Genomics and post-genomics of Staphylococcus aureus causing bullous impetigo

本研究与日本60多家全国性医院和开放诊所合作，采用种子拭子法采集大疱疮患者病变的细菌标本，并在实验室分离金黄色葡萄球菌。我们鉴定出1017株金黄色葡萄球菌。分离菌株经smi酶切、eta、ethb、etd基因的Southern杂交、凝固酶检测、SEA、SEB、SEC、SED凝集试验、eta、ethb、etd、SEA、SEB、SEC、SED、tsst-1、femA、mecA的PCR检测进行PFGE鉴定。分离菌株的总体PFGE模式采用Gel compare进行聚类分析。分离菌株对cefem、大环内酯、镰刀酸、四环素、喹诺酮类药物的MIC测定。根据这些特征，我们发现日本引起大疱性脓疱病的菌株聚集在5个克隆群中，它们的剥脱毒素、凝固酶类型的产率与聚类结果有很好的相关性，即有3个产eta的聚类，1个产etb的聚类和1个产etd的聚类。三个产生eta的集群分别是凝固酶V型、III型和VII型。etd生产集群为I型，etd生产集群为II型。令人惊讶的是，100%的凝固酶III型eta产生集群为mecA阳性，70%的etb产生集群为mecA阳性。多位点测序结果与聚类分析结果具有良好的相关性，表明其克隆性。MIC试验明确表明，mecA阳性菌株对β-内酰胺具有临界耐药水平。这些结果强烈表明，现在在日本引起大疱疮的克隆群正在扩大为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.