Staphylococcus lugdunensis臨床菌株之致病因子及抗Methicillin SCCmec基因分析

Abstract

Staphylococcus lugdunensis為一凝固酶陰性葡萄球菌(coagulase-negative Staphylococcus, CoNS)，但相較於一般CoNS，S. lugdunensis在感染時有更強的致病力，細菌表現特性也更接近金黃色葡萄球菌，但目前為止對大部分抗微生物物質仍沒有抗性，基因變異性低於其他葡萄球菌，另外在生物膜的表現機制上也和葡萄球菌中的ica-dependent mechanism有所不同，雖然臨床上感染案件鮮少，但其中不乏高破壞性及致死率的感染症狀。 本研究針對臺大醫院2006~2008年分離的12株S. lugdunensis進行agr基因及pulsotype分型，分析各菌株生物膜和agr相關致病因子表現量，探討致病因子表現特性和agr genotype/pulsotype不同分型間的關係。12株臨床菌株可分為agr-I (4/12)及agr-II (8/12)兩種基因型，及A~F六個pulsotype，其中分離菌株以pulsotype A為主(5/12)，其次為pulsotype F (3/12)。生物膜表現量在各分型間無顯著差異，但agr-I的菌株生物膜受到鹽類抑制的現象比較明顯；溶血素在agr-I中表現量皆明顯偏弱，agr-II菌株溶血素則和蛋白水解酶表現量有正相關。 在pulsotype F中，有兩株為抗oxacillin菌株；因此亦針對此兩株oxacillin抗藥菌株分析其抗藥基因組SCCmec之序列及基因結構，兩株S. lugdunensis皆具有SCCmec type V插入LSU methyltransferase RlmH/ybeA基因下游(ofrX-like gene)，並完成orfX至ccrC約17 kb之基因定序；但在抗藥菌株中同時也發現一插入位置尚不清楚之non-mec containing SCC結構，其中包含了完整的ccrA2B2基因。除此之外在另一非抗藥S. lugdunensis中也發現類似SCCmec type V之non-mec containing SCC。 最後本實驗中建構了抗oxacillin S. lugdunensis NTUH-4179之Fosmid library，以利未來進行non-mec containing SCC定序，及更了解S. lugdunensis之基因結構。

Staphylococcus lugdunensis is a member of coagulase-negative staphylococci (CoNS). It causes a variety of severe infections, although most CoNS were considered opportunistic pathogens with mild virulence. Compared with other staphylococci, S. lugdunensis remains susceptible to most antimicrobial agents and has relatively low genomic diversity. Despite the presence of ica locus, S. lugdunensis forms biofilm via an unique, ica-independent mechanism. The isolation rate of S. lugdunensis is markedly lower than other staphylococci, but it does have potential to cause aggressive, destructive and high-mortality infections, especially endocarditis. Here we focused on 12 S. lugdunensis clinical isolates from National Taiwan University Hospital. The 12 clinical isolates could be divided into two agr genotypes by DraI-RFLP and six pulsotypes by PFGE. Four of 12 clinical isolates belonged to agr-I, and eight isolates belong to agr-II. Agr-I isolates displayed weak heamolysin expression level, and the biofilm formation ability was remarkably decreased after NaCl treatment, but bot in agr-II isolates.In 8 agr-II isolates, we observed positive correlation between hemolysin and protease expression level. But the biofilm formation ability didn’t show significant difference between two genotypes. There were two oxacillin-resistant S. lugdunensis isolates containing SCCmec type V that inserted into downstream of LSU methyltransferase RlmH/ybeA (orfX-like gene), and we also identified a non-mec containing SCC with an intact ccrA2B2 gene in both oxacillin-resistant isolates. Besides, another non-mec containing SCC found in an oxacillin-susceptible isolate, carring ccrC and a part of SCCmec type V ORFs, but further characterization is needed.