We analyzed eight strains of Clostridium perfringens belonging to distinct Hobb's types for their enterotoxin gene, and of them, we analyzed six strains for C. perfringens enterotoxin (CPE) production. All strains possessed the enterotoxin gene, but enzyme-linked immunosorbent assay (ELISA) showed a wide variation in the level of CPE in 24hr sporulation cultures. This finding suggests that phenotypic determination is essential for the confirmation of enterotoxigenicity of C. perfringens. Flow cytometry (FCM) using enterotoxin-specific monoclonal antibody was applied to identify enterotoxin-producing C. perfringens, especially the sporangia. FCM profiles of sporangia harvested from 6-8hr sporulation cultures in Duncan and Strong (DS) medium showed a positive reaction for CPE. Of six different strains, the higher enterotoxin-producing strains showed higher peak channel numbers under FCM analysis, and this finding was coincident with ELISA results. Preparation of the sporangia was easier and faster than that of the spores. FCM using monoclonal antibody was found to be applicable to the rapid and specific detection of enterotoxin-producing C. perfringens at an early stage of sporulation.
Listeria monocytogenes have been attracting recent attention worldwide as an important causative agent of foodborne infections. We attempted to adapt the PCR method for quick and specific detection of this bacteria from foodstuff, and carried out basic studies on seven PCR primers (C-D, MonoA-B, hly1-2, prfAI-2, LM1-2, SH2A-B and SI3A-B) regarding the detection by specificity and differences in serovars. The detection of specificity and different serovars against L. monocytogenes were the highest for primer MonoA-B. The detection sensitivities were good for primers prfAI-2, hly1-2 and LM 1-2. As for specificity, cross-reacting with some strains was observed and bands other than the target PCR products were obtained. Non-specific products disappeared and specificity increased with some primers for bands other than the targets by varying the dilution concentration.
Viability of Escherichia coli O157:H7 during frozen storage at -20°C was measured by plating methods using Tryptic Soy Agar (TSA) and Cefixime-Tellurite-Sorbitol-MacConkey (CT-SMAC) agar. Viability of unwashed cells of exponentially-growing E. coli O157: H7 gradually decreased with an increase in storage period when the cells were suspended in water, 50 mM phosphate buffer (pH 7.0) and Tryptic Soy Broth. However, the viability decreased to only about 10% of that of the unfrozen cells after a 14-day frozen-storage in physiological salt solution. In contrast, the viability of the cells washed with water was the lowest when the cells were frozen-stored in physiological salt solution. Viability during frozen storage of both washed and unwashed cells of E. coli O157: H7 harvested at the stationary phase of growth was higher than that of the exponentially growing cells. When the cells in the exponential phase of growth were frozen-stored in phosphate buffers (pH6 .0) of various concentrations, the viability was the highest in 200mM buffer. Effects of pH on the viability of E. coli O157: H7 were examined after frozen-storage at-20°C for 3 days. Viability was the highest at pH6.5 in the cells of both exponential and stationary phases of growth after frozen storage. The viability of the washed cells was higher than that of the unwashed cells. The viability of E. coli O157: H7 was also measured by microscopy using LIVE/DEAD BacLightTM Bacterial Viability Kit. In the unwashed cells of the stationary phase of growth, the number of viable cells measured by the plating methods using TSA and CT-SMAC agar was 32% and 23% of that measured by microscopy using the kit, respectively, after storage in 50mM citric acid-phosphate buffer (pH6.5) at-20°C for 3 days. In contrast, in the washed cells, these numbers were 69% and 66% of that measured by microscopy, respectively.
We evaluated the most effective media for isolating hydrogen sulfide (H2S) non-producing Salmonella. Hydrogen sulfide non-producing Salmonella is a common cause of of food poisoning and the incidence seems to be increasing. At present, it is popular to use medium for isolating Salmonella that is based on Salmonella spp. producing H2S. However, it is difficult to detect H2S non-producing Salmonella and it is very easy to miss them. We evaluated several popular media in food testing using 27 strains of H2S nonproducing Salmonella that were isolated from feces samples. Many H2S non-prpducing Salmonella were not detectable by traditional media nor Rambach Agar or Brilliant Green Agar. The latter two media do not depend on H2S production. A new chromogenic medium CHROMagar Salmonella (CHROMagar Ltd; France) uses a specific enzymatic reaction of Salmonella, and was able to distinguish all Salmonella colonies clearly irrespective of H2S production, therefore, the CHROMagar Salmonella is very valuable in screening for Salmonella in food and environmental specimens. However, the media did not support the growth of Shigella and Salmonella Typhi and thus is not suitable for clinical specimens. We believe that this chromogenic media should be widely used as isolating medium for the screening of Salmonella to increase the reliability of food sanitary control.