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:

We studied 107 isolates of Escherichia coli O153 from sporadic diarrhea cases in Fukui, Toyama, Aichi, and Saga prefectures from 1991 to 2005 for antimicrobial susceptibility and mechanisms of fluoroquinolone resistance, based on standard disk diffusion. Of 12 drugs tested, ampicillin displayed resistance to 72.9% of isolates, streptomycin to 48.6%, tetracycline to 46.7%, sulfisoxazole to 46.7%, trimethoprim/sulfamethoxazole to 29.9%, nalidixic acid (NA) to 29.9%, and ciprofloxacin (CPFX) to 24.3%. Ten of 32 isolates resistant to 3-6 drugs and 16 of 18 isolates resistant to 7-10 drugs were resistant both to NA and CPFX. Mutations of amino acid in quinolone resistance-determining regions of gyrA and parC genes were detected in 24 isolates resistant both to NA and CPFX, and in 1 isolate resistant to NA. The former possessed a combination of double substitution (S83L and D87L) in GyrA and a single substitution (S80I) in ParC. Some 12 of 24 isolates possessed another single substitution (E84V or E84G or A108T) in ParC. The 25 isolates were classified into 4 types as follows. 1 isolate as type 1: GyrA (S83L) and ParC (S80I); 12 isolates as type 2: GyrA (S83L and D87N) and ParC (S80I); 8 isolates as type 3: GyrA (S83L and D87N) and ParC (S80I and E84G/S80R and E84V); and 4 isolate as type 4: GyrA (S83L and D87N) and ParC (S80I and A108T). In the relationship between amino acid mutations and minimal inhibitory concentrations (MIC) of fluoroquinolone, MICs of CPFX, ofloxacin, and norfloxacin showed 1microg/mL, 2microg/mL and 8microg/mL in type 1; 8 approximately 32microg/mL, 8 approximately 32microg/mL and 16 approximately 256microg/mL in type 2; and 32 approximately 256microg/mL' 32 approximately 128microg/mL and 128-->512microg/ mL in types 3 and 4. These results suggest that most of multiple-antimicrobial-resitant E. coli O153 isolates from sporadic diarrhea cases were resistant to fluoroquinolones and possessed mutations at gyrA and parC genes associated with fluoroquinolone resistance.

We developed a rapid genetic approach for screening bfpA variants of enteropathogenic E. coli (EPEC) using a heteroduplex mobility assay (HMA). A total of 204 human EPEC strains were isolated in Thailand and Japan. Of 34 bfpA‐positive EPEC strains, bfpA variants were classified into 5 HMA‐types. Different HMA‐types were found in EPEC of the same serotypes. The results suggest that HMA is a simple and easy method to analyze polymorphism of bfpA gene, and can be used in laboratories without large apparatus such as sequencers.

The prevalence of virulence-related genes of localized- and aggregated-adherent Escherichia coli (EPEC and EAggEC), such as eaeA, aggR and astA was compared between E. coli isolated from 0 to 5 year old children with and without diarrhea in Saga Prefecture. In the case of eaeA, 233 cases in Aichi Prefecture were included. The subjects were 74 diarrheal patients from which no diarrheagenic bacteria were detected besides E. coli. The control subjects were 304 nursery school children without diarrhea, and E. coli was isolated from 278 children in which 105 strains were of 0-serotype. EaeA-positive E. coli was isolated from nine (12.2%) Saga cases, 19 (8.2%) Aichi cases and 6 (5.7%) control subjects; aggR-positive E. coli was isolated from 10 (13.5%) cases and 6 (5.7%) control subjects and astA-positive E. coli from 10 (13.5%) cases and 14 (13.3%) control subjects. No significant difference (p > 0.05) was observed in the prevalence of eaeA, aggR and astA between healthy and diarrheal children, even in age-matched and 0-serotypable E. coli limited comparisons. The pathogenicity of EPEC and EAggEC should be investigated, considering other known or unidentified factors.