Application of molecular biological technologies to epidemiology of new staphylococcal enterotoxins

近年来，许多新的葡萄球菌肠毒素(Ses)被报道。为了调查这些新发现的SE的病理生物学和流行病学，我们进行了如下1项研究。为了解金黄色葡萄球菌肠毒素A~I(SEA~SEI)基因(SEA~SEI)在金黄色葡萄球菌中的分布情况，采用多重聚合酶链式反应技术对从日本食物中毒暴发、健康人、牛乳房炎和牛生奶中分离到的146株金黄色葡萄球菌进行了检测。113株(77.4%)金黄色葡萄球菌的一个或多个Se基因阳性。Se基因可分为14种基因类型。在所有来源的金黄色葡萄球菌中均检测到seg、seh和sei基因，seg和sei基因共存于同一株金黄色葡萄球菌中。最新开发的夹心ELISA法显示，大多数含有SEH的金黄色葡萄球菌分离株都能够产生大量的SEH。然而，大多数金黄色葡萄球菌分离株含有SEG，约60%的分离株具有…更枯燥的SEI没有产生可检测到的SEG或SEI水平，而RT-PCR分析证明SEG和SEI的mRNA在含有seg和sei基因的金黄色葡萄球菌中转录。这些结果表明了定量评估食品中SEG和SEI产生的重要性，以澄清这些新的SE与食物中毒之间的关系(发表在《临床微生物学杂志》上)。研究了葡萄球菌肠毒素(SE)A对家麝鼠(Suncus Murinus)的SEE和SEG诱导的呕吐反应。SEA、SEE和SEI对家麝鼠有较高的催吐活性。SEB、SEC、SED、SEG和SEH也可在该动物模型中诱导呕吐反应，但需要相对较高的剂量。家麝鼠似乎是研究葡萄球菌肠毒素引起呕吐反应机制的有价值的模型(发表在《感染与免疫》杂志上)。我们鉴定并鉴定了一种新的葡萄球菌超抗原，命名为SER，它与葡萄球菌肠毒素(SE)具有亲缘关系。SER编码基因ser是从金黄色葡萄球菌分离株中克隆的，该分离株与日本发生的一起不明原因的食物中毒疫情有关。该分离株含有另一个Se基因sej。Ser和Sej基因位于同一2.8kbp的EcoRI片段上，这些开放阅读框的转录方向相反。核苷酸序列分析表明，SER与SEG的同源性最高，核苷酸序列同源性为65.9%，氨基酸序列同源性为56.4%。Northern杂交证实了Ser和Sej的转录。Ser基因产物SER在大肠杆菌表达系统中以重组蛋白形式表达，并制备了抗重组SER的单特异性多克隆抗体。利用该抗体，我们能够检测到免疫印迹法检测携带金黄色葡萄球菌的菌株产生的SER。在大肠杆菌中表达的重组SER蛋白具有明显的超抗原性。此外，Ser至少由两种类型的质粒编码。特别是，一种编码Ser基因的质粒被称为Sed-和Sej-编码pIB485相关质粒(已提交)。较少

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