Application of molecular biological technologies to epidemiology of new staphylococcal enterotoxins

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In recent years, many new staphylococcal enterotoxins (SEs) have been reported. To Investigate pathobiology and epidemiology of these newly identified SEs, we had undertaken studies shown in below.1. To investigate the distribution of staphylococcal enterotoxin A to I (SEA to SEI) genes (sea to sei) in Staphylococcus aureus, 146 isolates obtained in Japan from food poisoning outbreaks, from healthy humans, from bovine mastitis, and from bovine raw milk were analyzed by multiplex PCR. One hundred and thirteen (77.4%) S. aureus isolates were diagnosed as positive for one or more se genes. The se genotype was classified into 14 genotypes. The seg, seh and sei genes were detected in S. aureus isolates obtained from all of the sources, seg and sei coexisted in the same S. aureus. The newly developed sandwich ELISA showed that most seh-harboring S. aureus isolates were able to produce a significant amount of SEH. However, most S. aureus isolates harboring seg and about 60% of the isolates ha … More rboring sei did not produce a detectable level of SEG or SEI, while RT-PCR analysis proved that the mRNA of SEG and SEI was transcribed in S. aureus strains harboring seg and sei genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning (published in Journal of Clinical Microbiology).2. The emetic responses induced by staphylococcal enterotoxin (SE) A to SEE and SEG to SEI in house musk shrew (Suncus murinus) were investigated. SEA, SEE and SEI showed higher emetic activity in house musk shrew. SEB, SEC, SED, SEG and SEH also induced emetic responses in this animal model but required relatively high dose. House musk shrew appears to be a valuable model for studying of the mechanisms of emetic reaction caused by staphylococcal enterotoxins (published in Infection and Immunity).3. We identified and characterized a novel staphylococcal superantigen, named SER, which is phylogenetically related to staphylococcal enterotoxin (SE). The SER encoding gene, ser, was cloned from Staphylococcus aureus isolate that was involved in an SE-unidentified food poisoning outbreak that occurred in Japan. This isolate harbored another se gene, sej. Both ser and sej genes were located on the same 2.8 kbp EcoRI fragment and these open reading frames are transcribed in opposite directions. Nucleotides sequencing analysis revealed that SER was most closely related to SEG, with 65.9% (nucleotide sequence) and 56.4% (amino acid sequence) identity. Both ser and sej transcriptions were confirmed by Northern hybridization. The ser gene product, SER, was successfully expressed as recombinant protein in an E. coli expression system, and a monospecific polyclonal antibody was prepared against recombinant SER. Using this antibody, we were able to detect SER production by immunoblot in ser harboring S. aureus strains. Recombinant SER protein expressed by E. coli showed significant superantigenic activities. Moreover, ser was seen to be encoded by at least two types of plasmids. In particular, one kind of plasmid encoding the ser gene has been known as sed- and sej-encoding pIB485-related plasmid (submitted). Less