DIASALM was tested for its suitability for the simple, rapid screening and examination of typical and atypical Salmonella. In an additional recovery test by the DIASALM method that used minced chicken meat, 8 strains of atypical Salmonella including S. Enteritidis not producing hydrogen sulfide, and lysine-negative S. Chester were successfully recovered. Using the DIASALM method and the FSIS method which shows a high isolation rate, 32 samples of minced chicken meat were examined. As a result, 17 (53.1%) of the samples were judged positive by both examination methods. The detection sensitivities of these 2 exmination methods were the same, but screening of bacteria with the DIASALM method achieved significantly better results than with the FSIS method. This was because, using the DIASALM method, pure Salmonella could be incubated, whereas significant contamination with other bacteria occurs with the FSIS method. If the DIASALM is used, the latex agglutination test after incubation for 24 hours could rapidly confirm Salmonella. DIASALM is an excellent examination method for epidemiological investigations and the detection of typical and atypical Salmonella can be rapidly evaluated.
We analyzed the microbial communities in kusaya gravy obtained from three manufacturers by a combination of cultivating and molecular typing methods, PCR-RFLP and PCRDGGE. Viable counts were 1.2×108-1.5×109/g on TSA medium, BP5G medium and ABCM medium. With PCR-RFLP, forty isolates obtained from BP5G medium and ABCM medium were classified into six to nine groups. Representative strains of the isolates were identified as Pseudomonas, Marinobacter, Peptostreptococcus, Enterococcus etc. with 16S rDNA sequencing. DGGE analysis of 16S rDNA fragments amplified directly from each of three kinds of kusaya gravy showed a similar banding pattern each other. Several main fragments were recovered from DGGE-gel and were cloned and sequenced. The existence of Bacteroides, Flavobacterium, Fusobacterium, Eggerthela lenta and Clostridium was indicated from the analysis of fragments. Therefore, there are great differences between the dominant organisms obtained by culturing and by PCR-DGGE, suggesting that unculturable organisms prevail in kusaya gravy.
In this study, we compared the performance of a variety of systems to detect Escherichia coli 0157 inoculated into different kinds of foods. Samples were subjected to enrichment culture followed by plating onto sorbitol MacConkey agar (SMAC) or SMAC containing cefixime and potassium tellurite (CT-SMAC) with or without immunomagnetic separation (IMS). Finally E. coli O157 was detected by using commercial immunoassay kits after enrichment culture. The enrichment cultures were done for 18 hr at 42°C, using four different media such as trypticase soy broth (TSB), TSB containing cefixime, tellurite and vancomycin (CTV-TSB), modified EC broth (mEC) and mEC containing novobiocin (N-mEC) for experiment1 (raw ground beef), or using CTV-TSB and N-mEC for experiment 2 (raw ground beef, potato salad and tossed salad). Immunoassays were performed uSing five different commercial kits such as VIP, Reveal, PATH-STIC, EZ Coli and Now E. coli. These experiments were performed simultaneously by six different laboratories with the same materials and methods. The IMS procedure following enrichment culture increased the detection rate of E. coli O157, irrespective of the enrichment media. E. coli O157 was recovered in 49 and 117 samples out of 144 and 216, respectively, while only 38 and 73 samples were positive when the IMS was not applied, irrespective of experiments 1 and 2. In experiment 1, CTV-TSB showed the best recovery rate among the four enrichment media, while TSB was the only media that recovered the E. coli O157 at a level of 5.5×10-2-5.5×10-4 cfu/25 g. In experiment 2, CTV-TSB showed a better recovery rate than N-mEC from raw ground beef, but the recoveries from potato salad and tossed salad were slightly better in N-mEC than in CTVTSB. The fishing of E. coli O157 from CT-SMAC increased the detection rate of E. coli 0157 in both experiments. From the results we recommend the use of CTV-TSB and CT-SMAC for the detection of E. coli O157 especially in raw ground beef contaminated by competitive bacteria. The immunoassays detected E. coli O157, irrespective of commercial kit used, and the recovery rate was almost as good as culture diagnosis without IMS procedure. The immunoassays used in this study are simple, and the test results are less influenced by individual techniques than the isolation method. They are convenient to use in screening procedure for the detection of E. coli 0157 from large quantities of food samples.
Nineteen cases of food poisoning caused by Salmonella Enteritidis (SE) occurred in Kobe City between 1989 and 2002. The causative food was identified in nine cases, while it was not clarified in the remaining 10 cases. All causative agents was egg-containing foods. Most of the phagovar of SE causing the food poisoning were PT1, PT4, PT6 and PT 34, which was consistent with the transition of phagovar in Japan. However, PT14b detected in five cases in Kobe City since 2000 was a rare type, which had been isolated from only two cases in 11 years (1990-2000) in Japan. In our survey of unpasteurized eggs examined from 1993 to 1998, PT1 and PT4 were detected throughout the country, but PT14b was not found at all. Since three cases of food poisoning due to SE had occurred in a short period (from October 1999 to June 2000) in Kobe City, we carried out genotype analysis employing the AFLP method for rapid epidemiological investigation. The genotypes of SE derived from the three cases were identical, but the phagovar were different. It was impossible to apply the AFLP method to epidemiological analysis as with the PFGE method, which could specialize an identical serotype of enterohaemorrhagic Escherichia coli O157. Therefore, further investigation of the AFLP method is required.