Thermostable direct hemolysin (TDH) produced by Vibrio parahaemolyticus has been supposed to be the greatest causative factor for the enteropathogenicity. However, there have been no reports concerning the existence of TDH in any foods. We tried to detect it in some foods which are well known as the causative foodstuffs of this foodborn disease, using the method of the human red blood cell lysis system. Fresh shrimp showed the lytic activity only when the strain was inoculated. Shirasu-boshi (salted, small and half-dried sardines) already showed the lytic activity without the strain, and increased activity was observed after the inoculation. The lytic activity of fresh oyster was not changed significantly after the inoculation. The pre-incubation with trypsin or pancreatin for 1 hr at 37°C increased the lytic activity in fresh shrimp. These findings auggest that TDH is produced not only in some foods, but also in our small intestine by V. parahaemolyticus.
An outbreak of mixed food borne infection with 2 serotypes of enterotoxigenic Escherichia coli occurred twice at a wedding hall in Osaka during the period from September 11 through 23, 1993. During the first outbreak, 776 out of 1242 persons were affected, while 350 out of 627 persons were affected during the second outbreak. The major clinical symptoms observed among patients in both cases were diarrhea and abdominal pain. E. coli O25:H42 and O169:H41 were isolated from the feces of the patients (first outbreak), and were confirmed to produce heat stable toxin (ST). We succeeded in detecting E. coli O169:H41 from the incriminated food (Kaiseki Ryouri). This may be the first report of an enterotoxigenie E. coli isolated from incriminated food as the cause of outbreak in Japan.
The polymerase chain reaction (PCR) technique was applied to detect L. monocytogenes in cheese samples. Two types of soft cheese were inoculated with various numbers of L. monocytogenes (serovar 1/2a and 4b). Then, 10g of each cheese sample was mixed 50ml of modified EB broth (MEB), buffered by the addition of MOPS buffer, and incubated at 30°C for 20 hours. An 0.5ml aliquot of each MEB culture was plated onto Oxford agar. After incubation at 35°C for 24 hours, the growth was removed and suspended in distilled water. This suspension was boiled, centrifuged and the resultant supernatant fluid was used as a DNA sample for PCR. Primers used in the study were designed to amplify a 702-bp region of the L. monocytogenes listeriolysin O (hly) gene, as previously reported by Border et al (1990). Fewer than 10 CFU of L. monocytogenes per g of cheese was detectable by this procedure, irrespective of cheese type and Listeria stain. Seventy-six retailed cheese samples including naturally contaminated ones which had been stored in a freezer were assayed by the PCR procedure as described above. A conventional culture procedure (IDF standard) was also performed on the same cheese samples In 76 cheese samples, 12 were identified as being positive by both the PCR procedure and the conventional culture procedure. Four samples identified as being positive by the PCR procedure were negative by the conventional culture procedure. Among 60 samples that were negative in the PCR assay, 9 were shown by culture procedure to contain L. innocua. These findings demonstrated that the PCR assay following a two-day broth and plate enrichment is useful for the rapid detection of L. monocytogenes in cheese samples.
The contamination of coliform groups were investigated on 1,969 and 220 samples of individual well water in Tokyo from May to December in 1991 and from May to November in 1992, respectively. Coliform groups were isolated in 672 from 1,969 samples (34.1%) in 1991 and in 85 from 220 samples (38.6%) in 1992. The numbers of coliform groups were detected from 1-2,000 CFU per 100ml of individual well water samples. One hundred thirteen strains isolated were identificated in Enterobacter (27.5%), Klebsiella (26.6%), Serratia (15.0%), Citrobacter (8.9%), Kluyvera (6.1%), Escherichia (4.4%), Rahnella (3.5%), Leclercia (0.9%) and Unknown (7.1%). The most predominant species of coliform groups in individual well water samples were K. pneumoniae (14.2%), which were followed by E. cloacae (11.5%) and K. oxytoca (10.6%) 5 strains of E. coli was isolated from 113 strains (4.4%).