In laboratory tests of outbreaks of food poisoning, it is important to isolate the same causative microorganisms from stools of patients and the causative food samples. However, in Vibrio parahaemolyticus food poisoning outbreaks, it is difficult to isolate Kanagawa phenomenon-positive (KP+) strains from causative food samples, whereas they are easily isolated from the patients. Therefore, studies were made for a method for effective isolation of KP+strain from the causative seafoód samples. Twelve seafood samples collected from five outbreaks of food poisoning due to V. parahaemolyticus in Hiroshima prefecture were investigated. Each sample was homogenized and inoculated into equal volume of double strength polymyxin broth. After incubation, the polymyxin broth culture was subcultured onto three TCBS agar plates, which were incubated at 35°C for 24 hr. Then, a total of 100 colonies from TCBS agar plates were inoculated onto three modified Wagatsuma agar plates. KP+strains were isolated from all samples by this method. The rate of isolation of KP+strains was 5.96% (75/1, 261) and that from individual sea food sample varied widely from 1.33 to 18.1%. In the experiments using artificially prepared mixtures of KP+and KP- strains, the recovery rate (Y) of the KP+strain from a mixture and the dilution rate (X=log10 (KP-/KP+)) of KP-: KP+was significantly correlated (r=-0.903, p<0.01) and a regression formula of Y=14.393X-1.536 was obtained. By this formula, the incidence ratio of KP-: KP+strains in the seafood samples investigated was calculated at 1: 1/60. This method is considered to be effective for recovery of KP+strains from seafood samples in food poisoning outbreaks.
In an outbeak of Clostridium perfringens food poisoning, isolates from stool specimens from 29 patients represented five Hobbs' types, ten Tokyo Metropolitan Research Institue of Public Health's types (TW), and untypable from two patients. Isolation of the organisms was attempted from nine stool specimens treated under three different conditions; untreated and heat-treated for 10 min at 100°C and for 60 min at 100°C. The results obtained were as follows: Among 135 isolates, frequency of isolation of a particular sero-type differed depending upon the condition for heat treatment. After heating for 60 min at 100°C, H12 and H13 organisms were isolated in 11.1% and 77.8%, respectively. After heating for 10 min at 100°C, H6, H12, and H13 organisms were isolated in 55.6%, 22.2%, and 77.8%, respectively. From unheated specimens, H6 organisms were isolated in 100% and H12 in 11.6%. Only the H6 isolates among those of the five Hobbs' sero-types were shown to prouce enterotoxin and to form relatively heat-labile spores. From these results, we concluded that the H6 organisms were the true etiological agent of this outbreak. It seems justified to recommend that unheated in addition to heat-treated stool specimens should be examined for C. perfringens and the isolates be tested for the sero-type and enterotoxigenicity in laboratory diagnosis of C. perfringens food poisoning.
A large number of Photobacterium phosphoreum (6-7 log/g) was isolated from “tamagoyaki” (a kind of nigirisushi; Japanese food) that had been lumineferous in the dark. The isolates were smeared on the surfaces of sliced “tamagoyakis”. After the incubation at 10°C for 48 hr or at 25°C for 24 hr, the surfaces became luminous. It was indicated that this abnormality of “tamagoyaki” was caused by contamination with and multiplication by P. phosphoreum. On the surface of “tamagoyaki”, the bacteria in an early growth phase in such a small number as 4 log/g luminesced. Furthermore, the luminescence was observed when pieces of squid, boiled prawn or “yakichikuwa” (a kind of food made of fishes) with the bacteria were incubated, but not observed on pickled Japanese gizzard shad. Nevertheless the the bacteria grew on the surface of tuna, but no luminescence was observed on it. The bacteria produced a small amount of histamine on squid and tuna (less than 250μg/g), and their ability to putrefy food seemed to be low. The opitmum concentration of sodium chloride for growth of the bacteria in a medium was 3%, but they grew in food containing sodium chloride less than 0.5%. When sodium chloride in the medium was replaced by potassium chloride, calcium chloride, magnesium chloride, ammonium chloride or sodium phosphate, the bacteria were still able to grow but unable to grow when replaced by potassium phosphate or sucrose. The bacteria metabolized arginine by arginine decarboxylase but not by arginine dehydrolase.
Two outbreaks of food poisoning due to Salmonella sp. (1) serovars Enteritidis and Litchfield occurred at two hotels in Shizuoka Prefecture in July and September, 1989. Raw fish “shimasuzuki” served at the restaurants in the hotels were presumed to be the food responsible for both outbreaks. So, we examined “simasuzuki” cultured in freshwater and the environments of the fish pond for the presence of Salmonella. S. Enteritidis and S. Lithfield were isolated from “shimasuzuki”, the fish pond and patients and the isolates were examined for the drug susceptibilities, phage-types and plasmid profiles. Approximately 78% (7/9) of S. Enteritidis isolates were of phage type 8. All S. Litchfield isolates were resistant to erythromycin. Some of the plasmids were unique to each of the serovars implicated in these outbreaks and their molecular sizes were 36-MDa for S. Enteritidis and 1.4-and 2.2-MDa for S. Litchfield. The drug susceptibilities, phagetypes and plasmid profiles were useful epidemiological markers for these outbreaks.
To improve the sensitivity of microbiological detection of resiudual antibacterial agents in meat and fish, various factors affecting the formation of the inhibition zone were studied. For the media for detecting antibacterial agents, antibiotic medium (AM) 5 was useful for detecting penicillin (PCs), macrolide antibiotics (MLs), and aminoglycosides (AGs) ; AM 8 for tetracyclines (TCs) ; and sensitivity disk agar supplemented with trimethoprim (SD-T) for sulfa drugs (SAs). Optimal conditions were as follows: include the agar concentration, 1.5%. ; medium amount of 8 ml for a 86 mm petri dish; inoculum size, 105 cells per ml of the plate medium. Refrigertion of the plate medium for 30 min prior to imcubation was sufficient. The thin-layer plate/paper disk assay method was more sensitive than in the double-layer plate/cup assay method.