Simultaneous Screening of 24 Target Genes of Foodborne Pathogens in 35 Foodborne Outbreaks Using Multiplex Real-Time SYBR Green PCR Analysis.

A set of 8 multiplex real-time SYBR Green PCR (SG-PCR) assays including 3 target primers and an internal amplification control (IAC) primer was simultaneously evaluated in 3 h or less with regard to detection of 24 target genes of 23 foodborne pathogens in 7 stool specimens of foodborne outbreak using a 96-well reaction plate. This assay, combined with DNA extraction (QIAamp DNA Stool Mini kit), offered detection of greater than 10(3)-10(4) foodborne pathogens per g in stool specimens. The products formed were identified using melting point temperature (Tm) curve analysis. This assay was evaluated for the detection of foodborne pathogens in 33 out of 35 cases of foodborne outbreak, using 4 different PCR instruments in 5 different laboratories. No interference from the multiplex real-time SG-PCR assay, including IAC, was observed in stool specimens in any analysis. We found multiplex real-time SG-PCR assay for simultaneous detection of 24 target genes of foodborne pathogens to be comprehensive, rapid, inexpensive, accurate, of high selectivity, and good for screening probability.

[A case of cervical abscess caused by Salmonella enteritidis after intestinal infection].

Salmonella Enteritidis often causes food poisoning. In this study, an extremely rare case of cervical abscess caused by S. Enteritidis is reported. In January 2003, a 44-year-old man visited our hospital with swelling of the left submandibular region. He had been suffering from a severe diabetic condition but had neglected to seek medical attention. An incision was made at the abscess to drain the pus from which only S. Enteritidis was isolated. This finding led to the discovery that he had suffered from an episode of food poisoning 6 months earlier. However, the organism was not isolated from the stool. The patient recovered with the administration of panipenem/betamipron and gatifloxacin. The S. Enteritidis strain isolated from the pus obtained from this case and that detected from the samples originating from the other patients during the episode of food poisoning 6 months earlier were examined by using pulse-field gel electrophoresis (PFGE). The PFGE patterns of the strains were almost identical. The molecular epidemiological analysis by PFGE was useful in estimating the infection route. In an immuno-compromised host such as those suffering from diabetes mellitus, one must be reminded that unusual bacteria (including S. Enteritidis) may cause a cervical abscess.

[Isolation of Neisseria meningitidis from healthy persons in Japan].

Between September 2000 and March 2003 healthy subjects in 10 prefectures of Japan were investigated to identify carriers of Neisseria meningitidis. Twenty-five N. meningitidis strains were isolated from 5886 throat swab specimens collected from healthy persons, such as students, elderly, and foreigners. Of the 25 carriers, 9 were teenagers, 15 were in their twenties, and only one was in the fifties. The male-female ratio of the carriers was 17 to 8, showing male dominance. The serogroups of the 25 strains were B (9 strains), Y (4 strains) and non-groupable (12 strains). One of the strains was found to be deficient in gamma-glutamyl aminopeptidase activity, which is an identification marker for N. meningitidis.

Dissemination of the phage-associated novel superantigen gene speL in recent invasive and noninvasive Streptococcus pyogenes M3/T3 isolates in Japan.

In Japan, more than 10% of streptococcal toxic shock-like syndrome (TSLS) cases have been caused by Streptococcus pyogenes M3/T3 isolates since the first reported TSLS case in 1992. Most M3/T3 isolates from TSLS or severe invasive infection cases during 1992 to 2001 and those from noninvasive cases during this period are indistinguishable in pulsed-field gel electropherograms. The longest fragments of these recent isolates were 300 kb in size, whereas those of isolates recovered during or before 1973 were 260 kb in size. These 260- and 300-kb fragments hybridized to each other, suggesting the acquisition of an about 40-kb fragment by the recent isolates. The whole part of the acquired fragment was cloned from the first Japanese TSLS isolate, NIH1, and its nucleotide sequence was determined. The 41,796-bp fragment is temperate phage phiNIH1.1, containing a new superantigen gene speL near its right attachment site. The C-terminal part of the deduced amino acid sequence of speL has 48 and 46% similarity with well-characterized erythrogenic toxin SpeC and the most potent superantigen, SmeZ-2, respectively. None of 10 T3 isolates recovered during or before 1973 has speL, whereas all of 18 M3/T3 isolates recovered during or after 1992 and, surprisingly, Streptococcus equi subsp. equi ATCC 9527 do have this gene. Though plaques could not be obtained from phiNIH1.1, its DNA became detectable from the phage particle fraction upon mitomycin C induction, showing that this phage is not defective. A horizontal transfer of the phage carrying speL may explain the observed change in M3/T3 S. pyogenes isolates in Japan.