Survival of Escherichia Coli O157:H7, Salmonella Enteritidis, Staphylococcus Aureus, and Listeria Monocytogenes in Kimchi

1497

Journal of Food Protection, Vol. 67, No. 7, 2004, Pages 1497±1500 Copyright ᮊ, International Association for Food Protection

Research Note Survival of Escherichia coli O157:H7, Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes in Kimchi

Y. INATSU,* M. L. BARI, S. KAWASAKI, AND K. ISSHIKI

National Food Research Institute, Research Planning and Coordination Division, Food Hygiene Team, Kannondai-2-1-12, Tsukuba 305-8642, Japan

MS 03-227: Received 21 May 2003/Accepted 26 July 2003 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/67/7/1497/1676839/0362-028x-67_7_1497.pdf by guest on 30 September 2021

ABSTRACT

The survival of gram-positive and gram-negative foodborne pathogens in both commercial and laboratory-prepared kimchi (a traditional fermented food widely consumed in Japan) was investigated. It was found that Escherichia coli O157:H7, Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes could survive in both commercial and laboratory- prepared kimchi inoculated with these pathogens and incubated at 10ЊC for 7 days. However, when incubation was prolonged, the S. aureus level decreased rapidly from the initial inoculum level to the minimum detectable level within 12 days, whereas Salmonella Enteritidis and L. monocytogenes took 16 days to reach similar levels in commercial kimchi. On the other hand, E. coli O157:H7 remained at high levels throughout the incubation period. For laboratory-prepared kimchi, the S. aureus level decreased rapidly from the initial inoculum level to the minimum detectable level within 12 days, and L. monocytogenes took 20 days to reach a similar level. E. coli O157:H7 and Salmonella Enteritidis remained at high levels throughout the incubation period. The results of this study suggest that the contamination of kimchi with E. coli O157:H7, Salmonella Enteritidis, S. aureus, or L. monocytogenes at any stage of production or marketing could pose a potential risk.

Kimchi is a traditional fermented Korean food whose tation differ widely depending on the cabbage and the other preparation involves a series of processes, including pre- ingredients. The growth, activity, and role of the microor- treatment of Chinese cabbage (or radish), brining, blending ganisms involved in kimchi fermentation are in¯uenced with various spices and other ingredients, and fermentation. more by environmental conditions. There are many (Ͼ50) types of kimchi, depending on the From August to September 2001, a widespread out- raw ingredients and preparation methods used. Of these break of Escherichia coli O157:H7 in the Kanto area of types of kimchi, baechu kimchi (Chinese cabbage kimchi) Japan occurred, and in an epidemiological investigation, lo- is the most typical and is often referred to simply as ``kim- cally made kimchi was incriminated, raising special con- chi'' (2). cerns about the transmission of E. coli O157:H7 by kimchi Kimchi is usually prepared from Chinese cabbage (7, 10). (Brassica pekinensis Rupr.) and processed with a seasoning The present study was carried out to investigate the mixture consisting of red pepper (Capsicum annuum L.) survival of E. coli O157:H7, Salmonella Enteritidis, Staph- powder, garlic, ginger, and edible Allium varieties and is ylococcus aureus, and Listeria monocytogenes in both com- subjected to long-term fermentation at low temperatures in mercial and laboratory-prepared kimchis. winter (2). However, for Japanese-style kimchi, spices and seasonings are involved in the overnight brining of Chinese MATERIALS AND METHODS cabbage at low temperatures. This product is packed and Test strains. The strains studied and their sources were as distributed to the market immediately after preparation and follows. Enterohemorrhagic E. coli O157:H7 strains CR-3, MN- is consumed before much fermentation has taken place. 28, MY-29, and DT-66 were isolated from bovine feces. Salmo- This type of kimchi is very popular in Japan. The pHs of nella Enteritidis strains SE-1, SE-3, and SE-4 (from chicken feces) Japanese and Korean commercial kimchis range from 4.5 and SE-2 (from bovine feces) were provided by the Laboratory of Zoonosis, National Institute of Animal Health, Tsukuba, Japan. to 5.0 and from 3.8 to 4.2, respectively, depending on the Salmonella Enteritidis strain IFO-3313 (of an unknown origin) raw material and the recipe. In laboratory-prepared baechu was obtained from the Institute for Fermentation, Osaka, Japan. kimchi made with the same recipe but with different spices, S. aureus IFO 13276 was isolated from a human lesion, and S. high concentrations of nonvolatile organic acids like lactic aureus JCM 2413 was a clinical isolate. L. monocytogenes strains and succinic acids and major volatile organic acids like ace- ATCC 43256 (from Mexican-style cheese) and ATCC 49594 (de- tic and propionic acids are present. The numbers and spe- rived from L. monocytogenes Scott A) were obtained from the cies of major microorganisms involved in kimchi fermen- American Type Culture Collection, Manassas, Va., and L. monocytogenes strains JCM 7676 (from roast beef), JCM 7672 (from * Author for correspondence. Tel and Fax: ϩ81-29-838-8067; E-mail: salami sausage), and JCM 7671 (from lax ham) were obtained [email protected]. from the Japan Collection of Microorganisms. The rifampicin- 1498 INATSU ET AL. J. Food Prot., Vol. 67, No. 7 resistant derivatives of these strains were used in this study to acid bacteria were enumerated on plate count agar containing BCP facilitate the recovery of test strains from kimchi. (Nissui).

Preparation of inocula. Each strain of E. coli O157:H7, Sal- Statistical analyses. All trials were replicated four times. Re- monella, S. aureus, and L. monocytogenes was cultured in tryptic ported plate count data represent mean values obtained for four soy broth (10 ml) supplemented with 50 ␮g of rifampicin per ml individual trials, with each of these trials involving duplicate sam- (TSB/Rif) at 37ЊC. Cultures were transferred to TSB/Rif by loop ples. Signi®cant differences in plate count data were established at three successive 24-h intervals immediately before they were by the least signi®cant difference at the 5% level of signi®cance. used as inocula. Cells of each strain were collected by centrifu- gation (3,000 ϫ g, 10 min, 20ЊC) and resuspended in 5 ml of RESULTS AND DISCUSSION sterile distilled water. Equal volumes of cell suspensions of two Table 1 shows the survival of E. coli O157:H7, Sal- or ®ve strains of each pathogen were combined to give approxi- monella, S. aureus, and L. monocytogenes in commercial mately equal populations of each strain. The inoculum was main- Japanese kimchi and commercial Korean kimchi. The initial tained at 22 Ϯ 1ЊC and applied to the kimchi within 2 h of prep- counts of lactic acid bacteria for commercial Japanese kim- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/67/7/1497/1676839/0362-028x-67_7_1497.pdf by guest on 30 September 2021 aration. chi were 5.41 to 7.81 log CFU/g; however, after 1 week of Commercial kimchi. Commercially prepared kimchis pro- incubation, those values increased by ca. 1.0 to 2.0 log duced by different local manufacturers were purchased randomly CFU/g for all samples tested except that obtained from from different supermarkets in the Tsukuba area of Japan and were manufacturer D. On the other hand, irrespective of the ini- stored refrigerated for a maximum of 6 h before they were used tial populations of E. coli O157:H7, Salmonella, S. aureus, in experiments. Each kimchi sample was inoculated with the ap- and L. monocytogenes, these bacteria showed a tendency to propriate bacteria separately and incubated at 10ЊC for 7 days for decrease by ca. 1.0 to 2.0 log CFU/g (Table 1) over 7 days the determination of viable bacterial counts. in commercial Japanese kimchi. The lactic acid bacteria Laboratory preparation of kimchi. For laboratory-scale concentrations for Korean kimchi were higher than those preparation of kimchi, one whole Chinese cabbage (2.3 kg) was for Japanese products and the test kimchi produced for the cut into small pieces (4 by 4 cm), which were washed twice with study (Table 1). Viable counts of S. aureus and L. mono- chlorine water (100 ppm as effective chlorine) for 10 min and cytogenes after 7 days of incubation decreased signi®cantly were then washed with tap water. After the leaf pieces had been to near the detection limit (200 CFU/g) for some but not washed with tap water twice, they were soaked with salt (30 g/ all samples, but populations of the other organisms showed kg) and 7% sodium chloride water (300 ml/kg) overnight at 10ЊC little tendency to decrease for all samples of commercial for salt curing. The next day, salted leaves were washed twice for Korean kimchi (Table 1). 10 min with 3 liters of water to reduce the salt concentration. However, it was found that pathogenic bacteria like E. After the desalination, leaves were mixed with commercial sea- coli O157:H7 were able to survive under acidic conditions soning liquid (Ebara Co. Ltd., Yokohama, Japan), kept for 1 h at (at pHs of Ն4.0) for up to 54 days but were affected by 4ЊC for seasoning, and used for experiments. acidulants and temperature (3). Other serotypes of E. coli Procedure for inoculation. One hundred microliters of each have been shown to survive for at least 2 days in traditional cell suspension was added to 100 g of kimchi in a sterile plastic lactic acid±fermented foods (6, 8). Salmonella and Listeria cup separately, and the contents were mixed thoroughly with a also have systems that allow adaptation to low pHs (3.0 to sterile glass rod. The inoculated samples were then kept at 10ЊC 4.0) and survival at even lower pHs (4). A study by Si- for periodic microbiological analysis. mango and Rukure (9) on mahewu and sour porridge (traditional fermented foods in Zimbabwe) showed that all Survival study. The survival study involved determining strains of enteric pathogens except campylobacters survived numbers of pathogens that could be recovered from inoculated for 24 h after inoculation. kimchi after incubation at 10ЊC for 24 days. E. coli O157:H7, Salmonella, S. aureus, and L. monocytogenes were evaluated sep- Figures 1 and 2 show the survival of E. coli O157:H7, arately. Four replicate trials were performed for each pathogen, Salmonella, S. aureus, and L. monocytogenes in commer- and three or four samples were used for each trial. After inocu- cial Japanese kimchi and in test kimchi produced for the lation, 10-fold serial dilutions of the inoculated samples in 0.1% study. In commercial Japanese kimchi, the S. aureus level peptone water were plated every 4 days for the enumeration of decreased rapidly from the initial inoculum level to the surviving bacterial cells. Duplicate 0.1-ml quantities of appropri- minimum detectable level within 12 days, whereas Salmo- ately diluted suspensions of E. coli O157:H7, Salmonella, S. au- nella and L. monocytogenes took 16 days to reach similar reus, and L. monocytogenes were surface plated on tryptic soy levels in commercial kimchi. On the other hand, E. coli agar (TSA) supplemented with 50 ␮g of rifampicin per ml (TSA/ O157:H7 remained at high levels throughout the incubation Њ Rif). Inoculated enumeration media were incubated at 37 C for period (Fig. 1). In test kimchi, the S. aureus level also de- 24 to 28 h before presumptive colonies of each pathogen were creased rapidly from the initial inoculum level to the min- counted. At least ®ve presumptive colonies of E. coli O157:H7 imum detectable level within 12 days, and L. monocyto- were con®rmed with the E. coli O157 direct immunoassay test kit (Universal Health Watch, Columbia, Md.). Salmonella con®rma- genes took 20 days to reach similar levels (Fig. 2). Popu- tion was carried out by testing reactions on triple sugar iron (Nis- lations of E. coli O157:H7 and Salmonella increased little sui, Tokyo, Japan) slants and LIM medium (Nissui). Presumptive over the ®rst 4 days and decreased slightly over 8 days and/ colonies of Salmonella and S. aureus were con®rmed by biochem- or remained constant throughout the incubation periods, ical tests. Randomly picked presumptive colonies of L. monocy- whereas populations of S. aureus and L. monocytogenes togenes were con®rmed with API Listeria diagnostic kits. Lactic were found to decrease over time (Fig. 2). J. Food Prot., Vol. 67, No. 7 SURVIVAL OF PATHOGENS IN KIMCHI 1499

TABLE 1. Recovery of microorganisms from different commercial Japanese and Korean kimchi samples arti®cially inoculated with pathogenic bacteria and kept at 10ЊC for 7 days Population (log CFU/g) recovered fromb:

Japanese kimchi Korean kimchi

Manufacturer Microorganismsa Day 0 Day 7 Day 0 Day 7

A LAB 6.90 A 8.48 B 7.70 C 8.70 D E. coli 5.28 A 4.97 C 5.25 A 4.76 B SE 5.42 B 4.48 D 5.44 A 4.47 A S. aureus 4.98 D 3.20 A 5.01 A 2.73 C Listeria 5.52 A 3.97 A 5.59 C 3.44 C B LAB 6.37 E 7.32 A 5.15 E 7.40 A Downloaded from http://meridian.allenpress.com/jfp/article-pdf/67/7/1497/1676839/0362-028x-67_7_1497.pdf by guest on 30 September 2021 E. coli 5.26 A 3.69 A 5.28 A 4.33 B SE 5.34 A 3.76 B 5.40 A 4.73 A S. aureus 4.81 A 4.60 A 4.83 A 1.30 A Listeria 5.49 A 5.05 A 5.49 A 5.13 A C LAB 7.81 A 8.09 A 7.95 A 7.67 A E. coli 5.27 A 5.12 A 5.29 A 4.79 A SE 5.62 A 4.42 B 5.57 A 2.30 A S. aureus 5.10 A 3.48 A 4.95 A 1.36 B Listeria 5.48 A 4.55 A 5.38 A 1.36 C D LAB 5.41 B 4.69 D 5.93 B 8.72 B E. coli 5.29 A 5.10 A 5.30 A 5.06 B SE 5.40 A 4.64 A 5.34 A 4.21 A S. aureus 5.07 A 3.42 A 5.11 A 3.65 A Listeria 5.55 A 5.09 B 5.55 A 3.69 B E LAB 6.05 B 8.43 D 6.93 A 8.04 A E. coli 5.22 A 5.19 A 5.28 A 5.20 A SE 5.52 A 5.25 B 5.51 A 2.30 A S. aureus 5.19 B 2.50 B 5.25 A 2.08 B Listeria 5.60 A 3.74 D 5.53 A 5.53 A a LAB, lactic acid bacteria; E. coli, Escherichia coli O157:H7; SE, Salmonella Enteritidis; S. aureus, Staphylococcus aureus; Listeria, Listeria monocytogenes. b Average population for four replicate experiments (n ϭ 4). Values in the same column followed by the same letter are not signi®cantly different (␣Ͼ0.05).

FIGURE 1. Survival of inoculated Escherichia coli O157:H7 (ⅷ), Salmonella Enteritidis (Ⅵ), Staphylococcus aureus (᭡), and Lis- FIGURE 2. Survival of inoculated Escherichia coli O157:H7 (ⅷ), teria monocytogenes (ࡗ) strains in commercially prepared Jap- Salmonella Enteritidis (Ⅵ), Staphylococcus aureus (᭡), and Lis- anese-style kimchi kept at 10ЊC for 24 days. Periodic populations teria monocytogenes (ࡗ) strains in laboratory-prepared Japa- of lactic acid bacteria (∗) are also shown (n ϭ 4). nese-style kimchi kept at 10ЊC for 24 days (n ϭ 4). 1500 INATSU ET AL. J. Food Prot., Vol. 67, No. 7

Lactic acid fermentation of cabbage and other vegeta- REFERENCES bles is a common method for preserving fresh vegetables. 1. Beuchat, L. R. 1996. Pathogenic microorganisms associated with However, the most important role of lactic acid fermenta- fresh produce. J. Food Prot. 59:204±216. tion is the preservation of perishable vegetables and ®shes 2. Cheigh, H.-S., and K.-Y. Park. 1994. Biochemical, microbiological, and nutritional aspects of kimchi (Korean fermented vegetable prod- under safe and sanitary conditions. The organic acids ucts). Crit. Rev. Food Sci. Nutr. 34:175±203. (mainly lactic acid and acetic acid) produced by lactic acid 3. Conner, D. E., and J. S. Kotrola. 1995. Growth and survival of Esch- bacteria are effective antimicrobial agents, and they reduce erichia coli O157:H7 under acidic conditions. Appl. Environ. Mi- pHs in the foods to prevent the growth of undesirable microbiol. 61:382±385. 4. Foster, J. W. 2000. Microbial responses to acid stress, p. 99±115. In croorganisms (5). However, in the present study, we found G. T. Stortz and R. Hengge-Aronis (ed.), Bacterial stress responses. that pathogenic bacteria could survive if there was contam- ASM Press, Washington, D.C. ination at any stage of the fermentation process and/or in 5. Lee, C. H. 1997. Lactic acid fermented foods and their bene®ts in raw materials. The raw vegetables used for the production Asia. Food Control 8:259±269. 6. Sainz, T., C. Wacher, J. Espinoza, D. Centurion, A. Navarro, J. Mo- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/67/7/1497/1676839/0362-028x-67_7_1497.pdf by guest on 30 September 2021 of kimchi could be contaminated at the farm and serve as lina, A. Inzunza, A. Cravioto, and C. Eslava. 2001. Survival and vehicles for pathogenic bacteria (1, 11). Chinese cabbage characterization of Escherichia coli strains in a typical Mexican acid- and radishes are commonly used in the production of Jap- fermented food. Int. J. Food Microbiol. 71:169±176. 7. Saito, M., K. Ootsuka, T. Kurasono, Y. Ozeki, M. Yamaguchi, G. anese kimchi; the contamination of the cabbages or the rad- Kishimoto, and S. Aoba. 2001. An outbreak of Escherichia coli ishes and/or raw vegetables used in the production of kim- O157 caused by ``Japanese styled'' kimchi in Saitama. Infect. Agent chi with pathogenic bacteria at any stage of pre- or post- Surveillance Rep. 22:291. harvest production or marketing could pose a potential risk. 8. Simango, C. 1995. Effective acidi®cation of traditional fermented foods. J. Trop. Med. Hyg. 98:465±468. 9. Simango, C., and G. Rukure. 1991. Survival of Campylobacter jejuni ACKNOWLEDGMENTS and pathogenic Escherichia coli in mahewu, a fermented cereal gru- el. Trans. R. Soc. Trop. Med. Hyg. 85:399±400. This work was supported by a grant-in-aid (MAFF Food Research 10. Tazaki, T. 2002. A case of EHEC food poisoning caused by Japanese Project) from the Ministry of Agriculture, Forestry and Fisheries of Japan. styled kimchi in Tokyo. Infect. Agent Surveillance Rep. 23:139±141. We thank Dr. Nakazawa Muneo of the National Institute of Animal Health, 11. Viswanathan, P., and R. Kaur. 2001. Prevalence and growth of path- Tsukuba, Japan, for kindly providing the E. coli O157:H7 and Salmonella ogens on salad vegetables, fruits and sprouts. Int. J. Hyg. Environ. cultures. Health 203:205±213.