[Isolation of Escherichia coli O128:HNM harboring stx2f gene from diarrhea patients].

Shiga-like-toxin-producing Esherichia coli O128:HNM were isolated from feces of a one-year-old boy with diarrhea and abdominal pain on July, 2002, and a 11-month-old girl with diarrhea and fever on June, 1997. None of other enteropathogenic bacteria including Salmonella were isolated. E. coli O128:HNM isolates from both patients carry stx2f and eaeA gene, but not stx1, stx2, aggR, bfpA, esth, estp, invE, astA, ureC and hlyA gene. As far as we know, this may be the first report indicating that E. coli O128:HNM carrying stx2f gene were isolated from patients in Japan.

Rapid categorization of pathogenic Escherichia coli by multiplex PCR.

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 system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.

PCR identification of the plasmid-borne enterotoxin gene (cpe) in Clostridium perfringens strains isolated from food poisoning outbreaks.

It is generally accepted that Clostridium perfringens strains associated with food poisoning carry their enterotoxin gene, cpe, on the chromosome, while C. perfringens strains isolated from non-food-borne diseases, such as antibiotic-associated diarrhea and sporadic diarrhea, carry cpe on the plasmid. However, we recently encountered a food poisoning outbreak caused by C. perfringens bearing a plasmid cpe. We therefore investigated a total of 31 clinical and non-clinical C. perfringens strains to locate the cpe gene by PCR. The cpe of nine heat-sensitive (100 degrees C for 10min) strains isolated from three outbreaks of food poisoning were located on the plasmid, while those of six heat-resistant strains from other food poisoning outbreaks were located on the chromosome. Moreover, the cpe of 5 heat-sensitive strains isolated from healthy human feces and those of 11 heat-sensitive soil strains were also located on the plasmid. These findings indicate that heat-sensitive, cpe-plasmid-borne C. perfringens strains should not be disregarded as causative agents of food poisoning.

emm Typing of group A streptococcus clinical isolates: identification of dominant types for throat and skin isolates.

T and emm types were determined for group A streptococci isolated from patients with various infections during 1990-1999 in Toyama Prefecture, Japan. Out of 906 isolates, 872 isolates were divided into 20 T serotypes, and 34 isoltes were T nontypeable (TNT). T12, T1, and T4 were dominant among 699 throat isolates; on the other hand, T11, T28, TB3264, and TNT were dominant among 80 skin isolates. The emm types of 190 isolates were determined following specific PCR amplification and sequencing of the products. Twenty T serotypes were divided into 34 T type/emm type combinations. Thirty-four TNT isolates were divided into 14 emm types, in which emm58 was the most common (38%). Among 82 throat isolates randomly selected, predominant T types T12, T1, and T4 isolates were of the respective same numbers in emm type. T11/emm89, T28/emm28, TB3264/emm13w, and TNT/emm58 were predominant among 80 skin isolates. emm-type distribution observed in the present study was that usually reported in the western world. To our knowledge, 3 T/emm is a novel combination. These results show that emm typing allows the characterization of group A streptococci from various sources.