拉登葡萄球菌臨床菌株之加強溶血基因分析及分子分型

Abstract

Staphylococcus lugdunensis是一種凝固酶陰性葡萄球菌（coagulase-negative Staphylococcus, CoNS），可從對人體無害的皮膚表面共生菌到致命的病原菌都有。臨床上病例雖不是很多，但已知可引起許多感染，如傷口感染、心內膜炎等等。 目前對S. lugdunensis之毒力因子的了解並不多，已知95%的臨床菌株會分泌delta-like溶血素（δ-like hemolysin），delta-like溶血素由slush基因組所編碼，並調控加強溶血表現型。另一方面，細菌分子分型在病原性基因探討或流行病學上均很重要，但分型法各有優缺點，且不同菌種適合的分型法也不一樣。目前漸漸盛行的VNTR分型法已應用在許多病原菌上，但尚無應用在S. lugdunensis的研究，因此，本研究欲進一步探討slush基因與加強溶血表現型的相關性，並開發S. lugdunensis臨床菌株之VNTR分型法。 實驗結果顯示，94.7％（18/19）的S. lugdunensis臨床菌株有加強溶血的表現，且100％（19/19）的臨床菌株都有slush基因存在，表示slush基因在臨床菌株間保守性高。亦由RNA表現量證實了加強溶血表現型的差異是因為slush 基因轉錄程度不同所造成的。 分析兩株已發表全基因組序列S. lugdunensis之VNTR loci，篩選出九個保守度高且單位長度近60 bp的VNTR loci，且由PCR和核酸定序結果可知，九個VNTR loci在S. lugdunensis臨床菌株間的重複次數有差異，其中七個保守度較高的VNTR loci可將所有臨床菌株分成10種亞型（SID＝0.918），較分成七種亞型的PFGE分辨力好（SID＝0.789），且VNTR可再細分相同pulsotype的菌株，表示VNTR較PFGE更適合用於S. lugdunensis之分型。此外，編號5、6、7的VNTR loci在臨床菌株之間的差異性較大，因而利用這三個loci設計multiplex PCR，結果可將所有臨床菌株概分成9種亞型（SID＝0.901）。

Staphylococcus lugdunensis is a member of coagulase-negative staphylococci(CoNS) with the potential to cause many types of infection,ranging from superficial skin infections to life-threatening endocarditis.Nevertheless,in contrast to S.aureus,data on virulence factors and molecular typing for S. lugdunensis is scarce.In this study,94.7％（18/19）of S. lugdunensis clinical isolates produce a synergistic hemolytic activity (SLUSH),phenotypically similar to the delta-hemolysin of S. aureus.The slush gene was detected in all strains, indicating that this locus is highly conserved in S. lugdunensis.The differences of synergistic hemolytic phenotype may result from the activity of slush transcription.On the other hand,we analyzed two known-sequenced S. lugdunensis genome for the presence of variable-number tandem repeats(VNTR). Variations in repeat numbers were observed in nine VNTR loci within the S. lugdunensis clinical isolates. The criteria used were ≥94% conservation between repeat units and repeat units between 45 and 200 bp in length.Seven VNTR loci were found using these criteria and were able to separate 19 clinical isolates into 10 VNTR types（SID＝0.918）,whereas PFGE was able to separate 19 isolates into 7 pulsotypes（SID＝0.789）,indicating that VNTR typing had better discrimination than PFGE. Furthermore,the greater variations in repeat numbers were observed in locus 5,6,7.Based on these loci,we develop a multiplex PCR method for molecular typing and it was able to separate 19 isolates into 9 multiplex VNTR types（SID＝0.901）. Therefore,VNTR can be a useful addition to PFGE analysis for molecular epidemiological investigation of S. lugdunensis.