on Vero cells (degrees between 1 and 7).

4. Discussion E. coli O157 can causes severe disease and death in humans (Elder et al., 2000; Karmali et al., 1985; Su and Brandt, 1995) and has also emerged as an important foodborne pathogen for humans in Korea (NIH, Korea, 2001-2003). E. coli O157 is one of the most frequently isolated serotypes from human enterohe-

morrhagic E. coli infections in Korea (approximately 30% of the enterohemorrhagic E. coli infections are due to E. coli O157). Human infections of E. coli O157 have been mostly attributed or linked to food products from animals (Elder et al., 2000; Kim et al., 1998; Paton et al., 1996; Riley et al., 1983). Cattle especially have been implicated as the principal reservoir of E. coli O157 (Chapman et al., 1993). In the present study, the prevalence of E. coli O157 in cattle, pigs, chicken, and their respective meats in Korea was investigated using the method of immunomagnetic separation (Elder et al., 2000; Heuvelink et al., 1998; Wells et al., 1983). Of 2843 samples collected for a period of 28 months in the five different provinces of Korea, approximately 3% (86) of the samples harbored E. coli O157. Of 86 E. coli O157 isolates, 83 were isolated from fecal and meat samples of beef and dairy cattle. All the cattle isolates were positive for eaeA and EhlyA, and most of them were positive for stx1 and/or stx2. These indicate that cattle are important reservoirs of E. coli O157 in Korea, and the isolates are characteristic of E. coli O157 stains causing illness in humans. Prevalence surveys have been previously conducted on E. coli O157 from cattle fecal samples (Chapman et al., 1993; Elder et al., 2000; Hancock et al., 1997; Wells et al., 1983). The overall prevalence of E. coli O157 was variable depending on isolation methods and geographical location. The recent investigation revealed that 28% of cattle fecal samples harbored E. coli O157 in the Midwestern United States, using the similar isolation method (Elder et al., 2000). The E. coli O157 in this study were more frequently isolated from dairy cattle samples [66/990(6.7%)] than from beef cattle samples [15/864(1.7%)]. The samples of cattle were collected from mainly Holstein dairy cattle and Korean native beef cattle. These implied that Holstein dairy cattle may be prone to more producing this organism than the Korean native beef cattle in this environment although no definitive conclusions can be drawn from this study. Prevalence of E. coli O157 in pigs and chicken were relatively lower than that in cattle. Three E. coli O157 (0.7%) were isolated from 425 pig specimens; two from retail pork samples and one from a pig fecal sample, and no E. coli O157 were isolate from chicken specimens in this study. These indicate that pigs and chicken may be less suitable reservoir of E.

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coli O157 than cattle. Nonetheless, several recent studies revealed that pig and poultry are potentially important sources of human E. coli O157 infections (Doyle and Schoeni, 1987; Hakkinen and Schneitz, 1996; Heuvelink et al., 1999; Read et al., 1990; Tozzi et al., 1994). The two isolates from the pork specimens were positive for eaeA and EhlyA. These isolates also contained Shiga toxin genes and were verocytotoxigenic. These indicate that pork may be an important source of E. coli O157 causing illness in humans. It is uncertain, however, whether the two isolates originated directly from pigs since E. coli O157 was rarely isolated from pig fecal specimens. It is highly possible that the two pork isolates were originated by cross-contamination from beef products or by contamination with bovine feces at slaughter since both cattle and pigs are often processed at the same slaughter facilities in Korea. One isolate from the pig fecal specimens was positive for eaeA and EhlyA, but negative for stx genes. The Shiga toxin may not be obligatorily produced by E. coli O157 associated with human diseases since E. coli O157 that do not produce Shiga toxin can be associated with diarrhea and hemolytic-uremic syndrome in humans (Schmidt et al., 1999). Although the isolate from the pig fecal specimens did not harbor stx genes, it contained other virulence genes such as eaeA and EhlyA, and appeared to be verocytotoxigenic. Therefore, pigs also can be sources of potentially virulent E. coli O157 for humans in Korea. The occurrence of E. coli O157 was apparently affected by temperature since there was a trend toward higher prevalence in the warmer spring and summer months during this study. Other studies also supported higher prevalence in spring and summer (Elder et al., 2000; Hancock et al., 1997; Wells et al., 1983). These indicate that season is a risk factor for E. coli O157 outbreaks. The E. coli O157 were isolated from the five different provinces in Korea with similar prevalence patterns. This indicates that these organisms are widely spread in Korea. PCR is generally considered to be the most sensitive means of determining genes of specific virulence factors of E. coli O157 (Jackson et al., 1987a,b; Paton and Paton, 1999; Schmidt et al., 1995; Yu and Kaper, 1992). PCR analysis of E. coli O157 virulence markers revealed that all 86 E. coli

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O157 isolates were positive for EhlyA, eaeA, and rfbO157. Thus, all these strains are likely to produce accessory virulence factors such as intimin and the enterohemolysin (Barrett et al., 1992; Beutin et al., 1993, 1995; Schmidt et al., 1995). In addition, 68 of 86 isolates (79%) contained stx2 (Table 2). Vero cell assay is presently used as a confirmatory test to determine the actual virulence potential of E. coli O157 isolates (Konowalchuk et al., 1977; Roberts et al., 2001). Vero cell assay also showed that stx2 positive isolates revealed generally higher cytotoxicities than other isolates. Within the human diseaseassociated strains, those producing Shiga toxin type 2 appear to be more commonly responsible for serious complications such as HUS than those producing only Shiga toxin type 1 (Kleanthous et al., 1990; Ostroff et al., 1989). These indicate that the majority of E. coli O157 isolates in Korea may cause serious diseases in humans. On the other hand, Vero cells with the toxin preparations from six isolates positive for stx2 remained nearly unaffected. There was a report that a stx2 positive E. coli O157 isolate was not cytotoxic to Vero cells (Jinneman et al., 2000). A 1310 bp insertion sequence was identified from this isolate and the insertion interrupted the carboxyl end of the A subunit coding region of the stx2 gene. Since the isolate did not produce a fully functional Stx 2, the Vero cells were likely unaffected. It is presently uncertain why the six stx2 positive strains did not show cytotoxicity and why stx negative strains showed cytotoxicities. Further molecular and genetic studies are required to define factors associated with Vero cell cytotoxicity.

Acknowledgements This study was supported by the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, and the Brain Korea 21 Project in 2003, Republic of Korea.

References Allerberger, F., Rossboth, D., Dierich, M.P., Aleksic, S., Schmidt, H., Karch, H., 1996. Prevalence and clinical manifestations of

48

M.-Y. Jo et al. / International Journal of Food Microbiology 95 (2004) 41–49

Shiga toxin-producing Escherichia coli infections in Austrian children. Eur. J. Clin. Microbiol. Infect. Dis. 15, 545 – 550. Barrett, T.J., Kaper, J.B., Jerse, A.E., Wachsmuth, I.K., 1992. Virulence factors in Shiga-like toxin-producing Escherichia coli isolated from humans and cattle. J. Infect. Dis. 165, 979 – 980. Beutin, L., Geier, D., Steinruck, H., Zimmermann, S., Scheutz, F., 1993. Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. J. Clin. Microbiol. 31, 2483 – 2488. Beutin, L., Geier, D., Zimmermann, S., Karch, H., 1995. Virulence markers of Shiga-like toxin-producing Escherichia coli strains originating from healthy domestic animals of different species. J. Clin. Microbiol. 33, 631 – 635. Chapman, P.A., Siddons, C.A., Wright, D.J., Norman, P., Fox, J., Crick, E., 1993. Cattle as a possible source of verocytotoxinproducing Escherichia coli O157 in man. Epidemiol. Infect. 111, 439 – 447. Cordovez, A., Prado, V., Maggi, L., Cordero, J., Martinez, J., Misraji, A., Rios, R., Soza, G., Ojeda, A., Levine, M.M., 1992. Enterohemorrhagic Escherichia coli associated with hemolytic-uremic syndrome in Chilean children. J. Clin. Microbiol. 30, 2153 – 2157. Doyle, M.P., Schoeni, J.L., 1987. Isolation of Escherichia coli O157:H7 from retail fresh meats and poultry. Appl. Environ. Microbiol. 53, 2394 – 2396. Elder, R.O., Keen, J.E., Siragusa, G.R., Barkocy-Gallagher, G.A, Koohmaraie, M., Laegreid, W.W., 2000. Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing. Proc. Natl. Acad. Sci. 7, 2999 – 3003. Gannon, V.P., D’Souza, S., Graham, T., King, R.K., 1997a. Specific identification of Escherichia coli O157:H7 using a multiplex PCR assay. Adv. Exp. Med. Biol. 412, 81 – 82. Gannon, V.P., D’Souza, S., Graham, T., King, R.K., Rahn, K., Read, S., 1997b. 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. Griffin, P.M., 1995. Escherichia coli O157:H7 and other enterohemorrhagic Escherichia coli. In: Blaser, M.J., Smith, P.D., Ravdin, J.I., Greeenberg, H.B., Guerrant, R.L. (Eds.), Infections of the Gastrointestinal Tract. Raven Press, New York, NY, pp. 739 – 761. Hakkinen, M., Schneitz, C., 1996. Efficacy of a commercial competitive exclusion product against a chicken pathogenic Escherichia coli and E. coli O157:H7. Vet. Rec. 139, 139 – 141. Hancock, D.D., Rice, D.H., Thomas, L.A., Dargatz, D.A., Besser, T.E., 1997. Epidemiology of Escherichia coli O157 in feedlot cattle. J. Food Prot. 60, 462 – 465. Heuvelink, A.E., van den Biggelaar, F.L., Zwartkruis-Nahuis, J., Herbes, R.G., Huyben, R., Nagelkerke, N., Melchers, W.J., Monnens, L.A., de Boer, E., 1998. Isolation and characterization of verocytotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep. J. Clin. Microbiol. 36, 3480 – 3487. Heuvelink, A.E., Zwartkruis-Nahuis, J.T., van den Biggelaar, F.L., van Leeuwen, W.J., de Boer, E., 1999. Isolation and characterization of verocytotoxin-producing Escherichia coli O157

from slaughter pigs and poultry. Int. J. Food Microbiol. 52, 67 – 75. Hoyle, B., 2000. Renewed concerns over E. coli O157:H7 in ground beef. ASM News 66, 331 – 332. Jackson, M.P., Neill, R.J., O’Brien, A.D., Holmes, R.K., Newland, J.W., 1987a. Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli. FEMS Microbiol. Lett. 44, 109 – 114. Jackson, M.P., Newland, J.W., Holmes, R.K., O’Brien, A.D., 1987b. Nucleotide sequence analysis of the structural genes for Shiga-like toxin I encoded by bacteriophage 933J from Escherichia coli. Microb. Pathog. 2, 147 – 153. Jinneman, K.C., Weagant, S.D., Johnson, J.M., Abbott, S.L., Hill, W.E., Tenge, B.J., Dang, N.L., Ramsden, R., Omiecinski, C.J., 2000. A large insertion in the Shiga-like toxin 2 gene (stx2) of an Escherichia coli O157:H7 clinical isolate. Int. J. Food Microbiol. 57, 115 – 124. Karch, H., Meyer, T., 1989. Single primer pair for amplifying segments of distinct Shiga-like toxin genes by polymerase chain reaction. J. Clin. Microbiol. 27, 2751 – 2757. Karmali, M.A., 1989. Infection by verocytotoxin-producing Escherichia coli. Clin. Microbiol. Rev. 2, 15 – 38. Karmali, M., Petric, A., Lim, M., Fleming, C., Arbus, P.C., Lior, G.S., 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 151, 775 – 782. Kim, Y.B., Okuda, J., Matsumoto, C., Morigaki, T., Asai, N., Watanabe, H., Nichibuchi, M., 1998. Isolation of an Escherichia coli O157:H7 strain producing shiga toxin 1 but not shiga toxin 2 from patient with hemolytic uremic syndrome in Korea. FEMS Microbiol. 166, 43 – 48. Kleanthous, H., Smith, H.R., Scotland, S.M., Gross, R.J., Rowe, B., Taylor, C.M., Milford, D.V., 1990. Haemolytic uraemic syndromes in the British Isles, 1985-8: association with verocytotoxin producing Escherichia coli: Part 2. Microbiological aspects. Arch. Dis. Child. 65, 722 – 727. Konowalchuk, J., Speirs, J.L., Starvic, S., 1977. Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18, 775 – 779. Laegreid, W.W., Elder, R.O., Keen, J.E., 1999. Prevalence of Escherichia coli O157:H7 in range beef calves at weaning. Epidemiol. Infect. 123, 291 – 298. March, S.B., Ratnam, S., 1986. Sorbitol – MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J. Clin. Microbiol. 23, 869 – 872. McDonough, P.L., Rossiter, C.A., Rebhun, R.B., Stehman, S.M., Lein, D.H., Shin, S.J., 2000. Prevalence of Escherichia coli O157:H7 from cull dairy cows in New York State and comparison of culture methods used during preharvest food safety investigations. J. Clin. Microbiol. 38, 318 – 322. Minami, S., 1997. On measures for the control of enterohaemorrhagic E. coli O157 in Japan, background paper number 9. WHO Consultation on the Prevention and Control of Enterohaemorrhagic Escherichia coli (EHEC) Infections. World Health Organization, Geneva, Switzerland. National Institute of Health and Infectious Diseases Control Division, Ministry of Health and Welfare of Japan, 1996. Outbreak

M.-Y. Jo et al. / International Journal of Food Microbiology 95 (2004) 41–49 of enterohemorrhagic Escherichia coli O157:H7 infection. Infect. Agents Surveill. Rep. 174, 180 – 181. National Institute of Health Division, Ministry of Health and Welfare of Korea, 2001-2003. Outbreaks of enterohemorrhagic Escherichia coli O157:H7 human infections in Korea. Communicable Disease Information (http://dis.mohw.go.kr/). Ojeda, A., Prado, V., Martinez, J., 1995. Sorbitol-negative phenotype among enterohemorrhagic Escherichia coli strains of different serotypes and from different sources. J. Clin. Microbiol. 33, 2199 – 2201. Ostroff, S.M., Tarr, P.I., Neill, M.A., Lewis, J.H., Hargrett-Bean, N., Kobayashi, J.M., 1989. Toxin genotypes and plasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections. J. Infect. Dis. 160, 994 – 999. Paton, A.W., Paton, J.C., 1999. Direct detection of Shiga toxigenic Escherichia coli strains belonging to serogroups O111, O157, and O113 by multiplex PCR. J. Clin. Microbiol. 37, 3362 – 3365. Paton, A.W., Ratcliff, R.M., Doyle, R.M., Seymour-Murray, J., Davos, D., Lanser, J.A., Paton, J.C., 1996. Molecular microbiological investigation of an outbreak of hemolytic-uremic syndrome caused by dry fermented sausage contaminated with Shiga-like toxin-producing Escherichia coli. J. Clin. Microbiol. 34, 1622 – 1627. Read, S.C., Gyles, C.L., Clarke, R.C., Lior, H., McEwen, S., 1990. Prevalence of verocytotoxigenic Escherichia coli in ground beef, pork, and chicken in southwestern Ontario. Epidemiol. Infect. 105, 11 – 20. Riley, L.W., Remis, R.S., Helgerson, S.D., McGee, H.B., Wells, J.G., 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. N. Engl. J. Med. 308, 681 – 685. Roberts, P.H., Davis, K.C., Garstka, W.R., Bhunia, A.K., 2001. Lactate dehydrogenase release assay from vero cells to distinguish verotoxin producing Escherichia coli from non-verotoxin producing strains. J. Microbiol. Methods 43, 171 – 181. Samadpour, M., Liston, J., Ongerth, J.E., Tarr, P.I., 1990. Evaluation of DNA probes for detection of Shiga-like toxin producing

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Escherichia coli in foods and calf fecal samples. Appl. Environ. Microbiol. 56, 1212 – 1215. Schmidt, H., Beutin, L., Karch, H., 1995. Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL933. Infect. Immun. 63, 1055 – 1061. Schmidt, H., Scheef, J., Huppertz, H.I., Frosch, M., Karch, H., 1999. Escherichia coli O157:H7 and O157:H strains that do not produce Shiga toxin: phenotypic and genetic characterization of isolates associated with diarrhea and hemolytic-uremic syndrome. J. Clin. Microbiol. 37, 3491 – 3496. Smith, H.R., Scotland, S.M., 1993. Isolation and identification methods for Escherichia coli O157:H7 and other Vero cytotoxin producing strains. J. Clin. Pathol. 46, 10 – 17. Sowers, E.G., Wells, J.G., Strockbine, N.A., 1996. Evaluation of commercial latex reagents for identification of O157 and H7 antigens of Escherichia coli. J. Clin. Microbiol. 34, 1286 – 1289. Su, C., Brandt, L.J., 1995. Escherichia coli O157:H7 infection in humans. Ann. Intern. Med. 123, 698 – 714. Tamura, K., Sakazaki, R., Murase, M., Kosako, Y., 1996. Serotyping and categorisation of Escherichia coli strains isolated between 1958 and 1992 from diarrhoeal diseases in Asia. J. Med. Microbiol. 45, 353 – 358. Tozzi, A.E., Niccolini, A., Caprioli, A., Luzzi, I., Montini, G., Zacchello, G., Gianviti, A., Principato, F., Rizzoni, G., 1994. A community outbreak of haemolytic – uraemic syndrome in children occurring in a large area of Northern Italy over a period of several months. Epidemiol. Infect. 113, 209 – 219. Watanabe, Y.K., Ozasa, K., Mermin, J.H., Griffin, P.M., Masuda, K., Imashuku, S., Sawada, T., 1999. Factory outbreak of Escherichia coli O157:H7 infection in Japan. Emerg. Infect. Dis. 3, 424 – 428. Wells, J.G., Davis, B.D., Wachsmuth, I.K., Riley, L.W., Remis, R.S., Sokolow, R., Morris, G.K., 1983. Laboratory investigation of hemorragic colitis outbreaks associated with a rare Escherichia coli serotype. J. Clin. Microbiol. 18, 512 – 520. Yu, J., Kaper, J.B., 1992. Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7. Mol. Microbiol. 6, 411 – 417.