Kenji Ohta

Biochemical and Molecular Characterization of Salmonella Serovar Enteritidis Phage Type 4 Isolated from Food Poisoning Outbreaks in Tokyo

Since 1989, outbreaks of Salmonella ser. Enteritidis (S. Enteritidis) food poisoning have dramatically increased in Tokyo, and a total of 31 outbreaks has been reported in 1989. Twenty-one of these 31 outbreaks were caused by S. Enteritidis PT34, but 8 outbreaks were caused by S. Enteritidis PT4. After 1990 instead of SE PT34, food poisoning due to PT4, which was a very common phage type in the UK, has increased in Tokyo. Between 1989 and 1995, there were 144 food poisoning outbreaks caused by S. Enteritidis, and 64 of these outbreaks were by due to S. Enteritidis PT4, which was one of the main phage types in Tokyo. To characterize these strains of phage type (PT) 4,293 isolates from patients, and vehicle foods, eggs and environment in Tokyo were examined for plasmid DNA profiles, acid productivity from glycols (propylene and ethylene) and antimicrobial resistance patterns. Plasmid DNA was extracted by Kados method, and analyzed by agarose gel electrophoresis. The acid productivity from propylene glycol or ethylene glycol were tested using Barsicow medium with 1% propylene glycol or ethylene glycol. Antimicrobial susceptibility to AM, CP, TC, SM, KM, NA, ST, FOM and NFLX was tested by the K-B disc method. The strains of PT4 were further subdivided into 9 types by those epidemiologic marker analysis. The prevalent pattern of PT4 strains was type A plasmid profile carrying only one plasmid (60 kb) and there were 2 kinds of antibiograms. One was SM resistant, while the other was susceptible. A total of 56 (87.5%) of 64 outbreaks was found to have been caused by these types of S. Enteritidis. Several kinds of egg-related foods were suspected as the vehicles of transmission among 24 outbreaks. Especially, in 5 outbreaks, S. Enteritidis strains were isolated both from patients and suspected food which were cooked with egg. This strongly suggests that these foods may be the potential source of infection in S. Enteritidis PT4 outbreaks.

Enteropathogenicity and some Biological Features of Group F (Ef—6) Vibrio Isolates

The organism now referred to as group F or EF-6 vibrio had probably been put together with Aeromonas species in the past without drawing any special attention from the standpoint of gastrointestinal diseases.

Regarding the Shigella flexneri 4 strains isolated in recent years

A total of 38 strains of Shigella flexneri 4, 14 of which were subserotype 4a but 24 of which were of an undecided subserotype, isolated in recent years, were studied for their biochemical and serological characteristics, and drug susceptibility. Among the biochemical characteristics examined, 21 of the 38 strains (55.3%) were mannitol negative biotype and utilized sodium acetate, meanwhile, 17 of the mannitol positive strains did not utilize sodium acetate. The results of serological tests showed that 22 of the 24 strains, were of an undecided subserotype, basically subserotype 4a. Drug susceptibility was tested for nine drugs (CP, TC, SM, KM, ABPC, ST, NA, FOM and NFLX). Fifteen of the 17 mannitol positive strains (88.2%) and 12 of the 21 mannitol negative ones (57.1%), were found to be resistant to the 5 drugs, such as CP, TC, SM, ABPC or ST. None of the strains were resistant to KM, NA, FOM or NFLX.

Lysogenicity of Vibrio Cholerae

In 1963, Takeya and Shimodori described a certain temperate phage infecting a majority of strains of Vibrio cholerae biotype eltor isolated in the western Pacific region and named it as kappa type phage(1). Then, on the basis of the individual strains’ lysogenicity by, and their sensitivity to, this phage, they classified the eltor type vibrio into three types, namely (i) Celebes type, which is lysogenic, (ii) cured type, non-lysogenic and sensitive, and (iii) classical-Ubon type, non-lysogenic and insensitive(2).

Vital States of Sh. sonnei and S. typhi in Water and Various Kinds of Food

In order to obtain the basic data to prevent the outbreaks of Shigellosis and Typhoid fever caused by polluted drinking water or foods, the fate of Sh. sonnei and S. typhi in well water and various kinds of food were investigated.Both Sh. sonnei and S. typhi, which can be survived for at least 6 days or longer in well water, were killed within 6 hours by adding 0.4ppm of chlorine. When well water polluted with stool was disinfected with the same amount of chlorine, the both bacteria survived 18-96 hours. The polluted well water in this expemiments was prepared by adding stool in such an amount (0.001%), as to give no perceptible change in tint, turbidity and smell. Further detailed experiments revealed that in successful chlorine disinfection to both bacteria in the above mentioned polluted well water or standing water, such an amount of sodium hypochlorite as to produce 3ppm/ml of free chlorine is necessary.Experiments on food were performed with bread, salad, “Fukujinzuke” (a kind of Japanese pickles), raw cream, commercial milk and cheese, all of which can be taken without any treatment, and beefs untreated and heated at 100°C for 30 minutes. In this experiments, bread and “Fukujinzuke” were preserved at 25°C, and other foods were preserved at 5°C. All of these were inoculated with each 106 cells of Sh. sonnei and S. typhi, respectively, and the fate of these bacteria were pursued.In bread, both bocteria survived as long as 30 days. In salad, they showed slight growth at 4 hours after the inoculation, and remained constant level for the succeeding 20 days, after that the curves weme declined gradually, and in the case of S. typhi, they diminished at the 30th day, but in the case of Sh. sonnei, they could survive little longer. In case of raw cream, both of the bacteria were demonstrated until the end of the 4th week, and in commercial milk, S. typhi survived for 30 days and Sh. sonnei more than 75 days. In raw beef, they survived more than 10 days. In heat treated beef, Sh. sonnei survived for 2 weeks, and S. typhi for 4 weeks. In case of “Fukujinzuke”, the results were quite different from other above mentioned foods, i. e., both bacteria were killed within 48 hours after inoculation. This is considered to be the influence of food preservatives contained in this pickle.