On examining the phenotypic characteristics of the various EPEC strains, we found that aggregates of the strains isolated in Japan were smaller and weaker than those of strains isolated in Thailand. Further, when we examined adherence to HEp-2 cells, the results were similar to those of the autoaggregation assay. The EPEC strains which showed strong autoaggregation also showed a greater degree of contact hemolysis. It seemed that the contact hemolysis would

be promoted by the presence of BFPs, which would facilitate more effective adherence, so we tested these strains for the bfpA gene expression by RT-PCR and for BFP expression by performing Western blotting. mRNA of the bfpA gene and BFPs were not detected in strains which showed weak or no autoaggregation. mTOR inhibitor The EPEC strain, which showed weak aggregation and pili-like structure in Figure 3c, but not BFP (Fig. 5), might have been expressing the type I pili. While, it remains to be seen why the strain with truncated perA sequences showed strong autoaggregation. We observed frame shift mutant of perA even in the E2348/69 strain which changed to weak phenotype, so these region of perA might be liable to mutation.

We also tested the EPEC strains for the presence of BFP-related genes such as bfpF and perC, and detected them in most strains. We then converted the perA genes into amino acid sequences and found that the amino acid sequences of some of the perA genotypes had been truncated by a frame shift mutation of the perA gene. Strains with a truncated perA gene showed weak

or no autoaggregation and decreased HEp-2 cell adherence Olaparib order (Table 2). We did not find truncated amino acid sequences in bfpA genes. This study showed that most of the typical EPEC strains isolated in Japan did not express BFP, and it appeared that a truncated perA gene was connected with inhibition of BFP expression (Fig. 5). We performed PFGE analysis to show molecular typing of EPEC strains isolated from Japan (Fig. 7). There were no relationship between PFGE profiles and bfpA polymorphism. MRIP According to the recent studies, the prevalence of atypical EPEC has continued to increase not only in developed but also in developing countries (39). In Japan, most EPEC isolates have been classified as atypical EPEC, and even the supposedly typical EPEC strains from Japan used in this study could in fact be atypical EPEC, although bfpA genes were detected with PCR. As comparable results were obtained with HMA and DNA sequencing for bfpA and perA genes, this shows that genotyping by HMA was a useful method for classifying these genes. The distributions of bfpA and perA genotypes differed between the EPEC isolates from Japan and those from Thailand. A study of global polymorphisms of virulence genes and their phenotypic characteristics would yield more significant information on the pathogenesis of EPEC.