In this study, clonally related strains of E. coli accounted for a large proportion of blaCTX-M-positive E. coli. This high proportion of clonal groups identified in different regions of Japan suggests their recent spread by mechanisms other than healthcare-associated transmission. These observations imply that restricting antimicrobial use in human clinical settings may have limited impact on the spread of ESBL-producing E. coli.

We have previously cloned two distinct regions of the Shigella sonnei form I plasmid pSS120, a 37-kilobase-pair DNA region and a virF region, which were found to be essential for cell invasion in Escherichia coli K-12 (J. Kato, K. Ito, A. Nakamura, and H. Watanabe, Infect. Immun. 57:1391-1398, 1989). The 37-kilobase-pair DNA region was randomly inserted by use of transposon Tn3-lac. At least eight genes were found to be located within the region, as determined by analysis of Tn3-lac-generated lac fusions. Expression of six genes, ipaB, ipaC, invE, invG, invJ, and invK, was apparently regulated by the positive regulator virF. IpaB and IpaC proteins could not found in invE mutants even if the virF gene was present. This observation suggested that the invE region encoded a positive regulator different from the virF gene. The functional relationship between the invE and virF genes was then examined. Translational fusions ipaB::Tn3-lac and invJ::Tn3-lac were used as indicators for gene expression, and the following results were obtained. Full expression of the ipaB and invJ genes required the presence of both the invE and virF regions. virF positively regulated the expression of invE at the transcriptional level. An increase in the copy number of invE enhanced the expression of ipaB and invJ in the absence of virF. These findings strongly indicate that the invE gene product, whose expression is regulated by virF, acts positively on the invasion-associated genes. InvE is a 35,407-dalton protein and has significant homologies with ParB of plasmid P1 and SopB of plasmid F, which are DNA-binding proteins involved in plasmid partitioning.

SUMMARY:The previously identified Shiga toxin (Stx) 2f-producing Escherichia coli O115:HNM strain F08/101-31, isolated from a symptomatic human, was confirmed to be E. albertii in the present study by whole genome DNA-DNA hybridizations, by sequencing (cpn60, dnaJ, and 16S rRNA genes), and by multi-locus sequence typing. The F08/101-31 strain was originally identified as E. coli rather than the relatively new bacterial species E. albertii, which was first described in 2003, because it did not display any of the biochemical characteristics of E. albertii. This new classification will impact public health management strategies in Japan because the present study showed that some E. albertii strains, which are often misidentified as E. coli, produce Stx and likely cause diarrhea in humans. Therefore, further guidelines for the management and identification of Stx-producing E. albertii are required in Japan.The discovery of Shiga toxin (Stx)-producing Escherichia albertii strains was significant from a public health perspective in Japan (1). E. albertii was first described by Huys et al. in 2003 (2). This newly described enteropathogen is often misidentified as E. coli (3) or other members of the family Enterobacteriaceae because of its poorly defined biochemical characteristics and general obscurity. In addition, E. albertii closely resembles E. coli in its phenotypic characteristics. Retention of the major virulence factor intimin in E. albertii is well established (4,5), whereas little is known about the incidence of Stx-producing E. albertii (1). Stxs are the most significant virulence factors of Stx-producing E. coli (STEC) in human infections. However, STEC strains that produce intimin, a host-cell adhesin, often cause more critical symptoms in patients than STEC strains that do not produce intimin (6). Therefore, the misidentification of Stx-producing E. albertii strains that also produce intimin as E. coli may complicate public health monitoring strategies in many countries.The F08/101-31 strain, which was previously reported as Stx2f-producing E. coli O115:HNM harboring eae (7), was recently inferred to be an Stx2f-producing E. albertii strain on the basis of species-specific polymerase chain reaction (PCR) analysis (4,8). Therefore, in the present study, we attempted to definitively determine whether the strain is an E. albertii isolate by whole-genome DNA-DNA hybridization and DNA sequencing techniques. The purpose of this study was to describe the characteristics of the E. albertii strain for the benefit of public health.For genetic analysis, species-specific PCR analysis, whole-genome DNA-DNA hybridization, sequence analysis (16S rRNA genes, cpn60 (groEL), and dnaJ), and multi-locus sequence typing (MLST) were performed. The F08/101-31 strain was examined by species-specific PCR, as described previously, for the presence of E. albertii-specific sequences within lysP and mdh (4) and for E. coli, Shigella, and E. albertii/S. boydii lineage-detecting alleles of clpX (4,8). Whole-genome DNA-DNA hybridization...

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

The relationship to diarrhea of genes located on the pathogenicity islands (PAI) other than the locus of enterocyte effacement (LEE) was investigated. Enteropathogenic Escherichia coli (EPEC), the retention of espC on the EspC PAI, the OI-122 genes (efa1/lifA, nleB), the phylogenetic marker gene yjaA, and the bundle-forming pilus gene bfpA on the EPEC adherence factor (EAF) plasmid were studied. E. coli strains carrying the intimin gene (eae) without the Shiga toxin gene, isolated from patients with diarrhea (n ‫؍‬ 83) and healthy individuals (n ‫؍‬ 38) in Japan, were evaluated using PCR. The genotypes of eae and espC were identified by heteroduplex mobility assay (HMA). The proportions of strains isolated from individuals with and without diarrhea that carried these genes were as follows: bfpA, 13.3 and 7.9%, respectively; espC, 25.3 and 36.8%; efa1/lifA, 32.5 and 13.2%; nleB, 63.9 and 60.5%; yjaA, 42.2 and 55.3%. Statistical significance (P < 0.05) was achieved only for efa1/lifA. The proportion of strains lacking espC and carrying efa1/lifA was higher for patient-derived strains (30.1%) than for strains from healthy individuals (13.2%), but the difference was not significant. Strains carrying both espC and efa1/lifA were rare (2 strains from patients). Statistical analyses revealed significant relationships between espC and yjaA and between efa1/lifA and nleB, as well as significant inverse relationships between espC and efa1/lifA and between efa1/lifA and yjaA. espC was found in eae HMA types a1, a2, and c2, whereas efa1/lifA was found in types b1, b2, and c1. In addition, 6 polymorphisms of espC were found. The espC, yjaA, efa1/lifA, and nleB genes were mutually dependent, and their distributions were related to eae type, findings that should be considered in future epidemiological studies.