For screening Salmonella, enterohemorrhagic E. coli (EHEC) and Shigella positive samples from a lot of fecal samples, a method using pooled samples was examined. We chose multiplex PCR for the detection of these pathogenic enterobacteria, which does not require DNA extraction and purification, or pre-amplification by culture. 1) By using the multiplex PCR, one Salmonella-positive sample containing 7×104cfu/g could be detected from 100-pooled samples. Likewise, one EHEC-positive sample containing 7×104cfu/g and one Shigella-positive containing 4×104cfu/g could be detected from 100-pooled samples. Since the limit of detection in the culture method was 7×104cfu/g for Salmonella and EHEC and 4×105cfu/g for Shigella, they can be detected at the equal sensitivity by the multiplex PCR from a pooled sample less than 100. 2) In order to verify the possibility that dead bacteria are detected by the multiplex PCR, a model fecal sample in which genomic DNA was spiked was prepared. As a result, the genomic DNA spiked to the feces was not detected by the multiplex PCR. It was therefore considered less possible that the dead bacteria, of which genomic DNA was eluted into the feces, may be detected. 3) From 2,000 of fecal samples, 40 pools containing 50 samples each were prepared. These pools were subjected to the multiplex PCR for screening. Each sample in the pool evaluated to be positive was subjected to the culture method. On the other hand, each of the 2,000 samples was subjected to the culture method without screening. As concern the correlation between with and without the screening, a positive agreement ratio and negative agreement ratio were 100% and 99.95%, respectively. These results indicate that the method examined in this study was useful for screening positive samples from a lot of samples for fecal test.
Norovirus (NoV) is one of the most important causative agent of food-borne gastroenteritis outbreaks. Studies of NoV outbreaks associated with food-handlers who were infected with NoV have been frequently reported. However, precise reports on the duration of NoV shedding from food-handlers are not found. In this report, we examined 94 stool specimens from 28 food-handlers in 10 food-borne outbreaks for the duration of NoV RNA excretion during the period from April 2009 to March 2010. The median duration of viral shedding was 21.9 days after causative foods were prepared. 17 food-handlers out of 28 (61%) excreted NoV RNA in the stool until 18 days after causative foods were prepared. However, five food-handlers out of 28 (18%) excreted NoV RNA more than one month. When more than ten days have passed after causative foods were prepared, the quantity of NoV excreted in feces from all 5 food-handlers decreased in the range of 1.7×104 to 9.6×106/g, and became negative after the excretion continued in the same range for three to five weeks. We should monitor carefully food-handlars who excreted NoV RNA to prevent the spread of food poisoning associated with NoV.
A total of 75 Staphylococcus aureus isolates associated with food-borne diseases, food samples, healthy human feces and human nasal swabs were examined for the presence of 20 kinds of classical and newly identified staphylococcal enterotoxin (SE), and enterotoxin-like (SEl) genes (SE/SEl genes: sea-selu) by PCR. Multiple SE/SEl gene types were detected from 70.7% of isolates. All isolates associated with food-borne disease produced classical SE, but some of them harbored several newly identified SE and SEl genes in addition to classical SE gene. Isolates associated with food samples, healthy human feces and human nasal swabs showed multiple SE/SEl gene types, and some of them harbored only newly identified SE and/or SEl gene. Detection of SE/SEl genes by PCR was also shown to be useful for epidemiological study of S. aureus.