We developed a quick genetic approach to screen variants of the intimin gene (eae) by using a heteroduplex mobility assay (HMA) that targets the 5 conserved region of eae. The eae variants were categorized into 4 major HMA types and 10 minor subtypes.Enteropathogenic Escherichia coli (EPEC) and Shiga toxinproducing E. coli (STEC) produce characteristic attaching and effacing lesions using intimin encoded by eae (24). The eae genes of several strains have been cloned and sequenced and have a highly conserved 5Ј-terminal region but are variable in the 3Ј-terminal region (13). Allele-specific PCRs targeting eae genes in the variable 3Ј region have been employed to determine eae types (1,5,10,27,30,31,33) and subtypes in combination with restriction fragment length polymorphism (RFLP) (4, 27). Ramachandran et al. (29) designed universal PCR primers to amplify the Int280-encoding region and identified types by RFLP. Recently, methods using real-time PCR (25) and oligonucleotide microarray (14) have been developed. Eighteen types and nine subtypes of intimin, namely, ␣, ␣2, ␤1 to -3, ␥1, ␥2, ␦, ε, ε2 to

Diarrheagenic Escherichia coli are differentiated from non-pathogenic members with enterotoxin production, enteroinvasiveness and serotyping. However, the serotypic members are rarely sufficient to reliably identify a strain as diarrheagenic on E. coli. Recently, there are many definite articles which the adhesive E. coli strain against intestinal epithelial cells is enterovirulent. In this study, 1,748 E. coli isolates of diarrheagenic and non-diarrheagenic categories which belonged to EHEC, ETEC, EIEC EPEC and non-EPEC were examinated by PCR method for the presence of eaeA, aggR and bfpA regarding adherence factor genes, and astA of EAST1. The strains examined were recognized to variable carrying geno-patterns, and a large number of EHEC, EPEC and non-EPEC had carried either eaeA or aggR genes. In EHEC isolates, a carrying pattern with the most high frequency was only eaeA, and this type was recognized in the isolates of serotype O157, O26 and O111. EPEC and non-EPEC isolates were recognized eaeA or aggR which harboring with astA or not. Of 508 EPEC isolates from human, a total of 137 isolates (27.0%) carried aggR, and a total of 74 isolates (14.6%) had eaeA, while of the 91 isolates from non-human were recognized aggR and eaeA with 2.2% (2 isolates) and 12.1% (11 isolates), respectively. Also, of 266 non-EPEC isolates from human, a total of 16 isolates (6.0%) carried aggR, and a total of 58 isolates (21.8%) had eaeA. On the other hand, 22 (7.0%) of 316 isolates examined from non-human had eaeA, however no isolate had aggR. Thirteen isolates of EIEC and 218 ETEC isolates were screened, and only 6 ETEC isolates had either eaeA or aggR. The astA gene was recognized in the isolates of all categories, and ETEC strains had more frequently. The bfpA gene was recognized with more frequently in a serotype O157: H45, which is obtained from human with diarrhea, however, this strain was not recognized a member of the EPEC serotype. There is no diagnostic system for the strain of E. coli that cause diarrheal diseases, therefore more laboratories are unable to identify them. The authors had confirmed which PCR technique is a useful simple and rapid method for the detection of adherence factor genes on E. coli strains. From the these results, we showed a differentiation method using PCR technique which have relation with adherence factor, enterotoxin-production and invasiveness, and we firmly believe that application of the procedure is a reasonable and useful method for the identification of diarrheagenic E. coli.

Enteroaggregative Escherichia coli (EAggEC) are an important cause of diarrhea. Four types of AAF have been identified; however, their prevalence and association with virulence properties remain unclear. E. coli strains carrying the aggR gene as EAggEC that were isolated in Japan and Thailand (n ¼ 90) were examined for AAF subunit genes, two toxin genes (pet/astA), and clump formation. The most prevalent AAF gene was hdaA (28%), followed by aafA (20%), aggA (12%), and agg3A (4%), as well as a putative new AAF sequence (25.6%). Retention status of the toxin genes and intensities of clump formation appeared to vary according to the AAF type.Key words aggregative adherence (AA), aggregative adherence fimbriae (AAF) type, aggR-positive Escherichia coli, toxin gene.Enteroaggregative Escherichia coli (EAggEC) comprise an emerging group of pathogens that cause pediatric and adult diarrhea worldwide and are known to be heterogeneous in their symptoms (1, 2) and patterns of retaining virulence genes (3-5). EAggEC exhibits characteristic AA at the surface of cultured cells by means of AAF (2). Four distinct AAF variants have been identified to date based on the sequences of fimbrial AAF subunits encoded by aggA (6), aafA (7), agg3A (8), and hdaA (alias agg4A) (9).Many putative virulence factors have been reported in addition to AAF, including EAST1, a 104-kDa cytotoxin designated as a Pet, and a global transcriptional regulator (AggR). AggR is the central regulator of virulence functions in EAggEC; therefore, the term 'typical EAEC List of Abbreviations: AA, aggregative adherence; AAF, aggregative adherence fimbriae; EAggEC, enteroaggregative Escherichia coli; EAST1, enteroaggregative E. coli heat stable enterotoxin 1; Pet, plasmid encoded toxin.

Percentage of the outbreaks by O3:K6 Vibrio parahaemolyticus (V. p) in Aichi Prefecture Japan increased from 3% (3/86) for 1988-95 to 75% (33/44) for 1996-2001. The percentage of the sporadic diarrhea cases caused by O3:K6 V. p in a general hospital in Aichi Prefecture also increased from 0% (0/253) to 61% (135/221) during the same periods. Thermostable direct hemolysin (TDH)-positive O3:K6 were isolated from 95% (19/20) of the outbreak incidents and 100% (135/135) of the sporadic cases. Only one TRH (TDH-related hemolysin)-positive O3:K6 was isolated from one outbreak incident. Percentage of the outbreaks by O3:K6 V. p associated with the consumption of boiled shellfishes increased from 5% (6/117) for 1988-95 to 25% (15/59) for 1996-2001, in particular, boiled crabs and squillas associated outbreaks increased from 2% (2/117) to 17% (10/59) and from 2% (2/117) to 10% (6/59), respectively. From 1,548 raw sea foods sampled in the Nagoya Central Wholesale Market in Aichi Prefecture in 1995-99, one TDH-positive O3:K6 was isolated from one live squilla (1/30). Increase in the percentage of outbreaks associated with TDH-positive O3:K6 V. p after 1996 in Aichi Prefecture was revealed to correlate with the increase in the outbreaks associated with consumption of boiled sea foods, especially boiled crabs as well as squillas. Accordingly, it becomes clear that sanitary handling of these boiled foods is important to prevent outbreaks and sporadic cases of diarrhea caused by O3:K6 V. p infection.

To investigate the prevalence of attaching and effacing Escherichia coli, we examined 364 strains isolated from the feces of 9,684 patients with diarrhea at the Anjo Kosei Hospital in Japan for the presence of eaeA. Twenty-nine (8%) of the strains were eaeA positive. Of enteropathogenic E. coli (EPEC), 11 of the 87 (13%) strains were for the positive eaeA gene. The serotypes and the numbers of eaeA-positive strains among the strains tested were as follows: