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.

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) ...

SUMMARY: A microbial strain harboring the eae gene, which is known as the virulence gene of enteropathogenic Escherichia coli (EPEC) and most enterohemorrhagic E. coli, was isolated from a patient in a gastroenteritis outbreak that occurred in 22 patients in Akita Prefecture, Japan, in November 2011. The biochemical characteristics of the isolate were more similar to those of a novel Escherichia sp., E. albertii than E. coli. Partial 16S rRNA gene sequences of the isolate were identical to those of a certain E. albertii strain, but also showed a high degree of similarity to those of E. coli strains. Finally, we identified this isolate as E. albertii by performing PCR analysis that targeted the uidA, lysP, mdh, and cdtB genes in addition to stx and eae genes to differentiate between the EPEC and E. albertii strains.

To determine the significance of poultry and bovine as infectious sources of Campylobacter jejuni in Japan, the serotype distribution and pulsed-field gel electrophoresis (PFGE) patterns of poultry and bovine isolates were compared with those of isolates from patients with diarrhea in Akita (Japan). Serotypes O:2 and O:4-complex were common in human, poultry, and bovine isolates, and serotype O:23,36,53 was common in human and bovine isolates. SmaI PFGE patterns of isolates belonging to these serotypes were generated. Eight PFGE patterns were shared by poultry and human isolates and three patterns were shared by human and bovine isolates. Further analysis of the isolates having the same SmaI PFGE pattern by KpnI PFGE confirmed that four patterns and two patterns were still shared by poultry and human isolates, and bovine and human isolates, respectively. Thus, serotypic and genotypic data indicated a possible link between sporadic human campylobacteriosis and C. jejuni from retail poultry and bovine bile and feces, suggesting that bovine serves as an infectious source of C. jejuni in Japan, as is observed in other countries.

Four bla VIM-2 gene-harboring Pseudomonas aeruginosa strains were identified. These strains possessed a class 1 integron harboring ORF1, bla VIM-2 , and aacA4 gene cassettes. The transposon-mediated horizontal spread of the bla VIM-2 gene among these strains was suggested, which increases the threat that the bla VIM-2 gene will disseminate among diverse genera of bacteria.

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