Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdhpositive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.

Phages and plasmids play important roles in bacterial evolution and diversification. Although many draft genomes have been generated, phage and plasmid genomes are usually fragmented, limiting our understanding of their dynamics. Here, we performed a systematic analysis of 239 draft genomes and 7 complete genomes of Shiga toxin (Stx)-producing Escherichia coli O145:H28, the major virulence factors of which are encoded by prophages (PPs) or plasmids. The results indicated that PPs are more stably maintained than plasmids. A set of ancestrally acquired PPs was well conserved, while various PPs, including Stx phages, were acquired by multiple sublineages. In contrast, gains and losses of a wide range of plasmids have frequently occurred across the O145:H28 lineage, and only the virulence plasmid was well conserved. The different dynamics of PPs and plasmids have differentially impacted the pangenome of O145:H28, with high proportions of PP- and plasmid-associated genes in the variably present and rare gene fractions, respectively. The dynamics of PPs and plasmids have also strongly impacted virulence gene repertoires, such as the highly variable distribution of stx genes and the high conservation of a set of type III secretion effectors, which probably represents the core effectors of O145:H28 and the genes on the virulence plasmid in the entire O145:H28 population. These results provide detailed insights into the dynamics of PPs and plasmids, and show the application of genomic analyses using a large set of draft genomes and appropriately selected complete genomes.

EHEC O157:H7 clade 6 strains harboring stx2a and/or stx2c and clade 8 strains harboring stx2a or stx2a/stx2c were frequently associated with childhood HUS cases in Japan. Rapid and specific detection of such lineages are required for infection control measures.

The virulence traits of the Escherichia coli strain associated with a waterborne diarrhea outbreak were examined. Forty-one of 75 students (ages 12 to 15) in Akita Prefecture, Japan, showed clinical symptoms. Seven E. coli Ouk:K-:H45 isolates were isolated from the patients as the causative agent of this outbreak. One isolate (EC-3605) showed the presence of E. coli attaching-and-effacing (eaeA) and enteroaggregative E. coli heatstable enterotoxin-1 (astA) genes and the absence of Shiga toxin (stx1 and stx2) genes. A polymorphic enteropathogenic E. coli (EPEC) adherence factor plasmid was detected in EC-3605 with a major structural gene deletion and a regulatory gene frameshift mutation, revealing that EC-3605 represents an atypical EPEC strain harboring the astA gene. The role that atypical EPEC strains harboring the astA gene play in human disease is unclear. Our results, along with those of others, present a possibility that these strains comprise a distinct category of diarrheagenic E. coli and that astA affects the age distribution of atypical-EPEC infection.

Virulence characteristics of diarrheal outbreak-associated Escherichia coli O55:NM, O126:NM, and O111:NM were examined. The E. coli O55:NM strains were atypical enteropathogenic E. coli (EPEC), while the E. coli O126:NM and O111:NM strains should be classified as enteroaggregative E. coli (EAggEC). The contributions of EPEC and EAggEC to the human disease burden in Japan might be significantly greater than is currently appreciated.There are six categories of Escherichia coli that cause diarrhea: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli, enteroaggregative E. coli (EAggEC), enteroinvasive E. coli, and diffusely adherent E. coli (21). EPEC causes characteristic attaching-and-effacing lesions (A/E), which can be observed by intestinal biopsy in both human patient (19) and animal (29) models. A/E is characterized by loss of microvilli, intimate adherence of bacteria between epithelial cell membranes (27,30), and cytoskeletal changes such as actin polymerization directly beneath the adherent bacteria (15). Generally, EPEC causes infantile diarrhea in developing countries and sporadic diarrhea in developed countries (21). EAggEC, on the other hand, is an enteric pathogen defined by its distinctive aggregative or "stackedbrick" pattern of adherence to cultured human epithelial cells (22). EAggEC associates mainly with persistent diarrhea in developing countries (21). Only two reports in Japan have described diarrheal outbreaks caused by EAggEC or EPEC. Itoh et al. (11) reported the isolation of EAggEC from the stools of patients with severe diarrhea in elementary and junior high schools. Makino et al. (18) reported the isolation of EPEC from a mass outbreak. In this paper, we describe three cases of diarrheal outbreaks in Japan caused by E. coli belonging to the traditional EPEC serotype.Chromosomal DNA-embedded agarose plugs for pulsedfield gel electrophoresis (PFGE) analysis were prepared by using the CHEF Bacterial DNA Plug Kit (Bio-Rad, Hercules, Calif.) and were digested with XbaI (Nippon gene; Osaka, Japan) at a concentration of 30 U/plug for 4 h at 37°C. The plugs were applied to a 1% PFC Grade Agarose (Bio-Rad) gel. Electrophoresis was performed in 0.5ϫ Tris-Borate EDTA buffer at 14°C using a CHEF DR-II PFGE apparatus (BioRad) under the following conditions: voltage, 6 V/cm; block 1, 11 h, with initial switching time of 4 s to final switching time of 8 s; block 2, 9 h, with initial switching time of 8 s to final switching time of 50 s. The HEp-2 cell assay was performed following the method described by Craviotto et al. (4), with modifications involving 3 or 6 h of incubation (15). The E. coli isolates were examined for the presence of the following virulence genes by PCR: stx1 (Shiga toxin) and stx2 (16), eaeA (E. coli attaching and effacing) (12), bfpA (bundle-forming pilus) (9), perA (EPEC plasmid-encoded regulatory region) (8), astA (EAggEC heat-stable enterotoxin) (28), aggR (transcriptional activator for EAggEC aggregative adherence fimbria I expression) ...