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Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef
International Journal of Food Microbiology 46 (1999) 27–36
Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef Youichi Onoue a , Hirotaka Konuma b , Hiroshi Nakagawa c , Yukiko Hara-Kudo d , e d, Toshiharu Fujita , Susumu Kumagai * a
Kanagawa Prefuctural Public Health Laboratory, 52 -2 Nakao, Asahi-ku, Yokohama 241, Japan Division of Microbiology, National Institute of Health Sciences, 1 -18 -1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan c Tokyo Kenbikyoin Foundation, 44 -1 Nihonbashi hakozaki-cho, Chuo-ku, Tokyo 103, Japan d Department of Biomedical Food Research, National Institute of Infectious Diseases, 1 -23 -1 Toyama, Shinjuku-ku, Tokyo 162, Japan e Department of Epidemiology, National Institute of Public Health, Shirokanedai 4 -6 -1, Minatoku, Tokyo 108, Japan b
Received 22 April 1998; received in revised form 3 September 1998; accepted 27 October 1998
Abstract For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC 1 n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg / l) and potassium tellurite (2.5 mg / l) (CT-SMAC), and agars containing b-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu / 25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu / 25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu / 25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu / 25 g) compared with ground beef (23.9 cfu / 25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing b-glucuronidase substrate in combination with static enrichment incubation in mEC 1 n at 428C. 1999 Elsevier Science B.V. All rights reserved. Keywords: Escherichia coli O157:H7; Detection; Collaborative evaluation; Radish sprouts; Ground beef
*Corresponding author. Tel.: 1 81-3-52851111; fax: 1 81-3-52851176; e-mail: [email protected] 0168-1605 / 99 / $ – see front matter PII: S0168-1605( 98 )00174-3
1999 Elsevier Science B.V. All rights reserved.
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1. Introduction Since its recognition as a food borne disease agent in 1982 (Riley et al., 1983), Escherichia coli O157:H7 has emerged as a significant food borne pathogen. Most outbreaks have been linked to foods and water, beef being most frequently involved as a vehicle of infection (Doyle, 1991). Other foods such as lettuce, cantaloupe, cabbage, unpasteurized apple juice and alfalfa sprouts have also been implicated in some outbreaks (Besser et al., 1993; Ackers et al., 1996; Beuchat, 1996; US Department of Health and Human Services, 1997). A highly publicized E. coli O157:H7 outbreak occurred at Sakai-City, Japan in July, 1996. Hydroponically grown radish sprouts were implicated epidemiologically as the vehicle (World Health Organization, 1996), and again in a ‘‘diffuse outbreak’’ of E. coli O157:H7 infection in March, 1997, in Japan (World Health Organization, 1997). Thus, hydroponically grown radish sprouts are concluded to be one of the foods at high risk for E. coli O157:H7 infection, but methods for detecting E. coli O157:H7 in radish sprouts been little studied. Incubation in E. coli broth (EC), tryptic soy broth (TSB) or buffered peptone water (BPW) supplemented by such selective agents as bile salts, novobiocin, acriflavin, vancomycin, cefixime and cefsulodin at 35–378C or 42–438C for 6 or 18–24 h has been used for the selective enrichment of E. coli O157:H7 (Okrend et al., 1990; Padhye and Doyle, 1991; Kim and Doyle, 1992; Wright et al., 1994; Bolton et al., 1995, 1996; Flint and Hartley, 1995; Weagant et al., 1995; Chapman and Siddons, 1996; Restaino et al., 1997). The method of immunomagnetic separation (IMS) of E. coli O157:H7 from enrichment culture using paramagnetic particles coated with E. coli O157 antibodies has been developed for the selective isolation of this organism from foods contaminated with background flora (Fratamico et al., 1992; Okrend et al., 1992; Mortlock, 1994; Wright et al., 1994; Bolton et al., 1995). The purpose of the present study was to evaluate plating and immunological methods for the detection of E. coli O157:H7 in ground beef and radish sprouts, focusing on a comparison of various plating media and immuno-kits in combination with enrichment with modified EC broth supplemented with 25 mg / l novobiocin (mEC 1 n) and IMS. Different laboratories participated in this collaborative study
by attempting to detect E. coli O157:H7 from artificially inoculated samples, using the same isolation procedure.
2. Materials and methods
2.1. Design The study participants were 20 laboratories (national and local government laboratories and laboratories of food research institutions). The reagents for enrichment and plating media, immunobeads and immuno-kits, each from the same lot, were sent to the participants, followed at a later date by the ground beef and radish sprout samples. Within a few hours of receiving the E. coli O157:H7-inoculated or control uninoculated samples, the participants started to analyze them according to the protocol shown in Fig. 1. After performing the analysis, the participants sent the data to the reference laboratory, Department of Biomedical Food Research, National Institute of Infectious Diseases, Tokyo, Japan.
2.2. Media and kits Media and kits were obtained from the following suppliers: Tryptic soy broth (TSB) and tryptic soy agar (TSA), Difco, Detroit, MI, USA. mEC 1 n: Modified EC broth supplemented with 25 mg / l novobiocin; Code No. E-MB10; Eiken, Bunkyo-ku, Tokyo, Japan. Sorbitol MacConkey agar (SMAC) and Sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC); Code No. 540611070; Code No. 540-610066; Mast Diagnostics, Merseyside, UK. BCM O157 TM : Agar containing b-glucuronidase, Code No. 8- MA51; Eiken. CHROMagar O157 TM : Agar containing bglucuronidase, CHROMagar, Paris, France. Now E. coli TM : Binax, Portland, ME, USA. Path-Stik O157 TM : Code No. 95037; Lumac, Landgraaf, Netherlands VIP TM : BioControl System, Bellevue, WA, USA.
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Fig. 1. Protocol of the method used by the participants for the detection of E. coli O157:H7.
EHEC-Tek ELISA system TM : Organon Teknika, Durham, NC, USA. Rapiblot E. coli O157 TM : Kalyx Bioscience, Nepean, Ontario, Canada. CLIG agar TM : Cellobiose-lactose-indole-b-Dglucuronidase agar; Code No. 03401; Kyokuto, Chuo-ku, Tokyo, Japan.
2.3. Sample preparation The E. coli O157:H7 strains, ATCC 43894, purchased from the American Type Culture Collection (Rockville, MD, USA), and strains 19, 23 and 212 (provided by Dr. K. Tamura, National Institute of Infectious Diseases, Tokyo) isolated from patients affected by the outbreaks in Japan were separately grown overnight in TSB at 378C. Each strain was diluted with phosphate-buffered saline, PBS(2) (pH 7.4, Nissui, Toshima-ku, Tokyo, Japan) containing 0.1% peptone, and then approximately equal proportions of the four strains were combined together to give appropriate cell numbers. Ground beef and radish sprouts were purchased from a retail shop in Tokyo and a farmer in Saitama-Prefecture, respectively. Prior to the inoculation of E. coli O157:H7, these food samples were examined for natural contamination with E. coli O157:H7 and found negative
by the IMS-plating method described below in combination with enrichment with mEC 1 n at 428C for 18 h. An E. coli O157:H7 suspension, 0.2 ml, was spiked into 25 g ground beef or 25 g of the edible part (hypocotyl and cotyledon) of radish sprouts in a Stomacher bag. The inoculated cell numbers, which were counted on TSA after 24 h culture at 358C, were 138.9 cfu / 25 g (105–170 cfu / 25 g, n 5 20) and 23.9 cfu / 25 g (18–34 cfu / 25 g, n 5 20) for the ground beef samples at high and low levels of E. coli O157:H7 inoculation, respectively, and 20.4 cfu / 25 g (16–36 cfu / 25 g, n 5 20) and 1.7 cfu / 25 g (0–3 cfu / 25 g, n 5 20) for radish sprout samples at high and low levels of E. coli O157:H7 inoculation, respectively. For control uninoculated samples, 0.2 ml PBS containing 0.1% peptone was spiked into the food samples. Immediately after spiking, the ground beef and radish sprout samples were frozen at 2 208C or chilled at 48C, both for 3–4 h, and then packed with dry ice or ice packs, respectively. Within 24 h of this packing, each participant received five packed chilled and frozen ground beef and radish sprouts samples each of high and low inoculation levels and uninoculated controls. These 15 chilled or frozen samples of ground beef or radish sprouts were assigned a random number by a staff member who did not participate the analysis.
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2.4. Enrichment and direct plating
2.7. Confirmation of E. coli O157: H7
mEC 1 n (225 ml) was added to each 25 g sample, which was then homogenized by a Stomacher (Stomacher 400 TM , Seward Medical, London, UK) for 1 min and incubated statically at 428C for 18 h. A loopful (approximately 10 ml) of the mEC 1 n culture was plated directly onto SMAC, CT-SMAC, BCM O157 or CHROMagar O157.
All of the agar plates were incubated at 378C for 18–24 h. Three suspect colonies from each plate were used for an agglutination test with an E. coli O157 latex agglutination assay kit (UNI TM , Unipath) and inoculated onto CLIG agar which was then incubated at 378C for 18–24 h and examined for acid non-production from cellobiose and non-glucuronidase activity. The isolates on the agar were tested for positive indole production and negative cytochrome oxidase activity.
2.5. Plating after immunomagnetic separation ( IMS-plating method)
2.8. Statistics E. coli O157:H7 cells in the mEC 1 n culture were separated with Dynabeads anti-E. coli O157 according to the manufacturer’s instructions (Dynal, Oslo, Norway). Briefly, 1 ml of the mEC 1 n enrichment culture was mixed with 20 ml Dynabeads anti-E. coli O157 in an Eppendorf tube and incubated at room temperature for 20 min. After being separated from the aliquot with the use of a magnetic particle concentrator (MPC-M TM , Dynal), the beads were washed with 1 ml PBS containing 0.05% Tween 20, separated again and resuspended in 300 ml PBS–Tween 20. The beads suspension (20 ml) was then plated onto a SMAC, CT-SMAC, BCM O157 and CHROMagar O157 and spread over the agar surface with a sterile spreader. A loopful (approximately 10 ml) of the beads suspension was also streaked onto these types of agar plates for comparison with direct plating.
The data are expressed for the inoculated samples as the sensitivity rate, which is the number of positive samples divided by the total number of inoculated samples, and for the uninoculated samples as the specificity rate, which is the number of negative samples divided by the total number of uninoculated samples (McClure, 1990). The sensitivity and specificity rates obtained after omitting the results of outlying laboratories are shown in Section 3. The screening of the data of outlying laboratories was done by conducting ANOVA and the Newman–Keuls multiple range test (Keuls, 1952) on all of the submitted data. Significant differences among the data were examined by conducting the same statistical method on the data submitted by non-outlyers.
3. Results
2.6. Immunological detection method 3.1. Ground beef For both the ground beef and radish sprouts samples, a portion of the mEC 1 n enrichment culture was analyzed by participating laboratories with the use of three immunochromato kits, Now E. coli, Path-Stick O157 and VIP, and a portion of the beads suspension was sent to a company laboratory (Chisso, Yokohama, Japan) which performed the analysis with an EHEC-Tek ELISA system. For the radish sprout samples, a portion of the mEC 1 n enrichment culture was sent to the Eiken, Biochemical Research Laboratory (Tochigi-Prefecture, Japan) which performed the analysis using Rapiblot E. coli O157.
The non-outlyers were 18 and 16 laboratories for the chilled and frozen ground beef samples, respectively. E. coli O157:H7 was recovered well by all the methods from the chilled ground beef samples at high and low inoculation levels and from the frozen samples at the high inoculation level, as seen from the sensitivity rate value which was equal or near to 1.0 for either method (Figs. 2 and 3). For frozen beef samples at a low inoculation level, the direct plating method using SMAC gave a relatively low sensitivity rate, but the other methods achieved high sensitivity rates (Fig. 3). The lowest specificity rate was
Y. Onoue et al. / International Journal of Food Microbiology 46 (1999) 27 – 36
Fig. 2. Sensitivity rate for chilled ground beef samples at a low and high inoculation level, shown as mean6S.E of 18 participants. SMAC-, CT-SMAC-, BCM- and CHROM-Direct indicate direct plating onto SMAC, CT-SMAC, BCM O157 and CHROMagar O157, respectively. SMAC*, CT-SMAC*, BCM* and CHROM* indicate IMS-plating method (a loopful) using SMAC, CT-SMAC, BCM O157 and CHROMagar O157, respectively. SMAC**, CTSMAC**, BCM** and CHROM** indicate the IMS-plating method (20 ml) using SMAC, CT-SMAC, BCM O157 and CHROMagar O157, respectively. NOW, PATH, VIP and EHECTEK indicate Now E. coli, Path-Stick O157, VIP and EHEC-Tek ELISA System, respectively.
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Fig. 3. Sensitivity rate for frozen ground beef samples at a low and high inoculation level, shown as mean6S.E. of 16 participants. Methods indicated are the same as in Fig. 2.
noted for chilled radish sprout samples at the low inoculation level, except for the relatively low sensitivity rate of Rapiblot E. coli O157 (Fig. 5). All of the methods gave apparently lower recovery of E. coli O157:H7 from frozen samples than from
given by the EHEC-Tek ELISA system, and relatively low rates were shown by the other immunological methods and the IMS-plating method with CT-SMAC (Fig. 4).
3.2. Radish sprouts The non-outlyers were 16 and 14 laboratories for the chilled and frozen radish sprout samples, respectively. E. coli O157:H7 was detected from more than 90% of the chilled radish sprout samples at high inoculation levels by the IMS-plating methods using CT-SMAC, BCM O157 and CHROMagar O157, and by such immunological methods as Now E. coli, EHEC-Tek ELISA system and Rapiblot E. coli O157 (Fig. 5). The direct plating method gave relatively poor recovery, and the method using SMAC showed the lowest sensitivity rate (Fig. 5). Similar trends in the comparative efficiency of the methods was also
Fig. 4. Specificity rate for chilled and frozen ground beef samples, shown as mean6S.E. of 18 and 16 participants, respectively. Methods indicated are the same as in Fig. 2.
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Fig. 5. Sensitivity rate for chilled radish sprout samples at a low and high inoculation level, shown as mean6S.E. of 16 participants. Methods indicated are the same as in Fig. 2.
Fig. 6. Sensitivity rate for frozen radish sprout samples at a low and high inoculation level, shown as mean6S.E. of 14 participants. Methods indicated are the same as those in Fig. 2.
chilled samples. The comparative efficiency of the methods for frozen samples was similar to that noted for chilled samples; the IMS-plating methods using CT-SMAC, BCM O157 and CHROMagar and some of immunological methods showed relatively high sensitivity rates (Fig. 6). The lowest specificity rate was given by VIP, and relatively low rates were seen in some of the other immunological methods (Fig. 7). The statistical evaluation using combined data of sensitivity and specificity rates for each food sample revealed that the IMS-plating methods were superior to the other methods for either type of food sample, although some of the immunological methods were remarkably efficient for frozen foods (Fig. 8). There was a notable disparity in the results between the immunological methods and IMS-plating methods, especially at the low inoculation level. More than 11.5% and 33.3% of the chilled and frozen samples, respectively, were positive by IMSplating method using CT-SMAC, but negative by the immunological methods (Table 1). Conversely, some samples were positive by immunological methods, but negative by the IMS-plating method using CTSMAC (Table 2). There were several samples which were positive by BCM O157, but negative by CT-
SMAC (Table 3), indicating that the use of two or three of the selective agars could raise the detection rate.
Fig. 7. Specificity rate for chilled and frozen radish sprout samples, shown as mean6S.E. of 16 and 14 participants, respectively. Methods indicated are the same as in Fig. 2.
Y. Onoue et al. / International Journal of Food Microbiology 46 (1999) 27 – 36
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Fig. 8. Detection methods ranked by Newman–Keuls multiple range test. D1, D2, D3 and D4 indicate direct plating onto SMAC, CT-SMAC, BCMO157 and Chromagar O157, respectively. IL1, IL2, IL3 and IL4 indicate IMS-plating (a loopful) onto SMAC, CT-SMAC, BCMO157 and Chromagar O157, respectively. IS1, IS2, IL3 and IL4 indicate IMS-plating (20 ml) onto SMAC, CT-SMAC, BCMO157 and Chromagar O157, respectively. E1, E2, E3, E4 and E5 indicate Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA system and Rapiblot, respectively. The methods are listed from most to least efficient (left to right). Methods that are underscored by the same line do not differ significantly at the 1% level.
4. Discussion All of the methods used in this study except for the direct plating method using SMAC detected E.
coli O157:H7 from ground beef samples inoculated with 23.9 cfu / 25 g, with a sensitivity rate equal or near to 1.0. The specificity rates in the direct and IMS-plating methods were above 0.9, indicating that
Table 1 Radish sprout samples from which E. coli O157:H7 was detected by the immunological method, but not by the IMS plating method with CT-SMAC Chilled radish sprouts Low level a NOW E. coli Path-Stick O157 VIP EHEC-Tek ELISA Rapiblot E. coli O157 a
4 / 18 (22.2%) 7 / 21 (33.3) 5 / 18 (27.8) 3 / 17 (17.6) 0 / 11 (0.0)
b
Frozen radish sprouts High level
Low level
High level
1 / 65 (1.5) 1 / 64 (1.6) 1 / 63 (1.6) 2 / 63 (3.2) 4 / 67 (6.0)
4 / 8 (50.0) 14 / 17 (82.4) 5 / 10 (50.0) 7 / 13 (53.8) 0 / 3 (0.0)
3 / 32 (9.4) 5 / 38 (13.2) 3 / 31 (9.7) 7 / 38 (18.4) 4 / 27 (14.8)
Level of inoculated E. coli O157:H7 cells. Number of samples from which E. coli O157:H7 was detected by the immunological method but not by the IMS plating method with CT-SMAC, out of the numbers of all of the samples from which E. coli O157:H7 was detected by the immunological method.
b
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Table 2 Radish sprout samples from which E. coli O157:H7 was detected by the IMS plating method with CT-SMAC, but not by the immunological method Chilled radish sprouts Low level NOW E. coli Path-Stick O157 VIP EHEC-Tek ELISA Rapiblot E. coli O157
7 / 26 7 / 26 8 / 26 3 / 26 9 / 26
a
(26.9%) (26.9) (30.8) (11.5) (34.6)
b
Frozen radish sprouts High level
Low level
High level
5 / 74 6 / 74 7 / 74 2 / 74 6 / 74
2/6 2/6 2/6 2/6 3/6
5 / 34 (14.7) 1 / 34 (2.9) 7 / 34 (20.6) 2 / 34 (5.9) 11 / 34 (32.4)
(6.8) (8.1) (9.5) (2.7) (8.1)
(33.3) (33.3) (33.3) (33.3) (50.0)
a
Level of inoculated E. coli O157:H7 cells. Number of samples from which E. coli O157:H7 was detected by the IMS plating method with CT-SMAC, but not by the immunological method, out of the numbers of all of the samples from which E. coli O157:H7 was detected by the IMS plating method with CT-SMAC. b
the IMS-plating method using any of the agars and the direct plating method using selective agars other than SMAC in combination with enrichment in mEC 1 n at 428C for 18 h, are sensitive enough to detect E. coli O157:H7 at the level of 23.9 cfu / 25 g from ground beef. The positive reaction of some uninoculated samples obtained by some methods may reflect a false positive reaction, natural contamination of the samples with E. coli O157:H7 and / or cross contamination of the samples during testing. The specificity rates for the ground beef samples obtained by some of the immunological methods were lower than those by the IMS-plating method, indicating that false positive reactions with the immunological methods could raise their sensitivity rates. The sensitivity rates of the IMS-plating methods using selective agars other than SMAC for chilled
radish sprout samples at the inoculation level of 20.4 cfu / 25 g were above 0.9, showing that these methods in combination with mEC 1 n enrichment at 428C are effective for E. coli O157:H7 detection from radish sprouts contaminated at such a level. Some of the immunological methods gave a sensitivity rate similar to or higher than that of the IMS-plating method. However, this high sensitivity rate with the immunological methods may be attributed in part to false positive reactions, because some of them showed a specificity rate lower than that of the IMS-plating method. In addition, all of the immunological methods showed negative responses to several radish sprout samples in which the IMSplating method using CT-SMAC detected E. coli O157:H7. Such discrepancies in the results between the two methods were particularly large for the samples at the low inoculation level, indicating that
Table 3 Recovery of E. coli O157:H7 from radish sprouts by selective agars Chilled radish sprouts Low level Loop CT-SMAC 1 BCM 1 BCM 1 Chrom 20 ml spread CT-SMAC 1 BCM 1 BCM 1 Chrom
a
Frozen radish sprouts High level
Low level
High level
23 / 80 (28.8%)b 24 / 80 (30.0) 24 / 80 (30.0)
74 / 80 (92.5) 75 / 80 (93.8) 75 / 80 (93.8)
7 / 70 (10.0) 9 / 70 (12.9) 9 / 70 (12.9)
32 / 70 (45.7) 34 / 70 (48.6) 34 / 70 (48.6)
26 / 80 (32.5) 27 / 80 (33.8) 27 / 80 (33.8)
74 / 80 (92.5) 74 / 80 (92.5) 74 / 80 (92.5)
6 / 70 (8.6) 8 / 70 (11.4) 8 / 70 (11.4)
34 / 70 (48.6) 37 / 70 (52.9) 38 / 70 (54.3)
BCM:BCM O157; Chrom:CHROMagar O157. a Level of inoculated E. coli O157:H7 cells. b Number of detected samples out of total numbers.
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the immunological methods are less sensitive than the IMS-plating method using CT-SMAC. The large difference in the sensitivity rate between the chilled radish sprout samples at the low inoculation level and those at the high inoculation level may be due not only to the detection limit of the methods, but also to a failure to include the target organism in some of the samples at the low inoculation level. The lower sensitivity rates for the frozen radish sprout samples compared to the chilled samples may reflect the death and / or injury of E. coli O157:H7 cells by freezing. Taken together, the results of the radish sprout samples demonstrate the comparative efficiency of the methods in combination with mEC 1 n enrichment, i.e., the use of IMS can raise the sensitivity, and CT-SMAC, BCM O157 and CHROMagar are superior to SMAC for the isolation of the organism. Since some samples were shown as positive by BCM O157 or CHROMagar but not by CT-SMAC, the combined use of CT-SMAC and an agar containing a b-glucuronidase substrate may raise the detection rate. Incubation in mEC 1 n at 428C for 18 h was used for selective enrichment in this study, although other conditions have also been used (Padhye and Doyle, 1991; Kim and Doyle, 1992; Wright et al., 1994; Bolton et al., 1995, 1996; Flint and Hartley, 1995; Weagant et al., 1995; Chapman and Siddons, 1996; Restaino et al., 1997). We recently showed that E. coli O157:H7 inoculated into ground beef and radish sprouts is well recovered by incubation in mEC 1 n at 428C for 18 h, rather than in mEC 1 n at 378C for 6 or 18 h or at 428C for 6 h or 18 h in TSB or modified TSB. In agreement with this observation, the use of mEC 1 n and a temperature of 42–438C has been shown to be superior to that of other media (Johnson et al., 1995; Bennett et al., 1996) and temperatures (Szabo et al., 1990; Blais et al., 1997) for the selective enrichment of E. coli O157:H7. Heuvelink et al. (1997) have recommended the IMS-plating method using CT-SMAC as the plating medium combined with enrichment in mEC 1 n at 378C for 6–8 h with shaking, but a static incubation may be better from a practical viewpoint, as has been suggested by Blais et al. (1997). Based on the results of the present study, we recommend the IMS-plating method using CT-SMAC and agars containing a
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b-glucuronidase substrate in combination with static enrichment incubation in mEC 1 n at 428C for 18 h.
Acknowledgements This research was supported by the Health Science Research Grants of the Ministry of Health and Welfare in Japan. The authors wish to thank the following collaborators: Agata, N., Nagoya City Health Research Institute, Nagoya; Akahane, S., Japan Food Hygiene association Institute of Food Hygiene; Chou, N., Tochigi Prefectural Hygienic Institute, Tochigi; Goto, K., Niigata Prefectural Research Laboratory for Health and Environment; Horikawa, K., Fukuoka Environmental Research Center; Kai, A., Tokyo Metropolitan Research Laboratory of Public Health, Tokyo; Kaneko, M., Yamanashi Institute for Public Health, Yamanashi; Kumagai, M., Iwate Prefectural Institute of Public Health, Iwate; Mabara, S., Ibaraki Prefectural Institute of Health; Machigaki, E., General Testing Research Institute of Japan Oilstuff Inspectors’ Corporation, Kobe; Masaki, H., Saitama Institute of Public Health, Saitama; Nukina, M., Public Health Institute of Kobe City, Kobe; Saito, M., Japan Frozen Foods Inspection Cooperation, Tokyo; Tanno, K., Japan Food Research Laboratories, Tokyo; Terai, K., Shizuoka Prefectural Institute of Public Health and Environmental Science, Shizuoka; Uchimura, M., Public Health Laboratory Chiba Prefecture, Chiba; Uchiyama, S., Food and Drug Safety Center, Hatano-City. We also express thanks to Chisso Co., Yokohama, and Eiken Chemical Co., Biochemical Research Laboratory, Tochigi-Prefecture, Japan, for technical assistance with analyses by EHEC-Tek ELISA System and Rapiblot E. coli O157, respectively.
References Ackers, M., Mahon, B., Leahy, E., Damrow, T., Hutwagner, L., Barrett, T., Bibb, W., Hayes, P., Griffin, P., Slutsker, L., 1996. An outbreak of Escherichia coli O157:H7 infections associated with leaf lettuce. Abstr. K43. Prog. Abstr. 36th Intersci. Conf. Antimicrob. Agents Chemother., American Society for Microbiology, Washington, DC.
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Bennett, A.R., MacPhee, S., Betts, R.P., 1996. The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures. Lett. Appl. Microbiol. 22, 237–243. Besser, R.E., Lett, S.M., Weber, J.T., Doyle, M.P., Barrett, T.J., Wells, J.G., Griffin, P.M., 1993. An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157:H7 in fresh-pressed apple cider. J. Am. Med. Assoc. 269, 2217– 2220. Beuchat, L.R., 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59, 204–216. Blais, B.W., Booth, R.A., Phillippe, L.M., Yamazaki, H., 1997. Effect of temperature and agitation on enrichment of Escherichia coli O157:H7 in ground beef using modified EC broth with novobiocin. Int. J. Food Microbiol. 36, 221–225. Bolton, F.J., Crozier, L., Williamson, J.K., 1995. New technical approaches to Escherichia coli O157. PHLS Micrbiol. Dig. 12, 67–70. Bolton, F.J., Crozier, L., Williamson, J.K., 1996. Isolation of Escherichia coli O157 from raw meat products. Lett. Appl. Microbiol. 23, 317–321. Chapman, P.A., Siddons, C.A., 1996. Evaluation of a commercial enzyme immunoassay (EHEK-Tek) for detecting Escherichia coli O157:H7 in beef and beef products. Food Microbiol. 13, 175–182. Doyle, M.P., 1991. Escherichia coli O157:H7 and its significance in foods. Int. J. Food Microbiol. 12, 289–302. Flint, S.H., Hartley, N.J., 1995. Evaluation of teh TECRA Escherichia coli O157 visual immunoassay for tests on dairy products. J. Food Prot. 21, 79–82. Fratamico, P.M., Schultz, F.J., Buchanan, R.L., 1992. Rapid isolation of Escherichia coli O157:H7 from enrichment cultures of foods using immunomagnetic separation method. Food Microbiol. 9, 105–113. Heuvelink, A.E., Zwartkruis-Nahuis, J.T.M., Boer, E.D., 1997. Evaluation of media and test kits for the detection and isolation of Escherichia coli O157 from minced beef. J. Food Prot. 60, 817–824. Johnson, J.L., Rose, B.E., Sharar, A.K., Ransom, G.M., Lattuada, C.P., Mcnamara, A.M., 1995. Methods used for detection and recovery of Escherichia coli O157:H7 associated with a foodborne disease outbreak. J. Food Prot. 58, 597–603. Keuls, M., 1952. The use of the ‘‘studentized range’’ in connection with an analysis of variance. Euphytica 1, 112–122. Kim, M.S., Doyle, M.P., 1992. Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef. Appl. Environ. Microbiol. 59, 1764–1767.
McClure, F.D., 1990. Design and analysis of quantitative collaborative studies: minimum collaborative program. J. Assoc. Off. Anal. Chem. 73, 953–960. Mortlock, S., 1994. Recovery of Escherichia coli O157:H7 from mixed suspensions: Evaluation and comparison of pre-coated immunomagnetic beads and direct plating. Br. J. Biomed. Sci. 51, 207–214. Okrend, A.J.G., Rose, B.E., Bennett, B., 1990. A screening method for the isolation of Escherichia coli O157:H7 from ground beef. J. Food Prot. 53, 249–252. Okrend, A.J.G., Rose, B.E., Lattuada, C.P., 1992. Isolation of Escherichia coli O157:H7 using O157 specific antibody coated magnetic beads. J. Food Prot. 55, 214–217. Padhye, N.V., Doyle, M.P., 1991. Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food. Appl. Environ. Microbiol. 57, 2693–2698. Restaino, L., Frampton, E.W., Irbe, R.M., Allison, D.R.K., 1997. A 5-h screening and 24-h confirmation procedure for detecting Escherichia coli O157:H7 in beef using direct epifluorescent microscopy and immunomagnetic separation. Lett. Appl. Microbiol. 24, 401–404. Riley, L.W., Remis, R.S., Helgelson, 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. New Engl. J. Med. 308, 681–685. Szabo, R., Todd, E., MacKenzie, J., Parrington, L., Armstrong, A., 1990. Increased sensitivity of the rapid hydrophobic grid membrane filter enzyme-labeled antibody procedure for Escherichia coli O157 detection in foods and bovine feces. Appl. Environ. Microbiol. 56, 3546–3549. US Department of Health and Human Services, 1997. Outbreaks of Escherichia coli O157:H7 infection associated with eating alfalfa sprouts — Michigan and Virginia, June–July 1997. Morbidity Mortality Weekly Report, 46, 741–744. Weagant, S.D., Bryant, J.L., Jinneman, K.G., 1995. An improved rapid technique for isolation of Escherichia coli O157:H7 from foods. J. Food Prot. 58, 7–12. World Health Organization, 1996. Enterohaemorrhagic Escherichia coli infection. Weely Epidemiol. Rec., 30, 229–230. World Health Organization, 1997. Enterohaemorrhagic Escherichia coli (EHEC) infection. Weely Epidemiol. Rec., 25, 185. Wright, D.J., Chapman, P.A., Siddons, C.A., 1994. Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples. Epidemiol. Infect. 113, 31–39.
Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef Youichi Onoue a , Hirotaka Konuma b , Hiroshi Nakagawa c , Yukiko Hara-Kudo d , e d, Toshiharu Fujita , Susumu Kumagai * a
Kanagawa Prefuctural Public Health Laboratory, 52 -2 Nakao, Asahi-ku, Yokohama 241, Japan Division of Microbiology, National Institute of Health Sciences, 1 -18 -1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan c Tokyo Kenbikyoin Foundation, 44 -1 Nihonbashi hakozaki-cho, Chuo-ku, Tokyo 103, Japan d Department of Biomedical Food Research, National Institute of Infectious Diseases, 1 -23 -1 Toyama, Shinjuku-ku, Tokyo 162, Japan e Department of Epidemiology, National Institute of Public Health, Shirokanedai 4 -6 -1, Minatoku, Tokyo 108, Japan b
Received 22 April 1998; received in revised form 3 September 1998; accepted 27 October 1998
Abstract For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC 1 n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg / l) and potassium tellurite (2.5 mg / l) (CT-SMAC), and agars containing b-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu / 25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu / 25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu / 25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu / 25 g) compared with ground beef (23.9 cfu / 25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing b-glucuronidase substrate in combination with static enrichment incubation in mEC 1 n at 428C. 1999 Elsevier Science B.V. All rights reserved. Keywords: Escherichia coli O157:H7; Detection; Collaborative evaluation; Radish sprouts; Ground beef
*Corresponding author. Tel.: 1 81-3-52851111; fax: 1 81-3-52851176; e-mail: [email protected] 0168-1605 / 99 / $ – see front matter PII: S0168-1605( 98 )00174-3
1999 Elsevier Science B.V. All rights reserved.
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1. Introduction Since its recognition as a food borne disease agent in 1982 (Riley et al., 1983), Escherichia coli O157:H7 has emerged as a significant food borne pathogen. Most outbreaks have been linked to foods and water, beef being most frequently involved as a vehicle of infection (Doyle, 1991). Other foods such as lettuce, cantaloupe, cabbage, unpasteurized apple juice and alfalfa sprouts have also been implicated in some outbreaks (Besser et al., 1993; Ackers et al., 1996; Beuchat, 1996; US Department of Health and Human Services, 1997). A highly publicized E. coli O157:H7 outbreak occurred at Sakai-City, Japan in July, 1996. Hydroponically grown radish sprouts were implicated epidemiologically as the vehicle (World Health Organization, 1996), and again in a ‘‘diffuse outbreak’’ of E. coli O157:H7 infection in March, 1997, in Japan (World Health Organization, 1997). Thus, hydroponically grown radish sprouts are concluded to be one of the foods at high risk for E. coli O157:H7 infection, but methods for detecting E. coli O157:H7 in radish sprouts been little studied. Incubation in E. coli broth (EC), tryptic soy broth (TSB) or buffered peptone water (BPW) supplemented by such selective agents as bile salts, novobiocin, acriflavin, vancomycin, cefixime and cefsulodin at 35–378C or 42–438C for 6 or 18–24 h has been used for the selective enrichment of E. coli O157:H7 (Okrend et al., 1990; Padhye and Doyle, 1991; Kim and Doyle, 1992; Wright et al., 1994; Bolton et al., 1995, 1996; Flint and Hartley, 1995; Weagant et al., 1995; Chapman and Siddons, 1996; Restaino et al., 1997). The method of immunomagnetic separation (IMS) of E. coli O157:H7 from enrichment culture using paramagnetic particles coated with E. coli O157 antibodies has been developed for the selective isolation of this organism from foods contaminated with background flora (Fratamico et al., 1992; Okrend et al., 1992; Mortlock, 1994; Wright et al., 1994; Bolton et al., 1995). The purpose of the present study was to evaluate plating and immunological methods for the detection of E. coli O157:H7 in ground beef and radish sprouts, focusing on a comparison of various plating media and immuno-kits in combination with enrichment with modified EC broth supplemented with 25 mg / l novobiocin (mEC 1 n) and IMS. Different laboratories participated in this collaborative study
by attempting to detect E. coli O157:H7 from artificially inoculated samples, using the same isolation procedure.
2. Materials and methods
2.1. Design The study participants were 20 laboratories (national and local government laboratories and laboratories of food research institutions). The reagents for enrichment and plating media, immunobeads and immuno-kits, each from the same lot, were sent to the participants, followed at a later date by the ground beef and radish sprout samples. Within a few hours of receiving the E. coli O157:H7-inoculated or control uninoculated samples, the participants started to analyze them according to the protocol shown in Fig. 1. After performing the analysis, the participants sent the data to the reference laboratory, Department of Biomedical Food Research, National Institute of Infectious Diseases, Tokyo, Japan.
2.2. Media and kits Media and kits were obtained from the following suppliers: Tryptic soy broth (TSB) and tryptic soy agar (TSA), Difco, Detroit, MI, USA. mEC 1 n: Modified EC broth supplemented with 25 mg / l novobiocin; Code No. E-MB10; Eiken, Bunkyo-ku, Tokyo, Japan. Sorbitol MacConkey agar (SMAC) and Sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC); Code No. 540611070; Code No. 540-610066; Mast Diagnostics, Merseyside, UK. BCM O157 TM : Agar containing b-glucuronidase, Code No. 8- MA51; Eiken. CHROMagar O157 TM : Agar containing bglucuronidase, CHROMagar, Paris, France. Now E. coli TM : Binax, Portland, ME, USA. Path-Stik O157 TM : Code No. 95037; Lumac, Landgraaf, Netherlands VIP TM : BioControl System, Bellevue, WA, USA.
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29
Fig. 1. Protocol of the method used by the participants for the detection of E. coli O157:H7.
EHEC-Tek ELISA system TM : Organon Teknika, Durham, NC, USA. Rapiblot E. coli O157 TM : Kalyx Bioscience, Nepean, Ontario, Canada. CLIG agar TM : Cellobiose-lactose-indole-b-Dglucuronidase agar; Code No. 03401; Kyokuto, Chuo-ku, Tokyo, Japan.
2.3. Sample preparation The E. coli O157:H7 strains, ATCC 43894, purchased from the American Type Culture Collection (Rockville, MD, USA), and strains 19, 23 and 212 (provided by Dr. K. Tamura, National Institute of Infectious Diseases, Tokyo) isolated from patients affected by the outbreaks in Japan were separately grown overnight in TSB at 378C. Each strain was diluted with phosphate-buffered saline, PBS(2) (pH 7.4, Nissui, Toshima-ku, Tokyo, Japan) containing 0.1% peptone, and then approximately equal proportions of the four strains were combined together to give appropriate cell numbers. Ground beef and radish sprouts were purchased from a retail shop in Tokyo and a farmer in Saitama-Prefecture, respectively. Prior to the inoculation of E. coli O157:H7, these food samples were examined for natural contamination with E. coli O157:H7 and found negative
by the IMS-plating method described below in combination with enrichment with mEC 1 n at 428C for 18 h. An E. coli O157:H7 suspension, 0.2 ml, was spiked into 25 g ground beef or 25 g of the edible part (hypocotyl and cotyledon) of radish sprouts in a Stomacher bag. The inoculated cell numbers, which were counted on TSA after 24 h culture at 358C, were 138.9 cfu / 25 g (105–170 cfu / 25 g, n 5 20) and 23.9 cfu / 25 g (18–34 cfu / 25 g, n 5 20) for the ground beef samples at high and low levels of E. coli O157:H7 inoculation, respectively, and 20.4 cfu / 25 g (16–36 cfu / 25 g, n 5 20) and 1.7 cfu / 25 g (0–3 cfu / 25 g, n 5 20) for radish sprout samples at high and low levels of E. coli O157:H7 inoculation, respectively. For control uninoculated samples, 0.2 ml PBS containing 0.1% peptone was spiked into the food samples. Immediately after spiking, the ground beef and radish sprout samples were frozen at 2 208C or chilled at 48C, both for 3–4 h, and then packed with dry ice or ice packs, respectively. Within 24 h of this packing, each participant received five packed chilled and frozen ground beef and radish sprouts samples each of high and low inoculation levels and uninoculated controls. These 15 chilled or frozen samples of ground beef or radish sprouts were assigned a random number by a staff member who did not participate the analysis.
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2.4. Enrichment and direct plating
2.7. Confirmation of E. coli O157: H7
mEC 1 n (225 ml) was added to each 25 g sample, which was then homogenized by a Stomacher (Stomacher 400 TM , Seward Medical, London, UK) for 1 min and incubated statically at 428C for 18 h. A loopful (approximately 10 ml) of the mEC 1 n culture was plated directly onto SMAC, CT-SMAC, BCM O157 or CHROMagar O157.
All of the agar plates were incubated at 378C for 18–24 h. Three suspect colonies from each plate were used for an agglutination test with an E. coli O157 latex agglutination assay kit (UNI TM , Unipath) and inoculated onto CLIG agar which was then incubated at 378C for 18–24 h and examined for acid non-production from cellobiose and non-glucuronidase activity. The isolates on the agar were tested for positive indole production and negative cytochrome oxidase activity.
2.5. Plating after immunomagnetic separation ( IMS-plating method)
2.8. Statistics E. coli O157:H7 cells in the mEC 1 n culture were separated with Dynabeads anti-E. coli O157 according to the manufacturer’s instructions (Dynal, Oslo, Norway). Briefly, 1 ml of the mEC 1 n enrichment culture was mixed with 20 ml Dynabeads anti-E. coli O157 in an Eppendorf tube and incubated at room temperature for 20 min. After being separated from the aliquot with the use of a magnetic particle concentrator (MPC-M TM , Dynal), the beads were washed with 1 ml PBS containing 0.05% Tween 20, separated again and resuspended in 300 ml PBS–Tween 20. The beads suspension (20 ml) was then plated onto a SMAC, CT-SMAC, BCM O157 and CHROMagar O157 and spread over the agar surface with a sterile spreader. A loopful (approximately 10 ml) of the beads suspension was also streaked onto these types of agar plates for comparison with direct plating.
The data are expressed for the inoculated samples as the sensitivity rate, which is the number of positive samples divided by the total number of inoculated samples, and for the uninoculated samples as the specificity rate, which is the number of negative samples divided by the total number of uninoculated samples (McClure, 1990). The sensitivity and specificity rates obtained after omitting the results of outlying laboratories are shown in Section 3. The screening of the data of outlying laboratories was done by conducting ANOVA and the Newman–Keuls multiple range test (Keuls, 1952) on all of the submitted data. Significant differences among the data were examined by conducting the same statistical method on the data submitted by non-outlyers.
3. Results
2.6. Immunological detection method 3.1. Ground beef For both the ground beef and radish sprouts samples, a portion of the mEC 1 n enrichment culture was analyzed by participating laboratories with the use of three immunochromato kits, Now E. coli, Path-Stick O157 and VIP, and a portion of the beads suspension was sent to a company laboratory (Chisso, Yokohama, Japan) which performed the analysis with an EHEC-Tek ELISA system. For the radish sprout samples, a portion of the mEC 1 n enrichment culture was sent to the Eiken, Biochemical Research Laboratory (Tochigi-Prefecture, Japan) which performed the analysis using Rapiblot E. coli O157.
The non-outlyers were 18 and 16 laboratories for the chilled and frozen ground beef samples, respectively. E. coli O157:H7 was recovered well by all the methods from the chilled ground beef samples at high and low inoculation levels and from the frozen samples at the high inoculation level, as seen from the sensitivity rate value which was equal or near to 1.0 for either method (Figs. 2 and 3). For frozen beef samples at a low inoculation level, the direct plating method using SMAC gave a relatively low sensitivity rate, but the other methods achieved high sensitivity rates (Fig. 3). The lowest specificity rate was
Y. Onoue et al. / International Journal of Food Microbiology 46 (1999) 27 – 36
Fig. 2. Sensitivity rate for chilled ground beef samples at a low and high inoculation level, shown as mean6S.E of 18 participants. SMAC-, CT-SMAC-, BCM- and CHROM-Direct indicate direct plating onto SMAC, CT-SMAC, BCM O157 and CHROMagar O157, respectively. SMAC*, CT-SMAC*, BCM* and CHROM* indicate IMS-plating method (a loopful) using SMAC, CT-SMAC, BCM O157 and CHROMagar O157, respectively. SMAC**, CTSMAC**, BCM** and CHROM** indicate the IMS-plating method (20 ml) using SMAC, CT-SMAC, BCM O157 and CHROMagar O157, respectively. NOW, PATH, VIP and EHECTEK indicate Now E. coli, Path-Stick O157, VIP and EHEC-Tek ELISA System, respectively.
31
Fig. 3. Sensitivity rate for frozen ground beef samples at a low and high inoculation level, shown as mean6S.E. of 16 participants. Methods indicated are the same as in Fig. 2.
noted for chilled radish sprout samples at the low inoculation level, except for the relatively low sensitivity rate of Rapiblot E. coli O157 (Fig. 5). All of the methods gave apparently lower recovery of E. coli O157:H7 from frozen samples than from
given by the EHEC-Tek ELISA system, and relatively low rates were shown by the other immunological methods and the IMS-plating method with CT-SMAC (Fig. 4).
3.2. Radish sprouts The non-outlyers were 16 and 14 laboratories for the chilled and frozen radish sprout samples, respectively. E. coli O157:H7 was detected from more than 90% of the chilled radish sprout samples at high inoculation levels by the IMS-plating methods using CT-SMAC, BCM O157 and CHROMagar O157, and by such immunological methods as Now E. coli, EHEC-Tek ELISA system and Rapiblot E. coli O157 (Fig. 5). The direct plating method gave relatively poor recovery, and the method using SMAC showed the lowest sensitivity rate (Fig. 5). Similar trends in the comparative efficiency of the methods was also
Fig. 4. Specificity rate for chilled and frozen ground beef samples, shown as mean6S.E. of 18 and 16 participants, respectively. Methods indicated are the same as in Fig. 2.
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Y. Onoue et al. / International Journal of Food Microbiology 46 (1999) 27 – 36
Fig. 5. Sensitivity rate for chilled radish sprout samples at a low and high inoculation level, shown as mean6S.E. of 16 participants. Methods indicated are the same as in Fig. 2.
Fig. 6. Sensitivity rate for frozen radish sprout samples at a low and high inoculation level, shown as mean6S.E. of 14 participants. Methods indicated are the same as those in Fig. 2.
chilled samples. The comparative efficiency of the methods for frozen samples was similar to that noted for chilled samples; the IMS-plating methods using CT-SMAC, BCM O157 and CHROMagar and some of immunological methods showed relatively high sensitivity rates (Fig. 6). The lowest specificity rate was given by VIP, and relatively low rates were seen in some of the other immunological methods (Fig. 7). The statistical evaluation using combined data of sensitivity and specificity rates for each food sample revealed that the IMS-plating methods were superior to the other methods for either type of food sample, although some of the immunological methods were remarkably efficient for frozen foods (Fig. 8). There was a notable disparity in the results between the immunological methods and IMS-plating methods, especially at the low inoculation level. More than 11.5% and 33.3% of the chilled and frozen samples, respectively, were positive by IMSplating method using CT-SMAC, but negative by the immunological methods (Table 1). Conversely, some samples were positive by immunological methods, but negative by the IMS-plating method using CTSMAC (Table 2). There were several samples which were positive by BCM O157, but negative by CT-
SMAC (Table 3), indicating that the use of two or three of the selective agars could raise the detection rate.
Fig. 7. Specificity rate for chilled and frozen radish sprout samples, shown as mean6S.E. of 16 and 14 participants, respectively. Methods indicated are the same as in Fig. 2.
Y. Onoue et al. / International Journal of Food Microbiology 46 (1999) 27 – 36
33
Fig. 8. Detection methods ranked by Newman–Keuls multiple range test. D1, D2, D3 and D4 indicate direct plating onto SMAC, CT-SMAC, BCMO157 and Chromagar O157, respectively. IL1, IL2, IL3 and IL4 indicate IMS-plating (a loopful) onto SMAC, CT-SMAC, BCMO157 and Chromagar O157, respectively. IS1, IS2, IL3 and IL4 indicate IMS-plating (20 ml) onto SMAC, CT-SMAC, BCMO157 and Chromagar O157, respectively. E1, E2, E3, E4 and E5 indicate Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA system and Rapiblot, respectively. The methods are listed from most to least efficient (left to right). Methods that are underscored by the same line do not differ significantly at the 1% level.
4. Discussion All of the methods used in this study except for the direct plating method using SMAC detected E.
coli O157:H7 from ground beef samples inoculated with 23.9 cfu / 25 g, with a sensitivity rate equal or near to 1.0. The specificity rates in the direct and IMS-plating methods were above 0.9, indicating that
Table 1 Radish sprout samples from which E. coli O157:H7 was detected by the immunological method, but not by the IMS plating method with CT-SMAC Chilled radish sprouts Low level a NOW E. coli Path-Stick O157 VIP EHEC-Tek ELISA Rapiblot E. coli O157 a
4 / 18 (22.2%) 7 / 21 (33.3) 5 / 18 (27.8) 3 / 17 (17.6) 0 / 11 (0.0)
b
Frozen radish sprouts High level
Low level
High level
1 / 65 (1.5) 1 / 64 (1.6) 1 / 63 (1.6) 2 / 63 (3.2) 4 / 67 (6.0)
4 / 8 (50.0) 14 / 17 (82.4) 5 / 10 (50.0) 7 / 13 (53.8) 0 / 3 (0.0)
3 / 32 (9.4) 5 / 38 (13.2) 3 / 31 (9.7) 7 / 38 (18.4) 4 / 27 (14.8)
Level of inoculated E. coli O157:H7 cells. Number of samples from which E. coli O157:H7 was detected by the immunological method but not by the IMS plating method with CT-SMAC, out of the numbers of all of the samples from which E. coli O157:H7 was detected by the immunological method.
b
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Table 2 Radish sprout samples from which E. coli O157:H7 was detected by the IMS plating method with CT-SMAC, but not by the immunological method Chilled radish sprouts Low level NOW E. coli Path-Stick O157 VIP EHEC-Tek ELISA Rapiblot E. coli O157
7 / 26 7 / 26 8 / 26 3 / 26 9 / 26
a
(26.9%) (26.9) (30.8) (11.5) (34.6)
b
Frozen radish sprouts High level
Low level
High level
5 / 74 6 / 74 7 / 74 2 / 74 6 / 74
2/6 2/6 2/6 2/6 3/6
5 / 34 (14.7) 1 / 34 (2.9) 7 / 34 (20.6) 2 / 34 (5.9) 11 / 34 (32.4)
(6.8) (8.1) (9.5) (2.7) (8.1)
(33.3) (33.3) (33.3) (33.3) (50.0)
a
Level of inoculated E. coli O157:H7 cells. Number of samples from which E. coli O157:H7 was detected by the IMS plating method with CT-SMAC, but not by the immunological method, out of the numbers of all of the samples from which E. coli O157:H7 was detected by the IMS plating method with CT-SMAC. b
the IMS-plating method using any of the agars and the direct plating method using selective agars other than SMAC in combination with enrichment in mEC 1 n at 428C for 18 h, are sensitive enough to detect E. coli O157:H7 at the level of 23.9 cfu / 25 g from ground beef. The positive reaction of some uninoculated samples obtained by some methods may reflect a false positive reaction, natural contamination of the samples with E. coli O157:H7 and / or cross contamination of the samples during testing. The specificity rates for the ground beef samples obtained by some of the immunological methods were lower than those by the IMS-plating method, indicating that false positive reactions with the immunological methods could raise their sensitivity rates. The sensitivity rates of the IMS-plating methods using selective agars other than SMAC for chilled
radish sprout samples at the inoculation level of 20.4 cfu / 25 g were above 0.9, showing that these methods in combination with mEC 1 n enrichment at 428C are effective for E. coli O157:H7 detection from radish sprouts contaminated at such a level. Some of the immunological methods gave a sensitivity rate similar to or higher than that of the IMS-plating method. However, this high sensitivity rate with the immunological methods may be attributed in part to false positive reactions, because some of them showed a specificity rate lower than that of the IMS-plating method. In addition, all of the immunological methods showed negative responses to several radish sprout samples in which the IMSplating method using CT-SMAC detected E. coli O157:H7. Such discrepancies in the results between the two methods were particularly large for the samples at the low inoculation level, indicating that
Table 3 Recovery of E. coli O157:H7 from radish sprouts by selective agars Chilled radish sprouts Low level Loop CT-SMAC 1 BCM 1 BCM 1 Chrom 20 ml spread CT-SMAC 1 BCM 1 BCM 1 Chrom
a
Frozen radish sprouts High level
Low level
High level
23 / 80 (28.8%)b 24 / 80 (30.0) 24 / 80 (30.0)
74 / 80 (92.5) 75 / 80 (93.8) 75 / 80 (93.8)
7 / 70 (10.0) 9 / 70 (12.9) 9 / 70 (12.9)
32 / 70 (45.7) 34 / 70 (48.6) 34 / 70 (48.6)
26 / 80 (32.5) 27 / 80 (33.8) 27 / 80 (33.8)
74 / 80 (92.5) 74 / 80 (92.5) 74 / 80 (92.5)
6 / 70 (8.6) 8 / 70 (11.4) 8 / 70 (11.4)
34 / 70 (48.6) 37 / 70 (52.9) 38 / 70 (54.3)
BCM:BCM O157; Chrom:CHROMagar O157. a Level of inoculated E. coli O157:H7 cells. b Number of detected samples out of total numbers.
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the immunological methods are less sensitive than the IMS-plating method using CT-SMAC. The large difference in the sensitivity rate between the chilled radish sprout samples at the low inoculation level and those at the high inoculation level may be due not only to the detection limit of the methods, but also to a failure to include the target organism in some of the samples at the low inoculation level. The lower sensitivity rates for the frozen radish sprout samples compared to the chilled samples may reflect the death and / or injury of E. coli O157:H7 cells by freezing. Taken together, the results of the radish sprout samples demonstrate the comparative efficiency of the methods in combination with mEC 1 n enrichment, i.e., the use of IMS can raise the sensitivity, and CT-SMAC, BCM O157 and CHROMagar are superior to SMAC for the isolation of the organism. Since some samples were shown as positive by BCM O157 or CHROMagar but not by CT-SMAC, the combined use of CT-SMAC and an agar containing a b-glucuronidase substrate may raise the detection rate. Incubation in mEC 1 n at 428C for 18 h was used for selective enrichment in this study, although other conditions have also been used (Padhye and Doyle, 1991; Kim and Doyle, 1992; Wright et al., 1994; Bolton et al., 1995, 1996; Flint and Hartley, 1995; Weagant et al., 1995; Chapman and Siddons, 1996; Restaino et al., 1997). We recently showed that E. coli O157:H7 inoculated into ground beef and radish sprouts is well recovered by incubation in mEC 1 n at 428C for 18 h, rather than in mEC 1 n at 378C for 6 or 18 h or at 428C for 6 h or 18 h in TSB or modified TSB. In agreement with this observation, the use of mEC 1 n and a temperature of 42–438C has been shown to be superior to that of other media (Johnson et al., 1995; Bennett et al., 1996) and temperatures (Szabo et al., 1990; Blais et al., 1997) for the selective enrichment of E. coli O157:H7. Heuvelink et al. (1997) have recommended the IMS-plating method using CT-SMAC as the plating medium combined with enrichment in mEC 1 n at 378C for 6–8 h with shaking, but a static incubation may be better from a practical viewpoint, as has been suggested by Blais et al. (1997). Based on the results of the present study, we recommend the IMS-plating method using CT-SMAC and agars containing a
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b-glucuronidase substrate in combination with static enrichment incubation in mEC 1 n at 428C for 18 h.
Acknowledgements This research was supported by the Health Science Research Grants of the Ministry of Health and Welfare in Japan. The authors wish to thank the following collaborators: Agata, N., Nagoya City Health Research Institute, Nagoya; Akahane, S., Japan Food Hygiene association Institute of Food Hygiene; Chou, N., Tochigi Prefectural Hygienic Institute, Tochigi; Goto, K., Niigata Prefectural Research Laboratory for Health and Environment; Horikawa, K., Fukuoka Environmental Research Center; Kai, A., Tokyo Metropolitan Research Laboratory of Public Health, Tokyo; Kaneko, M., Yamanashi Institute for Public Health, Yamanashi; Kumagai, M., Iwate Prefectural Institute of Public Health, Iwate; Mabara, S., Ibaraki Prefectural Institute of Health; Machigaki, E., General Testing Research Institute of Japan Oilstuff Inspectors’ Corporation, Kobe; Masaki, H., Saitama Institute of Public Health, Saitama; Nukina, M., Public Health Institute of Kobe City, Kobe; Saito, M., Japan Frozen Foods Inspection Cooperation, Tokyo; Tanno, K., Japan Food Research Laboratories, Tokyo; Terai, K., Shizuoka Prefectural Institute of Public Health and Environmental Science, Shizuoka; Uchimura, M., Public Health Laboratory Chiba Prefecture, Chiba; Uchiyama, S., Food and Drug Safety Center, Hatano-City. We also express thanks to Chisso Co., Yokohama, and Eiken Chemical Co., Biochemical Research Laboratory, Tochigi-Prefecture, Japan, for technical assistance with analyses by EHEC-Tek ELISA System and Rapiblot E. coli O157, respectively.
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