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The PCR primers for O, H, and Vi antigen genes, tyv (rfbE), prt (rfbS), fliC-d, fliC-a, and viaB, were designed and used for the rapid identification of Salmonella enterica serovars Typhi and Paratyphi A with multiplex PCR. The results showed that all the clinical isolates examined of Salmonella serovars Typhi and Paratyphi A were accurately identified by this assay.Enteric fever remains an important public health problem in many countries of the world. Typhoid fever and paratyphoid fever are still serious public health problems in many geographic areas and are endemic in most countries, especially those of Southeast Asia and Africa. Recently, multiple-drugor fluoroquinolone-resistant strains of Salmonella enterica serovars Typhi and Paratyphi A have been emerging on the Indian subcontinent, spreading to, and becoming major problems, throughout the world (1, 4, 6). Typhoid fever and paratyphoid fever are sometimes-fatal infections of adults and children that cause bacteremia and inflammatory destruction of the intestine and other organs and that require urgent treatment by the administration of appropriate antibiotics. The diagnosis of typhoid fever or paratyphoid fever is made by ordinary culture methods and biochemical tests. The classic diagnosis method for typhoid fever or paratyphoid fever requires at least 4 or 5 days for positive results. A rapid, alternative method is needed for the diagnosis of typhoid fever or paratyphoid fever. Some researchers have already reported serovar Typhi detection methods with PCR that use the fliC-d gene (7), the Vi capsular antigen gene (3), and the 16S rRNA gene (9). As only one gene was targeted for the identification of serovar Typhi in these methods, strains of Salmonella serovars other than serovar Typhi were detected in some cases. In this study, we developed a more specific diagnosis method for both typhoid fever and paratyphoid fever based on a multiplex PCR technique that detected the Vi antigen gene (viaB), H antigen genes (fliC-d and fliC-a), and O antigen synthesis genes (tyv and prt). This system enabled us to identify and differentiate serovars Typhi and Paratyphi A, which are clinically important human pathogens, by only a single PCR, when we isolated the bacteria from blood or stool cultures from clinical patients.The bacterial strains used in this study were collected from the regional public health office in Japan, and all isolates were identified by biochemical and serological tests. A suspension of bacteria was heated at 100°C for 10 min. The samples were then used for the PCRs. We designed the primers tyv-s and tyv-as for detection of the tyvelose epimerase gene (tyv, previously called rfbE) and the primers fliCcom-s and fliCd-as for detection of the fliC-d gene (phase-1 flagellin gene for d antigen [H:d]) of Salmonella serovar Typhi. The primers parat-s and parat-as were designed for detection of a paratose synthase gene (prt, previously called rfbS), and the primers fliCcom-s and fliCa-as were designed for detection of a fliC-a gene (phase-1 flagellin...

Pulsed-field gel electrophoresis (PFGE) was applied for molecular typing of 825 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates, most of which were from 19 outbreaks and 608 sporadic cases in Japan, mainly in May to August 1996. By PFGE, the EHEC O157:H7 isolates were classified into six types (type I to V and ND [nondescript]) and UT untypeable isolates. Fifty isolates from seven outbreaks in May to June and 60 isolates from patients with sporadic cases of infection showed almost identical PFGE patterns which differed in only 1 of 22 DNA fragments. They were classified into type I. Ninety-nine isolates from 10 other outbreaks and 156 isolates from patients in the Kinki area with sporadic cases of infection obtained in the early summer of 1996 showed identical PFGE patterns, suggesting that they were derived from one huge outbreak. They were classified into type II. Type IV EHEC isolates, which had only the stx2 gene, caused another outbreak in a primary school in June. EHEC isolates of two other types, types III and V, were not related to the outbreak but were isolated in several parts of Japan. ND EHEC isolates included a variety of patterns which could not be classified into either of the types mentioned above. Twenty-five isolates could not be analyzed due to degradation of their genomic DNAs and were represented as UT. These results indicate that EHEC O157:H7 strains with various PFGE types have already spread to Japan and caused the multiple outbreaks and sporadic infections in Japan in the summer of 1996. Enterohemorrhagic Escherichia coli (EHEC) (or Shiga toxin-producing Escherichia coli) O157:H7 is an important foodborne pathogen causing abdominal cramps, diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS) (6). Recently, EHEC O157:H7 infection has frequently been reported and has been epidemiologically linked to undercooked ground beef (5), water (11), and other foods (1, 2, 4). In Japan, an EHEC O157:H7 outbreak occurred in a kindergarten in Urawa City, Saitama Prefecture, in 1990, and 2 of 268 patients died of HUS. This outbreak was attributed to well water contaminated with EHEC O157:H7. In May 1996, an outbreak of EHEC O157:H7 occurred in primary schools in Oku City, Okayama Prefecture, and involved 138 symptomatic patients, 2 of whom died of HUS.

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Shiga toxin-producing Escherichia coli (STEC) are important enteropathogens causing severe diseases such as hemorrhagic colitis and hemolytic-uremic syndrome in humans. The majority of STEC strains of serogroups O157, O26, or O111 associated with severe cases of these diseases possess a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE, which is responsible for the formation of attaching-and-effacing lesions on intestinal epithelial cells, is important for the full virulence of STEC. Nonetheless, LEE-negative STEC strains have repeatedly been reported to be associated with severe diseases in humans. In this study, we characterized adhesion to cultured epithelial cells of certain LEE-negative STEC isolated from humans with or without bloody diarrhea. Several LEE-negative STEC belonging to serogroup O91 showed an unusual, chain-like adhesion pattern to HEp-2 cells. Using Tn5-based transposon mutagenesis, we identified the gene essential for the chain-like adhesion phenotype of this O91 STEC strain. Sequence analysis of the Tn5-inserted allele identified a novel chromosomal open reading frame (ORF) encoding a polypeptide with a high degree of similarity to the E. coli immunoglobulin-binding (Eib) proteins EibA, -C, -D, -E, and -F. Therefore, the ORF was designated EibG. Laboratory E. coli strain MC4100 transformed with a multicopy plasmid carrying eibG showed chain-like adhesion to HEp-2 cells, and whole-cell lysates of the strain bound to human-derived immunoglobulin G (IgG) Fc and IgA. These results indicate that EibG acts as an IgG Fc-and IgA-binding protein, as well as an adhesin of LEE-negative STEC.

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