Escherichia coli, which causes diarrhea in humans, can be classified into the following heterogenous groups: enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAggEC), enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli (ETEC). These diarrheagenic E. coli categories are differentiated on the basis of their infection and pathogenic mechanisms. Serotyping, phenotypic assays, and molecular detection assays are very useful in identifying these diarrheagenic E. coli categories (2, 12).Since several virulence factors and DNA sequences of diarrheagenic E. coli have been identified, their molecular analyses have been performed using genetic detection assays, including PCR and DNA hybridization (5, 21, 26). The PCR method, which is a rapid gene detection assay, is particularly effective in identifying these diarrheagenic E. coli categories. Several PCR methods for detecting the various virulence factors have been reported elsewhere (1,6,22).However, conducting the separate PCR reactions that are required for the detection of the virulence factors in order to assign an isolated E. coli strain to one of the five categories is very laborious and time comsuming. Therefore, various multiplex PCR methods have been developed for the simultaneous detection of several pathogenic genes in one PCR reaction (15,18,20,23). Using the multiplex PCR method, we will be able to save the time and effort involved in analyzing various virulence factors. Some multiplex PCR systems have been reported for the rapid detection of specific virulence factors that distinguish EHEC O157 from other serotypes of EHEC (10,11,14,16,17). Cebula et al. (3) reported the multiplex PCR systems for the simultaneous detection of stx1, stx2, and uidA genes, which are specific to EHEC O157:H7. Wang et al. (27) have also reported the combination of certain multiplex PCR systems for the detection of stx1, stx2, stx2 variants, eaeA, EHEC hlyA, and rfbE O157 , which are specific to the O157 serotype, and fliC H7 , which is specific to the flagellum H7 serotype. Pass et al. (15) and Toma et al. (25) reported the use of multiplex PCR systems for the detection of 11 and 6 virulence genes, respectively. However, the systems described by them for simultaneous categorization of E. coli have not been completely effective for the simultaneous categorization of all diarrheagenic E. coli. The multiplex PCR system reported Abstract: A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli (EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli (EPEC); invE for enteroinvasive E. coli (EIEC); elt, estp, and esth for enterotoxigenic E. coli (ETEC); CVD432 and aggR for enteroaggregative E. coli (EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR ...

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx 2 and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx 2 isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx 1 , stx 2 , and stx 1 stx 2 ), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx 2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx 2 to an stx-negative strain may have occurred during infection.

A microagglutination (MA) assay to identify antibodies to Escherichia coli O111 and O157 was conducted in sera collected from 60 patients during a food-poisoning outbreak affecting 181 patients in Japan which was caused by the consumption of contaminated raw beef. Enterohemorrhagic E. coli (EHEC) O111:H8 and/or O157:H7 was isolated from the stools of some of the patients, but the total rate of positivity for antibodies to O111 (45/60, 75.0%) was significantly higher than that for antibodies to O157 (10/60, 16.7%). The MA titers of antibodies to O111 measured in patients with hemolytic-uremic syndrome and bloody diarrhea were higher than those measured in patients with only diarrhea. In patients from whose stool no isolates of E. coli O111 and O157 were obtained, the positive antibody detection rates were 12/19 (63.2%) for O111 and 2/19 (10.5%) for O157, and the MA titers of antibodies to O111 measured were higher than those to O157. Similarly, the MA titers of antibodies to O111 were significantly higher than those to O157, regardless of the other groups, including groups O111, O111 and O157, and O157. These serodiagnosis results suggest that EHEC O111:H8 stx 2 played a primary role in the pathogenesis of this outbreak. Furthermore, our findings suggest that the isolates from the patients' stool specimens were not always the major causative pathogen in patients with multiple EHEC infections, because the sera from patients from whose stools only O157 was isolated were positive for antibodies to O111. Measuring antibodies to E. coli O antigen is helpful especially in cases with multiple EHEC infections, even with a non-O157 serotype.

bWe investigated the prevalence of Legionella species isolated from puddles on asphalt roads. In addition, we carried out sequence-based typing (SBT) analysis on the genetic relationship between L. pneumophila serogroup 1 (SG 1) isolates from puddles and from stock strains previously obtained from sputum specimens and public baths. Sixty-nine water samples were collected from puddles on roads at 6 fixed locations. Legionella species were detected in 33 samples (47.8%) regardless of season. Among the 325 isolates from puddles, strains of L. pneumophila SG 1, a major causative agent of Legionnaires' disease, were the most frequently isolated (n ‫؍‬ 62, 19.1%). Sixty-two isolates of L. pneumophila SG 1 from puddles were classified into 36 sequence types (STs) by SBT. ST120 and ST48 were identified as major STs. Environmental ST120 strains from puddles were found for the first time in this study. Among the 14 STs of the clinical isolates (n ‫؍‬ 19), 4 STs (n ‫؍‬ 6, 31.6%), including ST120, were also detected in isolates from puddles on roads, and the sources of infection in these cases remained unclear. The lag-1 gene, a tentative marker for clinical isolates, was prevalent in puddle isolates (61.3%). Our findings suggest that puddles on asphalt roads serve as potential reservoirs for L. pneumophila in the environment.

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