Association between aggregative adherence fimbriae types including putative new variants and virulence-related genes and clump formation among aggR-positive Escherichia coli strains isolated in Thailand and Japan.

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 = 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.

Development of an immunochromatographic test strip for detection of cholera toxin.

Because cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae infection, detection of CT is critical for diagnosis of the disease. In this study, we constructed an immunochromatographic test strip for detection of CT (CT-IC) with polyclonal antibodies developed against purified recombinant whole CT protein. The detection limit of the CT-IC was 10 ng/mL of purified recombinant CT, and it could detect the CT in culture supernatant of all 15 toxigenic V. cholerae isolates examined, whereas no false-positive signal was detected in all 5 nontoxigenic V. cholerae isolates examined. The specificity of the CT-IC was examined with recombinant heat-labile toxin (LT), which shares high homology with CT, and it was revealed that the minimum detection limit for LT was 100 times higher than that for CT. In addition, lt gene-positive enterotoxigenic Escherichia coli (ETEC) was examined by CT-IC. The false-positive signals were observed in 3 out of 12 ETEC isolates, but these signals were considerably faint. The CT-IC did not develop false-positive signals with all 7 V. parahaemolyticus isolates. These results showed the high specificity of CT-IC and the feasible use of it for the detection and surveillance of toxigenic V. cholerae.

Emergence of enterohemorrhagic Escherichia coli serovar O157 strains in clade 8 with highly similar pulsed-field gel electrophoresis patterns.

Enterohemorrhagic Escherichia coli serovar O157 (O157) strains with highly similar pulsed-field gel electrophoresis (PFGE) patterns were isolated in Japan during 2007 and 2008. Several genetic features related to O157 evolution were investigated to indicate whether homoplasy might have contributed to the highly similar PFGE patterns in these strains. The O157 strains were classified in lineage I/II, as defined by a lineage-specific polymorphism assay-6 with an atypical allele in Z5935 (code: 231111). Analysis of the insertion sites of stx(2) phage in these strains showed that the sites were "occupied" in yehV and "intact" in wrbA, indicating that the strains were derived from "Cluster 1" of "Subgroup C." When a specific single-nucleotide polymorphism in ECs2357 in clade 8 strains was investigated, all of the strains in the present study were confirmed to be clade 8 strains. These results indicated that the O157 strains in this study had common genetic features, suggesting that the highly similar PFGE patterns of these strains were not due to homoplasy. Because no common source of these strains could be identified in 2007 to 2008 in Japan, these strains may have emerged from a unique O157 clade 8 clone and then spread by dissemination in Japan.

Classification of perA sequences and their correlation with autoaggregation in typical enteropathogenic Escherichia coli isolates collected in Japan and Thailand.

Enteropathogenic Escherichia coli (EPEC) strains produce a bundle-forming pilus (BFP) that mediates localized adherence (LA) to intestinal epithelial cells. The major structural subunit of the BFP is bundlin, which is encoded by the bfpA gene located on a large EAF plasmid. The perA gene has been shown to activate genes within the bfp operon. We analyzed perA gene polymorphism among typical (eae- and bfpA-positive) EPEC strains isolated from healthy and diarrheal persons in Japan (n=27) and Thailand (n=26) during the period 1995 to 2007 and compared this with virulence and phenotypic characteristics. Eight genotypes of perA were identified by heteroduplex mobility assay (HMA). The strains isolated in Thailand showed strong autoaggregation and had an intact perA, while most of those isolated in Japan showed weak or no autoaggregation, and had a truncated perA due to frameshift mutation. The degree of autoaggregation was well correlated with adherence to HEp-2 cells, contact hemolysis and BFP expression. Our results showed that functional deficiency due to frameshift mutation and subsequent nonsense mutation in perA reduced BFP expression in typical EPEC strains isolated in Japan.

Intimin types determined by heteroduplex mobility assay of intimin gene (eae)-positive Escherichia coli strains.

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.

Characterization of isolates of Salmonella enterica serovar typhimurium displaying high-level fluoroquinolone resistance in Japan.

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 microg/ml or more). Most of these isolates shared some features including definitive phage type (DT 12/193), 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 DT 104. Mutations in quinolone resistance-determining 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.

[Cefotaxime-resistant shiga toxin-producing Escherichia coli O26 : H11 isolated from a patient with diarrhea].

A shiga toxin-producing Escherichia coli (STEC) O26 strain resistant to cefotaxime (CTX) and cefpodoxime (but not ceftazidime) was isolated from the faecal sample of a 17-year-old outpatient with diarrhea. The double disk synergy test, twin test, polymerase chain reaction and sequence analysis confirmed that the strain produced CTX-M-3 type extended-spectrum beta-lactamase (ESBL). Conjugation experiment results suggested that the CTX resistance in this strain was determined by an approximately 85kbp plasmid that was readily transferable to a susceptible recipient E. coli strain. This is the first report from Japan of CTX-M-3type ESBL-producing STEC O26.

Comparison of three methods for epidemiological typing of Campylobacter jejuni and C. coli.

A total of 168 Campylobacter strains (154 C. jejuni and 14 C. coli) isolated from human clinical samples and chicken meat were typed using Penner serotyping, randomly amplified polymorphic DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) with four restriction enzymes (Sac II, Sal I, Sma I, Kpn I). The 168 strains were found to represent 13 different Penner-types and 72 different RAPD-types. However, the discriminatory potential of PFGE was dependent on the restriction enzymes used. The 168 strains were divided into 74 (Sac II), 73 (Sal I), 72 (Sma I) and 69 (Kpn I) types. The DNA of some strains was not digested by Sal I, Sma I and Kpn I. Although three RAPD-types were further subdivided by PFGE, RAPD showed good discriminatory power and a high level of agreement with PFGE patterns in terms of strain differentiation. To compare the similarities of PFGE patterns (Sac II) among the strains, a dendrogram was constructed based on the unweighted pair group method with averages (UPGMA). In most cases, DNA types of C. coli were different from those of C. jejuni. The similarities between human and meat isolates were less than 0.42 except for one outbreak in which the isolates from both patients and chicken meat showed the same DNA types.

[A diffuse outbreak of enterohemorrhagic Escherichia coli O157:H7 related to the Japanese-style pickles in Saitama, Japan].

We have experienced an outbreak of enterohemorrhagic E. coli O157:H7 (Shiga-like toxin 1 & 2 producing) in child independence support facilities in the all dormitory system, in Saitama August 2001. There were 13 patient and EHEC O157s were detected in a total of 29 specimens. As a result of epidemic inspection and microbiological investigation. We recognized that the causative food was Japanese-style pickles named "Wafu-Kimuchi" which had been sold in Saitama and Tokyo area. As the same period, several infections caused by EHEC O157 were considered the same origin in Saitama (8 patients in 5 families). Furthermore some infections happened also in Tokyo. It was made clear this outbreak was a part of a diffuse outbreak caused by Wafu-Kimuchi. In diffuse outbreaks, it is important to grasp a common feature of the individual cases in a wide area. The exchange of epidemic information between two or more municipalities and the guess of the identity in the DNA levels of strains were the key role to the elucidation of this case.

Passage in mice causes a change in the ability of Salmonella enterica serovar Oranienburg to survive NaCl osmotic stress: resuscitation from the viable but non-culturable state.

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.

Commonly conserved genetic fragments revealed by genome profiling can serve as tracers of evolution.

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.

Selective amplification of tyv (rfbE), prt (rfbS), viaB, and fliC genes by multiplex PCR for identification of Salmonella enterica serovars Typhi and Paratyphi A.

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.