Growth and Survival of Escherichia albertii in Food and Environmental Water at Various Temperatures.

Escherichia albertii is an emerging foodborne pathogen that causes diarrhea. E. albertii has been isolated from various foods, including pork and chicken meat, and environmental waters, such as river water. Although many food poisoning cases have been reported, there have been insufficient analyses of bacterial population behaviors in food and environmental water. In this study, we inoculated 2-5 log CFU of E. albertii into 25 g of pork, chicken meat, Japanese rock oyster, Pacific oyster, and 300 mL of well water and seawater at 4°C, 10°C, 20°C, and 30°C, and analyzed the bacterial population behavior in food and environmental water. After 3 days at 4°C, the population of E. albertii strain EA21 and EA24 in foods maintained approximately 4 log CFU/25 g. After 3 days at 10°C, the population of E. albertii strains in pork and oysters maintained approximately 4 log CFU/25 g, and that in chicken meat increased to approximately 5-6 log CFU/25 g. After 2 days at 20°C, E. albertii strains grew to approximately 6-7 log CFU/25 g in pork and chicken meat, and E. albertii strain EA21 but not EA24 grew to 4.5 log CFU/25 g in Japanese rock oyster, E. albertii strain EA21 but not EA24 slightly grew to 3.1 log CFU/25 g in Pacific oyster. After 1 day at 30°C, E. albertii strains grew to approximately 7-8 log CFU/25 g in chicken meat and pork, grew to approximately 4-6 log CFU/25 g in Japanese rock oyster, and 6-7 log CFU/25 g in Pacific oyster. These results suggest that E. albertii survives without growth below 4°C and grew rapidly at 20°C and 30°C in foods, especially in meat. E. albertii strains did not grow in well water and seawater at 4°C, 10°C, 20°C, and 30°C. The population of E. albertii strains in well water and seawater decreased faster at 30°C than at 4°C, 10°C, and 20°C, suggesting that E. albertii has low viability at 30°C in environmental water.

Comparison of the Serovars and Characteristics of Salmonella Isolated from Human Feces and Foods in the 1990s and 2010s in Tokyo.

Salmonella foodborne disease outbreaks have markedly decreased in recent years, and different Salmonella serovars have been isolated. To clarify the characteristics of Salmonella strains causing annual epidemics and to estimate the source, we conducted a serotyping test on 1,132 human-derived Salmonella isolates in the 1990s and 2010s, and 1,061 food-derived Salmonella isolates in the 2010s in Tokyo. The serovars commonly isolated from human feces in the 1990s and after 2012 were S. Enteritidis, S. Typhimurium, S. Infantis, S. Thompson, and S. Agona. The new main serovars isolated after 2012 were S. Schwarzengrund, S. Enterica serovar 4:i:-, and S. Chester. In contrast, the main serovars detected from foods after 2012 were S. Infantis, S. Schwarzengrund, S. Agona, S. Manhattan, S. Typhimurium, and S. enterica serovar UT: r:1,5. S. Schwarzengrund has recently been frequently isolated. These strains were mainly isolated from chicken meat and offal. It was suggested that the same serovars of human-derived isolates were also isolated from foods, especially chicken meat and offal, and that these were recently an important causative food of Salmonellosis.

Detection of Escherichia albertii in Retail Oysters.

ABSTRACT: Escherichia albertii is an emerging foodborne pathogen. Owing to its distribution in river water, it is important to determine the presence of E. albertii in aquaculture-related foods. In this study, we investigated the distribution of E. albertii in retail oyster samples. A total of 427 raw oyster samples (385 Pacific oysters and 42 Japanese rock oysters) were enriched in modified Escherichia coli broth (mEC) or mEC supplemented with novobiocin (NmEC) at 42°C. The cultures were used for E. albertii-specific nested PCR assay, as well as for E. albertii isolation using deoxycholate hydrogen sulfide lactose agar (DHL), DHL supplemented with rhamnose and xylose, and MacConkey agar supplemented with rhamnose and xylose. The population of E. albertii in nested PCR-positive samples was determined using the most-probable-number (MPN) method. E. albertii isolates were subjected to biochemical and genetic characterization. E. albertii was detected in 5 (1.6%) of 315 Pacific oyster samples (one piece each), 2 (2.9%) of 70 Pacific oyster samples (25 g each), and 2 (4.8%) of 42 Japanese rock oyster samples procured from four geographically distinct regions. A total of 64 E. albertii strains were isolated from eight of the nine nested PCR assay-positive oyster samples, and the MPN value was under the detection limit (<3 MPN/10 g). A specific season or month for detecting E. albertii was not observed in this study, suggesting that the pathogen is present in seawater. All the E. albertii isolates, except one, were positive for the virulence factor eae, indicating that these isolates have the potential to infect humans.

[Major Vehicles and O-Serogroups in Foodborne Enterotoxigenic Escherichia coli Outbreaks in Japan, and Effective Detection Methods of the Pathogen in Food Associated with An Outbreak].

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen in developing countries, and causes foodborne infections through contaminated vegetables and water. ETEC also caused some foodborne infections in developed countries, though the vehicles are often unclear. We analyzed ETEC foodborne outbreaks in Japan based on the National Food Poisoning Statistics. Vegetables and private well water accounted for 50% and 22.2% of vehicles, respectively. The main vehicles were similar to those in developing countries. Serogroups of ETEC were also analyzed, and O6, O25, O27, O148, O153, O159, and O169 were the seven major O-serogroups. We investigated suitable detection methods for the pathogen (O148) in food samples associated with an outbreak of ETEC in Japan in 2011. We show that ETEC O148 could be effectively detected in cut leeks by means of a two-step enrichment and real-time PCR assay targeting heat-stable enterotoxin gene. Our survey of the vehicles and the major O-serogroups of ETEC outbreaks in Japan indicates that ETEC survives in the environment in Japan.

Closely related methicillin-resistant Staphylococcus aureus isolates from retail meat, cows with mastitis, and humans in Japan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pervasive healthcare-acquired (HA) pathogen with recent emergence as a community-acquired (CA) pathogen. To elucidate whether meat mediates MRSA transmission between animals and humans in Japan, this study examined MRSA isolates from retail meat (n = 8), cows with mastitis (n = 7), and humans (HA-MRSA = 46 and CA-MRSA = 54) by molecular typing, virulence gene analyses, and antimicrobial susceptibility testing. MRSA isolates from retail meat were classified into sequence type (ST) 8/spa type t1767 (n = 4), ST8/t4133 (n = 1), ST59/t3385 (n = 1), ST88/t375 (n = 1), and ST509/t375 (n = 1). All seven MRSA isolates from cows with mastitis were ST8/t1767. 46 HA-MRSA were clonal complex (CC) 5, divided into t002 (n = 30), t045 (n = 12), and t7455 (n = 4). 54 CA-MRSA were classified into 6 different CCs: CC1 (n = 14), CC5 (n = 7), CC8 (n = 29), CC45 (n = 1), CC89 (n = 1), CC509 (n = 1), and into 16 different spa types including newly identified t17177, t17193, and t17194. The majority were CC8/t1767 (n = 16). CC of one CA-MRSA isolate (spa type t1767) was not classified. Among 41 CC8 MRSA (five from meat, seven from cows with mastitis, and 29 CA-MRSA), 14 ST8/SCCmec IVl isolates (three from meat, one from a cow with mastitis, and 10 CA-MRSA) had identical pulsed-field gel electrophoresis patterns and similar spa type (t1767, t4133, and t17177), and were typed as CA-MRSA/J (ST8/SCCmec IVl, positive for sec + sel + tst but negative for Panton-Valentine leukocidin and the arginine catabolic mobile element). These results suggest that there is a transmission cycle of CA-MRSA/J among meat, cows, and humans in Japan, although it is unclear whether the origin is cow.

Genomic analysis of the evolutionary lineage of Norovirus GII.4 from archival specimens during 1975-1987 in Tokyo.

This study aimed to analyze NoV GII.4 sequences from archival specimens obtained during 1975-1987 by comparing them with reference sequences. The first NoV GII.P4_GII.4 sequence was identified in 1980. NoV GII.4 collected in 1970 had a GII.P1_GII.4 sequence. These results indicate that the GII.P4_GII.4 sequence may be the result of a recombination that might have occurred around 1980. Amino acid substitutions based on this replacement were mainly accumulated in the NTPase, p22, and RdRp regions. The emergence of GII.P4_GII.4 sequence is considered to have ended the major prevalence of NoV GII.4. J. Med. Virol. 89:363-367, 2017. © 2016 Wiley Periodicals, Inc.

Detection of Enteric Viruses in Fecal Specimens from Nonbacterial Foodborne Gastroenteritis Outbreaks in Tokyo, Japan between 1966 and 1983.

We investigated the prevalence of 5 enteric viruses (norovirus [NoV], sapovirus, rotavirus, astrovirus, and adenovirus) in archived stool specimens collected from 70 foodborne gastroenteritis outbreaks in Tokyo, Japan, which occurred from 1966 to 1983, and genetically characterized these viruses. NoV was detected in 48 (68.6%) outbreaks, while SaV, group C rotavirus (RVC), and astrovirus were detected in 1 (1.4%) outbreak each. Based on the partial capsid sequences, the detected NoVs were classified into the following genotypes: 9 in genogroup I (GI; GI.1-6, GI.8, GI.9, and GI.NA), 13 GII (GII.1-9, GII.13, GII.16, GII.17, and GII.22), and one in GIV. The oldest NoV outbreaks occurred in 1966. No predominant genotype was found. One strain, classified as GI. NA based on the N/S region sequence, was subsequently classified as GI.8 based on the complete VP1 sequence. Nine types of recombinant NoV sequences, including 7 unreported combinations, were identified. Further genetic characterization of NoV GII.17 and GII.4 demonstrated that the NoV GII.17 strains detected from 1970 to 1982 clustered independently from previously reported NoV GII.17 strains. Phylogenetic analysis, using the complete VP1 region and the P2 domain, demonstrated that NoV GII.4 strains collected between 1975 and 1980 clustered with archival strains collected in the USA in the mid-1970s. In contrast, a NoV GII.4 strain collected in 1983 formed an independent branch from reference strains collected in the mid-1970s to 2012.

[Two Outbreaks of Yersinia enterocolitica O:8 Infections in Tokyo and the Characterization of Isolates].

Although the number of outbreaks caused by Yersinia enterocolitica has been very small in Japan, 4 outbreaks were occurred during the 2 years between 2012 and 2013. We describe herein 2 outbreaks which were examined in Tokyo in the present study. Outbreak 1: A total of 39 people (37 high school students and 2 staff) stayed at a hotel in mountain area in Japan had experienced abdominal pain, diarrhea and fever in August, 2012. The Y. enterocolitica serogroup O:8 was isolated from 18 (64.3%) out of 28 fecal specimens of 28 patients. The infection roots could not be revealed because Y. enterocolitica was not detected from any meals at the hotel or its environment. Outbreak 2: A total of 52 students at a dormitory had diarrhea and fever in April, 2013. The results of the bacteriological and virological examinations of fecal specimens of patients showed that the Y. enterocolitica serogroup O:8 was isolated from 24 fecal specimens of 21 patients and 3 kitchen staff. We performed bacteriological and virological examination of the stored and preserved foods at the kitchen of the dormitory to reveal the suspect food. For the detection of Y. enterocolitica, food samples. together with phosphate buffered saline (PBS) were incubated at 4 degrees C for 21 days. Then, a screening test for Y. enterocolitica using realtime-PCR targeting the ail gene was performed against the PBS culture. One sample (fresh vegetable salad) tested was positive on realtime-PCR. No Y. enterocolitica was isolated on CIN agar from the PBS culture because many bacteria colonies other than Y. enterocolitica appeared on the CIN agar. After the alkaline-treatments of the culture broth or the immunomagnetic beads concentration method using anti-Y. enterocolitica O:8 antibodies, Y. enterocolitica O:8 which was the same serogroup as the patients' isolates was successfully isolated from the PBS culture. The fresh vegetable salad was confirmed as the incrimination food of this outbreak.

Prevalence and contamination levels of listeria monocytogenes in ready-to-eat foods in Tokyo, Japan.

We surveyed prevalence and contamination levels of Listeria monocytogenes in ready-to-eat foods between 2000 and 2012 in Tokyo. L. monocytogenes was isolated from 52 (1.7%) out of 2,980 samples. Comparing the prevalence in the study period, 2.2% were positive in the former period (2000-2005) and 1.2% in the latter (2006-2012). Using the most probable number (MPN) technique, 32 samples were contaminated with fewer than 0.3 L. monocytogenes/g, 10 samples with 0.3-1.0/g and 4 samples with more than 1.0/g (the maximum was 2.3/g). The most common serovar was 1/2a, followed by 1/2b, 4b and 1/2c. We revealed that ready-to-eat foods in Tokyo were contaminated with L. monocytogenes, although the contamination levels were low.

Shedding of Rubella Virus among Infants with Congenital Rubella Syndrome Born in Tokyo, Japan, 2013-2014.

Rubella is usually a mild illness, with febrile rash being its main symptom. However, serious consequences of rubella infection can result when the infection occurs during the early stages of pregnancy. After the occurrence of a rubella outbreak in Japan that was observed from 2012 to 2013, 45 infants were reportedly born with congenital rubella syndrome (CRS). We prospectively followed the 15 CRS cases reported in Tokyo to determine the virus shedding periods by using nested reverse transcriptase-polymerase chain reaction to detect rubella virus genes. Throast swabs were used for virus detection. The virus shedding period was measured from birth until the time when the sample last tested positive followed by 2 consecutive negative samples. Kaplan-Meier method was used to estimate the proportion of cases remaining positive for rubella virus genes over time. The proportion of CRS cases shedding virus dropped steadily after birth, dropping to 33.8% at 6 months and 16.9% at 12 months. Our findings also suggested that the earlier the mother's onset of rubella during pregnancy, the longer the infant remained positive. Based on our findings, we believe that infants with CRS should be monitored for rubella virus shedding until 1 year of age.

Multiplex PCR serogrouping of Listeria monocytogenes isolated in Japan.

PCR serogrouping methods were used to examine strains of L. monocytogenes isolated in Japan. Among 187 strains, 99.5% were classified into 4 PCR serogroups corresponding to conventional serotypes. Only one isolate had a new PCR profile, which may be a variant of serogroup IVb.

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.

[Isolation of Neisseria elongata subsp. elongata Isolated from an Acute Myelogenous Leukemia Patient].

Gram-negative cocci with a rod-like shape were isolated from a blood sample of a patient with acute myelogenous leukemia (AML). The 16S rRNA sequence of the isolate was similar to that of Neisseria elongata. Because previous reports about N. elongata as a pathogen have been extremely rare, more reliable identification seemed to be needed. We thus additionally performed a Multilocus Sequencing Analysis (MLSA) based on another four regions (argF, rho, recA, glnA), and confirmed the identification of N. elongata. The results from the MLSA identified the species; however, we could not identify the isolates into subspecies from the sequences. Three subspecies of N. elongata (N. elongata subsp. elongata, N. elongata subsp. glycolytica and N. elongata subsp. nitroreducens) were classified based on three definitive characteristics (catalase possession, nitrite reducibility, and acid from glucose). The results of the tests of three characteristics supported the identification of the isolate as N. elongata subsp. elongata. Therefore we determined the isolate from the AML patient to be N. elongata subsp. elongata.

[Serovars and Drug-Resistance of Salmonella Strains Isolated from Domestic Chicken Meat in Tokyo (1992-2012)].

A total of 477 Salmonella strains isolated from retail domestic chicken meat during 1992-2012 in Tokyo, were examined regarding their serovars and drug-resistance. These strains were detected in 469 (29.8%) of 1,576 samples. The detection rate in every two years was 10.1% to 46.3% of the range. Serological typing results showed that 477 strains were classified into 22 serovars excepting 2 untypable strains. Among them, S. Infantis (312 strains) was the most prevalent, followed by II O4: b: [e, n, x] (S. II Sofia) (71 strains), S. Hadar (20 strains), S. Typhimurium (20 strains), S. Manhattan (12 strains), S. Schwarzengrund (9 strains), S. Agona (7 strains), and other 15 serovars (24 strains). Results of the antibacterial drug susceptibility test for 477 strains revealed that 89.9% was resistant to some of the 12 drugs tested, and multidrug-resistant strains accounted for 90.2% among them. The frequencies of resistance to each drug were 81.8%; 77.8%, 45.5%, 33.3%, 11.3%, 9.6%, 2.9%, 0.6%, 0.6% and 0.2%, in order with high frequency, for SM, TC, KM, ST, NA, ABPC, CP, FOM, CTX and CAZ, respectively. None of the strains was resistant to NFLX or IPM. Three CTX-resistant strains were CTX-M type extended-spectrum ß-lactamase (ESBL) producers, and the group of CTX-M type ESBL genes were CTX-M-2 group (2 strains) and CTX-M-9 group (1 strain). CAZ-resistant 1 strain was an ESBL producer, but the ESBL gene was not determined.

[An autochthonous outbreak of dengue type 1 in Tokyo, Japan 2014].

OBJECTIVES: An outbreak of autochthonous dengue fever was reported in August 2014, with cases suspected mainly from Yoyogi Park in Tokyo. This is the first epidemic of dengue fever in Japan since 1945. METHODS: From August to October 2014, the following measures were taken to control the outbreak: 1) risk communication and information sharing; 2) active case finding; 3) vector surveillance in affected sites; and 4) laboratory testing. We also reviewed the surveillance data as reported to the National Epidemiological Surveillance of Infectious Diseases during the 44 epidemiological weeks. results: An official dengue fever call center was set up temporarily for the general public and 3,005 calls were received. The Tokyo Metropolitan Government issued 39 press releases regarding patients and nine related to dengue virus (DENV) detection and vector control activities for the media. Confirmed autochthonous dengue fever cases were reported between the 35th and 44th epidemiological weeks. Out of 160 cases of outbreak, 108 (67.5%) confirmed cases were reported in Tokyo. The estimated illness onset dates were between August 9 and October 7, and estimated dates of infections were between August 3 and October 3, 2014. The data suggest that the infective mosquitoes had already been present in Yoyogi Park at the end of July 2014. During the weekly vector surveillance at Yoyogi Park, a total of 1,152 adult mosquitoes, of which 856 (73.3%) were Aedes mosquitoes, were collected over 11 weeks by a light trap with dry ice. DENV was detected from adult Aedes mosquito samples collected on the 2nd, 9th, and 16th of September, 2014. Serum samples from 240 suspected cases were examined at the Tokyo Metropolitan Institute of Public Health, and 78 were positive for the DENV NS1 antigen, DENV-specific IgM antibody, or DENV nucleic acid with reverse transcriptase polymerase chain reaction (RT-PCR) (NS1: 66 cases; IgM: 50 cases; PCR: 57 cases). Genetic analysis of DENV-positive serum and mosquito samples found all to be categorized as DENV-serotype 1 (gene type I). Phylogenetic analysis of the envelope protein genome sequence from patients and mosquitoes in Tokyo revealed more than 99% similarity with each other and with the strain from the first outbreak-associated patient in Saitama. CONCLUSION: Measures important for control of infectious disease epidemic were learned during this recent indigenous dengue outbreak in Tokyo. It also highlighted the importance of preparedness for epidemics of indigenous or imported infectious diseases, especially in light of the fact that Tokyo is in preparation for the Olympic and Paralympic Games in 2020.

Detection of the staphylococcal enterotoxin D-like gene from staphylococcal food poisoning isolates over the last two decades in Tokyo.

The plasmid is a very well-known mobile genetic element that participates in the acquisition of virulence genes, such as staphylococcal enterotoxins (SEs), via horizontal transfer. SEs are emetic toxins and causative agents in staphylococcal food poisoning (SFP). We herein identified the types of plasmids harbored by seven SFP isolates and examined their production of plasmid-related SE/SEl to determine whether the new types of plasmid-related SE or SE-like (SEl) toxins (i.e. SElJ and SER) were involved in SFP. These isolates harbored pIB485-like plasmids, and all, except for one isolate, produced SElJ and SER. The amount of SER produced by each isolate accounted for the highest or second highest percentage of the total amount of SE/SEl produced. These new types of plasmid-related SE/SEls as well as classical SE may play a role in SFP. The seven isolates were classified into two SED-production types; a high SED-production type (>500 ng/ml) and no SED-production type. A nucleotide sequencing analysis revealed that three plasmids harbored by the SED-non-producing isolates had a single-base deletion in the sed gene with a resulting stop codon (from 233 amino acids of the intact SED to 154 amino acids of the mutant SED (mSED)). A real-time reverse transcription-PCR analysis showed that the mRNA of the msed gene was transcribed in the isolates. If the msed gene was translated as a protein, mSED may act as an emetic toxin instead of intact SED.

Four foodborne disease outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens.

The epidemiological and bacteriological investigations on four foodborne outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens are described. C. perfringens isolated from patients of these outbreaks did not produce any known enterotoxin and did not carry the C. perfringens enterotoxin gene. However, the culture filtrates of these isolates induced the accumulation of fluid in rabbit ileal loop tests. The molecular weight of the new enterotoxin may be between 50,000 and 100,000, although the known C. perfringens enterotoxin is ca. 35,000. This new enterotoxin was heat labile, and its biological activities were inactivated by heating for 5 min at 60°C. The new enterotoxin was sensitive to pH values higher than 11.0 and protease treatment but was resistant to trypsin treatment. These results suggest that the new enterotoxin may be a protein. Although C. perfringens enterotoxin induced morphological changes in Vero cells, the changes induced by the new enterotoxin differed from those by the known C. perfringens enterotoxin. The new enterotoxin also induced morphological changes in L929 cells, whereas the known C. perfringens enterotoxin did not, because L929 cells lacked an appropriate enterotoxin receptor. Although C. perfringens enterotoxin is recognized as the only diarrheagenic toxin responsible for C. perfringens foodborne outbreaks, the results of the present study indicate that C. perfringens isolated from these four outbreaks produced a new type of enterotoxin.

Detection rate of diarrhoea-causing Kudoa hexapunctata in Pacific bluefin tuna Thunnus orientalis from Japanese waters.

Diffuse outbreaks of food poisoning with unknown aetiologies leading to diarrhoea and vomiting within a short time after ingesting flatfish (Paralichthys olivaceus), tuna (Thunnus spp.), or amberjack (Seriola dumerili) have occurred nationwide in Japan, including the Tokyo metropolitan area. In this study, we surveyed the detection rates of kudoid parasites in 12 tuna samples that caused clinical diarrhoea from 2009 to 2012; we assessed 104 samples of whole juvenile Pacific bluefin tuna (PBT, Thunnus orientalis) and 153 block samples of other tuna distributed in the Tokyo Metropolitan Central Wholesale Market. The survey revealed that more than 70% of clinical diarrhoea cases due to tuna ingestion occurred between June and September, and Kudoa hexapunctata were detected in 9 of 12 tuna samples associated with clinical diarrhoea cases. The numbers of spores and 18S ribosomal DNA (rDNA) copies per gram of fish in 8 of 9 samples were more than 1×10(6) spores and 1×10(9) copies, respectively. Market research revealed that the K. hexapunctata-positive rate in juvenile PBT from Japanese waters was 64.4% (67/104) but that in adult PBT was 10.4% (7/67). The numbers of K. hexapunctata 18S rDNA copies in 64.5% (20/31) samples and 72.7% (16/22) of <5kg fish samples collected between May and July were more than 1×10(9)copies/g. On the other hand, kudoid parasites were not detected from 73 tuna samples except for a single sample of Thunnus albacares. Cell monolayer permeability assays performed to examine the toxicity of K. hexapunctata against Caco-2 cells revealed that the transepithelial electrical resistance (TER) in 5×10(7)K. hexapunctata spores decreased by 80% within 2-4h. In conclusion, K. hexapunctata was commonly detected in juvenile PBT from Japanese waters and are a likely cause of the diarrhoea outbreaks.

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo, Japan.

Staphylococcal food poisoning (SFP), one of the commonest food-borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin-encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.