Jpn. J. Infect. Dis., 56, 137-139, 2003

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Laboratory and Epidemiology Communications

An Outbreak of Food-Borne Gastroenteritis Caused by Clostridium perfringens Carrying the cpe Gene on a Plasmid　

Daisuke Tanaka1,3*, Junko Isobe1, Shiho Hosorogi1, Keiko Kimata1, Miwako Shimizu1, Koji Katori1, Yotaku Gyobu1, Yoshiyuki Nagai1, Takayoshi Yamagishi2, Tadahiro Karasawa3 and Shinichi Nakamura3

1Department of Bacteriology, Toyama Institute of Health, Toyama 939-0363, 2Department of Laboratory Science, School of Health Sciences, Faculty of Medicine, Kanazawa University, Ishikawa 920-0942 and 3Department of Bacteriology, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-8640

Communicated by Yutaka Miyazaki

(Accepted July 24, 2003)

*Corresponding author: Mailing address: Department of Bacteriology, Toyama Institute of Health, Nakataikoyama 17-1, Kosugi, Toyama 939-0363, Japan. Tel: +81-766-56-5506, Fax: +81-766-56-7326, E-mail: daisuke.tanaka@pref.toyama.lg.jp

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for C. perfringens type A food poisoning. The cpe gene was present on a chromosome in food poisoning isolates but on a plasmid in non-food-borne human gastrointestinal disease isolates (1,2). Here we describe an outbreak of food-borne gastroenteritis caused by C. perfringens isolates carrying a plasmid-borne cpe gene.

On 31 May 2001, an outbreak of gastroenteritis occurred in a nursing home for the aged in Toyama Prefecture, Japan. Of 192 persons who ate a lunch prepared at a catering facility, 90 became ill. The predominant symptoms were diarrhea (99%) and abdominal pain (41%). The mean incubation period was 15.5 h.

A total of 107 stool specimens collected on 1 and 2 June were examined bacteriologically. Stool cultures were negative for Salmonella, Vibrio, Campylobacter, and Bacillus. However, C. perfringens was cultured from 90 samples. Twenty-four isolates selected randomly were examined for CPE production by using modified DS (mDS) medium (3) and PET-RPLA (Denka Seiken, Tokyo); 22 isolates (92%) were shown to be CPE positive. Pulsed-field gel electrophoresis (PFGE) with SmaI digestion was performed for eight of these 22 CPE-positive isolates (Fig.1). The eight isolates showed identical PFGE patterns. These eight isolates were serotype TW54.

Boiled bean was the most highly suspected vehicle because it was cooked in large quantities, cooled slowly after cooking, and served without adequate reheating. However, no boiled beans remained. Specimens of seven other foods remaining from lunch and 13 environmental swabs were all negative for C. perfringens.

PFGE Southern blot analysis was performed to establish the chromosomal or plasmid location of the cpe gene, following a procedure previously described (4-6). The principle of this method was as follows. C. perfringens chromosomal DNA is too large to enter a pulsed-field gel without any restriction enzyme digestion, whereas most plasmid DNA enters a pulsed-field gel without any restriction enzyme digestion. In addition, since I-CeuI sites are located exclusively on the C. perfringens chromosome but not on the plasmid, digestion of DNA samples with I-CeuI produces chromosomal DNA fragments that can enter pulsed-field gels but does not affect the migration of plasmid DNA. We discovered that isolate T102 from this outbreak carried a plasmid cpe (Fig. 2). Similar PFGE Southern blot results were obtained with two isolates from different patients (data not shown). In contrast, strain Osaka4205 from other food poisoning outbreaks carried a chromosomal cpe gene.

The heat sensitivities of spores produced by C. perfringens isolates were examined. Three isolates from this food poisoning outbreak and strain Osaka4205 from another food poisoning outbreak were cultured anaerobically overnight at 37℃ in mDS medium. One milliliter of each mDS culture was heated at 80 and 100℃, respectively, for 10 min. A 0.1-ml aliquot of each mDS medium culture was then inoculated into 10 ml of cooked meat medium (Difco, Detroit, Mich., USA), incubated overnight at 37℃, then examined for growth. The spores of the three isolates from this food poisoning outbreak showed resistance to heating at 80℃ for 10 min, but not at 100℃ for 10 min. In contrast, strain Osaka4205 showed resistance to heating at both 80℃ for 10 min and 100℃ for 10min. Therefore, the present food poisoning isolates were thought to be the heat-sensitive spore-forming C. perfringens. This finding is in agreement with that of Sarker et al. (6), who reported that the spores of chromosomal cpe isolates were more heat resistant than those plasmid cpe isolates. Tortora et al. (7) reported that enterotoxigenic heat-resistant spore-forming C. perfringens isolates could not ferment trehalose and inositol, whereas enterotoxigenic heat-sensitive spore-forming C. perfringens isolates were trehalose- and inositol-fermentative. The food poisoning isolates presented here fermented these two sugars. To our knowledge, this is the first case report of a food-borne disease outbreak caused by C. perfringens isolates carrying a plasmid cpe gene.

We thank the staff of the regional health office for the epidemiological information, and Chie Monma (Tokyo Metropolitan Research Laboratory of Public Health) for serotyping.

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