Genomics and post-genomics of Staphylococcus aureus causing bullous impetigo

在本研究中，我们与日本60多家全国性医院和开放诊所合作，采集了大疱性脓疱病患者皮损的细菌标本，并在本实验室进行了金黄色葡萄球菌的分离。我们鉴定了1017株金黄色葡萄球菌。对分离的菌株进行SmaI酶切、Eta、ETB、ETD基因Southern杂交、凝固酶试验、SEA、SEB、SEC、SED凝集试验、ETA、ETB、ETD、SEA、SEB、Sec、Se、tsst-1、FEMA、mecA的聚合酶链式反应检测。用凝胶比较法对分离菌株的全部PFGE图谱进行了聚类分析。同时测定分离菌株对头孢菌素、大环内酯类、呋喃西酸、四环素、喹诺酮类药物的MIC。根据这些特征，我们发现引起日本大疱性脓疱病的菌株被聚为五个克隆群，它们的脱落毒素产量、凝固酶类型与聚类结果很好地相关，即有三个产生ETA的簇，一个产生ETB的簇和一个产生ETD的簇。三个产生ETA的簇分别是凝固酶V、III和VI型。ETB产生簇为I型，ETD产生簇为II型。令人惊讶的是，凝固酶III型ETA产生簇100%为mecA阳性，70%ETB产生簇为mecA阳性。多位点测序结果与聚类分析结果具有很好的相关性，表明它们具有克隆性。MIC检测清楚地表明，这些mecA阳性菌株对β-内酰胺类药物具有临界水平的耐药性。这些结果强烈表明，在日本，现在引起大疱性脓疱病的TEO克隆群正在扩大为MRSA。

In this study, we collected bacterial specimens by seed swab from lesions of bullous impetigo patients with the cooperation of more than 60 nation-wide hospitals and open clinics in Japan and isolated Staphylococcus aureus in our laboratory. We identified 1,017 S.aureus strains. The isolated strains were subjected to PFGE by SmaI digestion, Southern hybridization for genes of eta,etb,etd, coagulase testing, agglutination test for SEA,SEB,SEC,SED,PCR testing for eta,etb, etd,sea, seb,sec,sed,tsst-1,femA,mecA. The overaII PFGE patterns of isolated strains weresubjected to clustering analysis by Gel Compar. Isolated strains were also subjected for measurement of MIC against cefem, macrolide, fusigic acid, tetracycline, quinolone. Upon these characterization, we discovered that strains causing bullous impetigo in Japan were clustered in five clonal groups and their producibility of exfoliative toxin, coagulase types well correlated with clustering results, i.e.there are three ETA-producing clusters, one ETB-producing and one ETD-producing clusters. Three ETA-producing clusters are coagulase type V,III, and VII respectively. ETB-producing cluster is type I and ETD-producing cluster is type II. Surprisingly, 100% of coagulase type III ETA-producing cluster is mecA positive and 70% of ETB-producing cluster is mecA positive. Multilocus sequencing results well correlated with clustering analysis indicating their clonality. MIC testing clearly indicated that those mecA positive strains are borderline level resistant to β-lactams. These results strongly suggested that in Japan now teo clonal groups causing bullous impetigo are expanding as MRSA.