Prevalence of the main food-borne pathogens in retail food under the national food surveillance system in Japan.

The National Food Surveillance System in Japan was formed in 1998 to monitor the contamination of retail foods with bacterial pathogens. Approximately 2000-3000 samples were tested annually, and the data from food categories that had more than 400 samples collected during 1998-2008 were analysed. With regard to meat, the frequency of positive samples for Salmonella in chicken for raw consumption and ground chicken was 12.7% and 33.5%, respectively. Moreover, Shiga toxin-producing Escherichia coli (STEC) O157 was found in ground meat, organ meat and processed meat, although at a low frequency (0.1%). The prevalence of Campylobacter jejuni/coli was 13.3% and 20.9% in chicken for raw consumption and ground chicken, respectively. In vegetables and fruit, Salmonella was detected in cucumber, lettuce, sprout and tomato samples at a frequency of around 0.1-0.2%. With regard to seafood, Salmonella was found in 0.5% of oysters for raw consumption. Seafood was not contaminated with STEC O157 or Shigella. Serotype Infantis was the most frequently detected serotype of Salmonella in seafood, followed by the serotypes Typhimurium, Schwarzengrund and Manhattan. In ground chicken, 72.2% of the strains were identified as the serotype Infantis. E. coli, as an indicator of food hygiene, was detected in all food categories. The results show the prevalence of the above-mentioned pathogens in the retail food supplied in Japan; further, they indicate that consumption of raw food carries the risk of contracting food-borne infections.

Endoscopic full-thickness resection of a lateral spreading rectal tumor after unplanned injection of dilute hyaluronic acid into the subserosal layer (with video).

A 74-year-old woman underwent colonoscopy for investigation of a liver tumor. A lateral spreading tumor of the non-granular type (LST-NG), 25 mm in diameter, was detected at the rectosigmoid junction. As magnifying image-enhanced colonoscopy suggested a tubulovillous adenoma, endoscopic mucosal resection (EMR) was chosen for removal of the LST-NG. The lesion was effectively and evenly lifted after injection of 0.4% hyaluronic acid diluted with glycerol in the ratio of 1:1. A small amount of indigo-carmine dye was also added for coloration of the plane of resection. The lesion was completely removed en bloc. Although a blue-colored layer was identified in the resection defect, a small amount of a whitish layer was detected above the blue layer. The muscle layer was clearly located on the underside of the resected polyp. A total of 14 endoclips were used to close the defect completely. The patient was successfully treated conservatively without surgery. Histology of the resected specimen showed that it contained a tubulovillous adenoma with the submucosal layer and both layers of the muscularis propria. The surgical margin was free of neoplastic change horizontally and vertically. To the best of our knowledge, this is the first case report of full-thickness resection associated with EMR after unplanned injection of dilute hyaluronic acid into the subserosal layer rather than the intended submucosal layer. We describe how to promptly recognize this complication during colonoscopy, in order to achieve immediate closure of the defect, with the identification of a "mirror target sign" on the colonic wall.

Inactivation effects of UV irradiation and ozone treatment on the yeast and the mold in mineral water.

In recent years, bottled mineral water has undergone inactivation by methods other than the traditional heat treatment during the production process; there are fewer reports of the effectiveness of these inactivation methods on yeasts and molds in mineral water than on bacteria and protozoan oocysts. In this study, we evaluated the effects of UV irradiation and ozone treatment compared with heat treatment at 85 degrees C on yeast cells and mold spores inoculated into mineral water. A 5-log reduction occurred at a UV radiation dose of 31,433 microJ/cm2 for Saccharomyces cerevisiae and at 588,285 microJ/cm2 for Penicillium pinophilum. The treatment time for 5-log reduction estimated for UV irradiation was about 0.6 min for S. cerevisiae and about 10.7 min for P. pinophilum; at an ozone concentration of 0.1 ppm, it was 1.75 min for S. cerevisiae and 2.70 min for P. pinophilum, and at a concentration of 0.6 ppm, it was 0.32 min for S. cerevisiae and 0.57 min for P. pinophilum. Comparison of the inactivation effects among the three methods showed that UV irradiation and ozone treatment were less effective than heat treatment at 85 degrees C. Thus, when UV irradiation and ozone treatment are used for inactivation of mineral water, it seems that they need to be combined with heat treatment to achieve a definite effect. Yeast cells are more sensitive to all three inactivation methods than are mold spores, and the sensitivity of yeast cells and mold spores to these inactivation methods may vary among genera.

Differences in survival of Escherichia coli O157:H7 under various conditions that re-enact the cooking of lunches implicated in an outbreak of haemorrhagic diarrhoea.

Two elementary schools were served lunches that were cooked in the same kitchen. An outbreak of Escherichia coli O157:H7 occurred at one school where the dishes that were prepared for the school were lukewarm and kept for 33 min at an average temperature of 45 degrees C before serving. However, no outbreak occurred at the other school where dishes were hot and were kept for 60 min at an average temperature of 50 degrees C before serving. In a series of experiments on the survival of E. coli O157:H7 in the liquid portion of similarly prepared food, the population of E. coli O157:H7 was reduced by 10-3 by heating at 50 degrees C for 60 min and by only 10-1 by heating at 45 degrees C for 40 min. Further, E. coli O157:H7 survived at 45 degrees C for 40 min but not at 50 degrees C for 60 min at pH 4.0 with a 4.0% salt concentration that was similar to that of the liquid part of the food. These results indicate that pH and salt concentration of cooked food markedly affect the survival of E. coli O157:H7 and help to explain the occurrence of the disease outbreak at only one of the schools.

Improved method for detection of Vibrio parahaemolyticus in seafood.

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.

Laying season and egg shell cracks on the growth of Salmonella enteritidis in the egg albumen during storage.

We studied the effects of laying seasons and egg shell cracks on the ability of egg albumen to support the growth of Salmonella Enteritidis (SE) in eggs. Hens eggs used were those laid in February, June, and October in a farm in Japan and stored at 10, 20, and 30 degrees C, and at 30 degrees C after storage at 10 degrees C, immediately after receipt or after cracking the shell. At several-day intervals during storage, the egg contents were poured into a dish, SE was inoculated into albumen, and then the growth of SE during 3 days incubation at 18 degrees C was measured. The results demonstrated that storage temperature and laying season affected the growth of SE in the egg albumen. The proportion of eggs upon which albumen allowed the growth of SE was higher in the eggs stored at 30 degrees C than those stored at 10 degrees C. The growth of SE in eggs was lowest in the following order of laying: February, October, and June. SE grew preferably in albumen of cracked eggs than intact eggs.

Detection of Salmonella enteritidis in shell and liquid eggs using enrichment and plating.

Detection methods using various enrichment and plating media and immunoconcentration for Salmonella enteritidis in shell and liquid eggs were evaluated. For liquid egg samples naturally contaminated with S. enteritidis, pre-enrichment in 225 ml of buffered peptone water with cysteine followed by selective enrichment in 10 ml of tetrathionate broth was the superior, resulting in the detection of S. enteritidis in all samples on six of the seven types of selective agar substrate investigated. This enrichment procedure also enabled detection of S. enteritidis in most of artificially inoculated shell egg and pasteurized liquid egg samples.

[Detection of TDH-producing Vibrio parahaemolyticus O3:K6 from naturally contaminated shellfish using an immunomagnetic separation method and chromogenic agar medium].

We attempted to isolate TDH-producing Vibrio parahaemolyticus O3:K6 from shellfish. Asari samples were incubated with TSB supplemented with 2% (w/v) NaCl for 6 h, and then the 6-h cultures were incubated with salt polymyxin broth for 18 h. After the two-step enrichment, a 1 ml portion of the culture was treated with magnetic beads coated with K6 antibody for immunoconcentration of V. parahaemolyticus O3:K6. The immunoconcentrated and untreated cultures were plated onto a chromogenic agar and TCBS agar media for isolation of V. parahaemolyticus. TDH-producing V. parahaemolyticus O3:K6 was isolated from 3 out of 66 lots (4.5%) of naturally contaminated Asari. Six of 4,265 colonies suspected as V. parahaemolyticus (0.14%) were TDH-producing V. parahaemolyticus O3:K6.

Detection of freeze-injured Escherichia coli O157:H7 cells from foods by resuscitation prior to selective enrichment.

We tried to detect Escherichia coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 using an enrichment method with modified EC broth supplemented with novobiocin (mEC + n). When the samples were cultured for enrichment immediately after inoculation of freeze-injured cells, E. coli O157:H7 was not detected in 13 out of 18 samples. However, allowing the food samples to stand for 3 h at room temperature prior to enrichment in mEC + n remarkably improved recovery of E. coli O157:H7 except for some acidic foods. E. coli O157:H7 was detected in the acidic foods by introducing a resuscitation step of 3-h of incubation in a non-selective broth at room temperature prior to enrichment with mEC + n.

Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods.

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at -20 degrees C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25 degrees C for 2 h and then selectively enriched at 42 degrees C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25 degrees C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.

Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method.

We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.

Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef.

For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC + n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC), and agars containing beta-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu/25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu/25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu/25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu/25 g) compared with ground beef (23.9 cfu/25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing beta-glucuronidase substrate in combination with static enrichment incubation in mEC + n at 42 degrees C.

Escherichia coli O157 strains which caused Japanese outbreaks have residues of bacteriophage sequences.

Twelve strains of Escherichia coli O157 which caused outbreaks in Japan were used as DNA sources. The sequences of the gene encoding the Shiga toxin 2 in all 12 strains were almost identical and the sequences downstream of this gene were similar to that of bacteriophage 933W.

Combined use of bacteriophage typing and pulsed-field gel electrophoresis in the epidemiological analysis of Japanese isolates of enterohemorrhagic Escherichia coli O157:H7.

A total of 236 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates in Japan were investigated by bacteriophage typing, and the results were compared with those of pulsed-field gel electrophoresis (PFGE). Seven phage types (PTs) were observed in 71 isolates which were derived from 22 outbreaks. All of the isolates from ten outbreaks in the Kinki region (midwestern part of Japan) in July-August 1996 were grouped into the same PFGE type (IIa) and PT 32, while among total isolates, there were such varieties as PFGE type IIa containing five phage types and PT32 containing two PFGE types. These results suggest that the ten outbreaks should be considered to be a single outbreak, and show that the combined use of bacteriophage typing and PFGE enhances reliability in epidemiological surveys.

Enterohemorrhagic Escherichia coli O157:H7 present in radish sprouts.

Using cultivation, immunofluorescence microscopy, and scanning electron microscopy, we demonstrated the presence of viable enterohemorrhagic Escherichia coli O157:H7 not only on the outer surfaces but also in the inner tissues and stomata of cotyledons of radish sprouts grown from seeds experimentally contaminated with the bacterium. HgCl2 treatment of the outer surface of the hypocotyl did not kill the contaminating bacteria, which emphasized the importance of either using seeds free from E. coli O157:H7 in the production of radish sprouts or heating the sprouts before they are eaten.

Potential Hazard of Radish Sprouts as a Vehicle of Escherichia coli O157:H7.

We studied the contamination of radish sprouts after exposure to Escherichia coli O157:H7-inoculated water in the laboratory. The edible parts, the cotyledons and hypocotyl, became heavily contaminated with E. coli O157:H7 when they were grown from seeds soaked in E. coli O157:H7-inoculated water. These same parts became contaminated with E. coli O157:H7 when their roots were dipped into E. coli O157:H7-inoculated water. These findings suggest that E. coli O157:H7 contamination in the edible parts of radish sprouts could pose a serious hazard if the seeds or hydroponic water are contaminated with the bacterium.

Emesis of rhesus monkeys induced by intragastric administration with the HEp-2 vacuolation factor (cereulide) produced by Bacillus cereus.

To study the correlation between emetic toxin and HEp-2 vacuole activity produced by Bacillus cereus isolated from an outbreak of vomiting-type food poisoning, some properties and emetic activities of both purified HEp-2 factor (cereulide) and partially purified factor to rhesus monkeys were determined. The results indicate that both cereulide and partially purified factor were very stable to digestion with proteolytic enzymes, different pH, and heating. Vomiting was induced in the rhesus monkeys orally administered with both substances. From these findings, cereulide (or HEp-2 vacuole factor) is strongly suggested to be an emetic toxin itself.

Rates of detection of Salmonella and Campylobacter in meats in response to the sample size and the infection level of each species.

Pork, beef and chicken meat samples were collected from slaughter houses, poultry-processing plants and meat shops. Rates of incidence of Salmonella spp., Campylobacter jejuni and C. coli with respect to the sample size were compared and the most probable number for these species were determined. Salmonella spp. were detected in 69 (24.1%) of 286 chicken meat samples, in three (3.2%) of 94 pork samples, and in one (1.9%) of 52 beef samples. With chicken meat, the rates of detection were: 19.9% in 25-g, 15.7% in 10-g, and 12.2% in 1-g samples. The populations in most probable numbers, that gave positive results in 31 (20.8%) of 149 samples, ranged from 30 to 10(4) per 100 g, the majority (93.5%) being between 30 and 10(3) per 100 g. C. jejuni and C. coli were detected in 106 (67.9%) of 156 chicken meat samples, in two (2.1%) of 94 pork samples, and none of 52 beef samples. The results obtained with different sample sizes of chicken were compared. Positive rates were 55.8%, 39.7%, 27.6% in 10 g, 1 g, and 0.1 g, respectively. The most probable numbers in 107 (68.6%) positives out of 156 chicken samples examined ranged from 30 to 10(6) per 100 g: 46 (29.5%) contained between 10(2) and 10(3) per 100 g, 22 (14.1%) between 10(3) and 10(4) per 100 g, and the other 19 samples (12.2%) between 10(4) and 10(5) per 100 g.