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...

EHEC O157:H7 clade 6 strains harboring stx2a and/or stx2c and clade 8 strains harboring stx2a or stx2a/stx2c were frequently associated with childhood HUS cases in Japan. Rapid and specific detection of such lineages are required for infection control measures.

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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

Strains of the multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium isolated in Japan were examined for high-level fluoroquinolone resistance. Since the first isolation in 2000 (described in reference 13), we have identified 12 human and 5 nonhuman isolates with high-level fluoroquinolone-resistance (ciprofloxacin MIC of 24 g/ml or more). Most of these isolates shared some features including definitive phage type (DT12/193), resistance type (ACSSuTNCp; resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, nalidixic acid, and ciprofloxacin), and genotype on pulsed-field gel electrophoresis that were different from those of the MDR S. enterica Typhimurium DT104. Mutations in quinolone resistancedetermining regions of gyrA and parC were also conserved in almost all of the isolates despite the absence of any apparent epidemiological relationships among cases. This suggests that a specific clonal group of the serovar Typhimurium with high levels of fluoroquinolone resistance is disseminating among animals and humans in Japan.

An outbreak caused by dried processed squids contaminated with Salmonella Oranienburg occurred in Japan in 1999. Isolates obtained from the causative food were resistant to NaCl osmotic stress, but isolates from the patients were sensitive to NaCl. Although strains from both sources were almost identical in their virulence in mice, a NaCl-resistant strain from food (Sa9911T) became NaCl-sensitive after passage in mice and a NaCl-sensitive strain from one patient (Sa99004) retained NaCl sensitivity after such passage. When dried squid was contaminated experimentally with both strains during processing, only Sa9911T was recovered directly from the final product. Nevertheless, the viability of the Sa99004 cells was over 90% found by fluorescent staining. We suggested that Sa99004 might become viable but non-culturable (VNC) by NaCl stress. This hypothesis was confirmed by resuscitation by efficient enrichment. We concluded that VNC S. Oranienburg would be potentially dangerous contaminants of NaCl-preserved foods and that measures to ensure their detection should be taken at the time of food inspection.

We developed a method to produce, identify and analyze DNA fragments for the purpose of taxonomic classification. Genome profiling (GP) is a strategy that identifies genomic DNA fragments common to closely related species without prior knowledge of the DNA sequence. Random PCR, one of the key technologies of GP, is used to produce fragments and may be used even when there are mutations at the priming site. These fragments can then be distinguished based on the information of mobility and melting pattern when subjected to temperature gradient gel electrophoresis (TGGE). Corresponding fragments among several species, designated as commonly conserved genetic fragments (CCGFs), likely have the same genetic origin or correspond to the same gene. The criteria for identification of CCGFs has been defined and presented here. To assess this prediction, some of the fragments were sequenced and were confirmed to be CCGFs. We show that genome profiles bearing evolutionarily conserved CCGFs can be used to classify organisms and trace evolutionary pathways, among other profound applications.

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.