Surveillance of pathogens in outpatients with gastroenteritis and characterization of sapovirus strains between 2002 and 2007 in Kumamoto Prefecture, Japan.

Infectious acute gastroenteritis is an important public health problem worldwide. A total of 639 stool specimens were tested for the presence of diarrhea pathogens. The specimens were from outpatients with acute gastroenteritis who consulted the pediatric clinic in Kumamoto Prefecture, Japan, from June 2002 to December 2007. Of these, 421 (65.9%) were positive for diarrhea pathogens. Among them were norovirus (NoV) in 260 (61.8%), sapovirus (SaV) in 81 (19.2%), rotavirus in 49 (11.6%), adenovirus in 19 (4.5%), enterovirus in 13 (3.1%), astrovirus in 9 (2.1%), kobuvirus in 1 (0.2%), and bacterial pathogens in 11 (2.6%). Mixed infection (co-infection of viruses) was found in 22 (5.2%) of the 421 pathogen-positive stool samples. NoV was the most prevalent pathogen throughout the study period; however, the SaV detection rate was unexpectedly high and was found to be the secondary pathogen from 2005 to 2007. Genetic analysis of SaV with 81 strains demonstrated that SaV strains belonging to genogroup IV emerged in 2007, and dynamic genogroup changes occurred in a restricted geographic area. This study showed that SaV infection is not as rare as thought previously.

Efficiency of real-time polymerase chain reaction assay to detect Vibrio vulnificus in seawater.

The growth of Vibrio vulnificus in an enriched culture of seawater during the summer in Japan was monitored by a plating technique used as the culture method and a real-time polymerase chain reaction (PCR) assay as the molecular method. V. vulnificus was detected by the real-time PCR assay in the samples of August and September but not by the culture method. Vibrio parahaemolyticus, however, was detected among all of the samples with both the culture method and real-time PCR assay. In the analysis of the bacterial populations in enrichment culture, it was demonstrated that the growth of V. vulnificus on agar media was inhibited by the rapid growth of V. parahaemolyticus after 4h of incubation and the 100 times larger initial populations of bacteria other than V. vulnificus and V. parahaemolyticus. These findings demonstrate that V. vulnificus detection by culture methods is a failure, and molecular methods are effective and detect V. vulnificus accurately.

Epidemiological survey of Vibrio vulnificus infection in Japan between 1999 and 2003.

The frequency of Vibrio vulnificus infection is very rare and there are many questions regarding its epidemiology in Japan. To investigate the clinical course and epidemiology of V. vulnificus infection in Japan, we performed a retrospective questionnaire survey in which 1693 hospitals from all over Japan were surveyed, including advanced life saving emergency centers and dermatology institutions. Of the 1693 hospitals, we received answers from 1045. Ninety-four cases were confirmed as V. vulnificus infections during 1999 and 2003. Sixty-eight (72.3%) of the 94 patients had the septic type infection with a mortality rate of 75.0% (51/68 patients died). The prognosis of patients with the septic type was worse than that of the wound type (P < 0.001). V. vulnificus infections occurred from June to November and none occurred in winter. Many infections occurred in western Japan with the majority of infections (50/94) occurring in Kyushu. In particular, 43 infections occurred in marine coastal areas of the Ariake and Yatsushiro Seas, which have many tidelands. Seventy-seven of 89 patients (86.5%) had liver function impairment as an underlying disease, and 53 (59.6%) had liver cirrhosis, of whom nine (10.1%) suffered from liver cancer. The incidence of V. vulnificus infection was different according to districts. Geographic and climatic factors also contributed to the occurrence of V. vulnificus infection.

Rapid and specific identification of 5 human pathogenic Vibrio species by multiplex polymerase chain reaction targeted to dnaJ gene.

A multiplex polymerase chain reaction (PCR) method, specifically designed for application in routine diagnostic laboratories, was developed for identifying 5 human pathogen Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus, and Vibrio alginolyticus. This assay directed toward the dnaJ gene was tested on a total of 355 strains representing 13 Vibrio species and 17 non-Vibrio species. Specific PCR fragments were produced in isolates belonging to the 5 target species and were absent from all strains other than these 5 species and non-Vibrio strains, indicating a high specificity of this multiplex PCR. The multiplex PCR for the detection of Vibrio pathogens in clinical specimens was experimentally applied to spiked stool samples. Only 1 specific amplicon was observed, corresponding to the pathogen spiked into the stool sample. The detection limitation was 10(5) to 10(6) cells per milliliter stool. Our data showed that this method represented a robust tool for the specific and rapid detection of the 5 major pathogenic Vibrio species.

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species.

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.

Development of a quantitative real-time polymerase chain reaction targeted to the toxR for detection of Vibrio vulnificus.

The TaqMan assay, a quantitative real-time polymerase chain reaction (PCR), was developed to target the ToxR gene (toxR) of Vibrio vulnificus. The toxR of V. vulnificus was cloned and sequenced. Based on these results, we designed specific primers and a probe for use in the quantitative PCR assay. Twenty-nine strains of V. vulnificus that were obtained from various sources produced a single PCR product. The amount of final amplification product and threshold cycle number were the same among the strains. We used the method to detect V. vulnificus in seawater and oyster samples. We developed standard curves to quantitate V. vulnificus numbers using the PCR from seawater and oyster samples. The standard curves were not different from that of the pure culture of V. vulnificus. We found the assay was very sensitive detecting as few as 10 microbes per milliliter of seawater and oyster homogenate. Moreover, we evaluated the TaqMan assay to detect V. vulnificus in seawater samples. The numbers of V. vulnificus counted by the TaqMan assay were similar to those by a culture method in almost samples. The TaqMan assay was performed within 2 h compared to days using the culture method. The results indicate the TaqMan assay method used in this study was rapid, effective and quantitative for monitoring V. vulnificus contamination in seawater and seafoods such as oysters.

[Quantitative detection of Vibrio vulnificus in seafood].

To quantify the number of Vibrio vulnificus in shellfish, we compared the most probable number (MPN) combined with a culture (MPN-culture) or polymerase-chain reaction (PCR) assay (MPN-PCR) to a quantitative PCR assay. Enrichment in alkaline peptone water by MPN was conducted at 25 and 35 degrees C. Enrichment at 35 degrees C was superior or similar to enrichment at 25 degrees C in over 65% of samples by MPNculture and in more than 75% of samples by MPN-PCR assay. V. vulnificus was more easily isolated on chromogenic agar medium during culture, MPN-PCR assay was superior or similar to MPNculture in over 90% of samples by enrichment at 25 degrees C and to over 88% of samples by enrichment at 35 degrees C. The number of V. vulnificus by quantitative PCR assay was similar to that of MPN-PCR assay in 6 of 8 samples but not from MPNculture. V. vulnificus contamination was frequently detected in samples from Kyushu Island.

[Analysis of survival of enterohemorrhagic Escherichia coli in the steps during cooking on a Japanese barbecue].

Foodborne infections with enterohemorrhagic Escherichia coli (EHEC) related to food in each step of the cooking of a Japanese barbecue have been reported in Japan. We examined the survival of EHEC during various types of cooking on a Japanese barbecue. The number of EHEC in barbecue sauce remained stable during short-term storage at low temperature. In a series of experiments on survival of EHEC on beef during cooking on an electric griddle or a gas cooktop, the population was reduced by at least 1/1,100. Although these results suggested that EHEC are effectively killed by adequate cooking, the degree of reduction of EHEC varied among types of meat and was affected by uneven cooking. Furthermore, when the same cooking equipment was used to handle meats before and after cooking, 1/500 to 1/300,000 of EHEC population of contaminated uncooked meat cross-contaminated the cooked meat. Adequate cooking of beef, including internal organs, and use of separate cooking equipment for uncooked and cooked beef are important to avoid EHEC infection caused by Japanese barbecues.

[Control of toxicity of Sarcocystis fayeri in horsemeat by freezing treatment and prevention of food poisoning caused by raw consumption of horsemeat].

More than 27 outbreaks per year of food poisoning caused by consuming horse meat were reported in Kumamoto Prefecture (including Kumamoto City) from January 2009 to September 2011. It was found that the causative agent of the outbreaks was a protein with a molecular weight of 15 kDa that had originated from bradyzoites of Sarcocystis fayeri parasitizing the horse meat. Rabit ileal loop tests showed that pepsin treatment of homogenates of frozen horse meat containing the cysts of S. fayeri induced loss of toxicity, presumably by digestion of the proteinous causative agent(s). Slices of horse meat containing the cysts were frozen at below -20°C for various periods. The cysts were collected after thawing the slices, then treated in an artificial stomach juice containing pepsin. The bradyzoites of the cysts kept at -20°C for 48 hr or more completely disappeared. Simultaneously, the 15 kDa protein also disappeared in the frozen cysts. After notifying the public and recommending freezing treatment of horse meat, no subsequent cases of food poisoning were reported. This indicates that freezing of horse meat is effective to prevent the occurrence of food poisoning caused by consuming raw horse meat containing S. fayeri.

The growth of Vibrio vulnificus and the habitat of infected patients in Kumamoto.

In Japan, Vibrio vulnificus (V. vulnificus) infection is very rare, and most infections have occurred in Kumamoto Prefecture (1), and especially around the Ariake and Yatsushiro seas. To investigate the relationship between the occurrence of V. vulnificus infection and environmental factors, including the salinity of seawater and the amount of rain in the Ariake and Yatsushiro seas, we measured the most probable number (MPN) of V. vulnificus in seawater and sea mud. In the Ariake Sea, we also observed the temperature and salinity of seawater at one site located on an estuary where the salinity is easily affected by river water and another site located offshore where seawater is little affected by river water. Furthermore, we investigated the MPN of V. vulnificus and observed the temperature and the salinity of seawater in 25 sites in the Ariake and Yatsushiro seas from July to August 2003 and 2004. In addition, we collected data on patients with V. vulnificus infections in Kumamoto from 1990 to 2006. The MPN of V. vulnificus differed by sampling site. More V. vulnificus were detected around the inland sea than the open sea, and the increase in V. vulnificus levels was affected by rainfall around inland sea areas with many rivers. V. vulnificus increases significantly in brackish water areas, and the salinity of seawater was as important as the seawater temperature. In other words, an area's topography and amount of rain are believed to be important factors for the occurrence of V. vulnificus infection. V. vulnificus infection has been regarded as an infection of hot districts. However, the salinity of seawater may be more important than temperature for the growth of V. vulnificus. Therefore, investigating these geographical and meteorological factors can help predict areas with a higher number of V. vulnificus infection outbreaks.