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.
Thirteen Streptococcus dysgalactiae subsp. equisimilis isolates possessing Lancefield's group A antigen recovered from people in Japan during 2000 to 2004 were genotyped. The results indicate that a conserved clone has persisted and spread within Japan, and two different emm types were observed within members of this clone.
A large outbreak of enterohaemorrhagic Escherichia coli (EHEC) O111 and O157 occurred in Japan in April 2011. We conducted an unmatched case-control study and trace-back investigation to determine the source of EHEC O111 infection and risk factors for severe complications. Pulsed-field gel electrophoresis was performed to help define cases. A total of 86 individuals met the case definition. Of these, 40% experienced haemolytic uraemic syndrome (HUS), 24% acute encephalopathy, and 6% died. Illness was significantly associated with eating the raw beef dish yukhoe (odds ratio 19·64, 95% confidence interval 7·03-54·83), the likely food vehicle. EHEC O111 and its closely related stx-negative variants were found in the beef. HUS occurred most frequently in individuals aged 5-9 years, and this age group was significantly associated with acute encephalopathy. The prevalence of HUS and acute encephalopathy was higher than in previous non-O157-related outbreaks, indicating a high risk of severe complications.
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