抗methicillin溶血葡萄球菌臨床菌株之SCCmec基因分析

Abstract

溶血葡萄球菌(Staphylococcus haemolyticus)為凝固酶陰性葡萄球菌(coagulase-negative staphylococci)，為人體表面之正常菌叢，但近年來發現其對於臨床感染症的重要性，特別是針對使用侵入性醫療器材的病人，容易產生院內感染。溶血葡萄球菌常具有多重抗藥性及高程度的抗藥特性，也是很早被發現具有glycopeptides抗藥性的菌株，更增加其治療的困難度。 本研究針對台大醫院2007-2009年所分離之106株溶血葡萄球菌進行methicillin抗藥性分析，其中有99株溶血葡萄球菌具有methicillin抗藥性，且均偵測到其mecA基因，分析99株溶血葡萄球菌之SCCmec基因分型，發現溶血葡萄球菌不適用MRSA之分型方法，除了常見的SCCmec type V之外，本研究發現溶血葡萄球菌具有幾個特別的SCCmec分型，將其命名為UT1~5 (unnamed type)。比較SCCmec分型與最低抑菌濃度，發現SCCmec UT1~4相較於SCCmec type V有較高的抗藥程度，此現象原因目前還不明。 另以PFGE進行菌株分型，發現此99株共可分為21種pulsotypes，以pulsotype A (42/99)所含菌株最多，其次為pulsotype L(11/99)及pulsotype G (10/99)。將SCCmec分型與PFGE分型比較，發現pulsotype A大部分屬於SCCmec UT4 (39/42)，顯示此群菌株具有Clonal的跡象。 接著以南方墨點法搭配LA-PCR及Inverse-PCR分析pulsotype A，SCCmec UT4中菌株NTUH20085513之SCCmec基因結構，發現此ΨSCCmec基因結構不具有ccr基因，且與中國大陸分離出之溶血葡萄球菌WCH1與MRSA JCSC6945 SCCmec type X結構最相近，可能具有相同的演化來源，接著再利用NTUH20085513ΨSCCmec所分析出的基因序列進行PCR mapping及南方墨點法，以分析其他溶血葡萄球菌臨床菌株之SCCmec結構，發現SCCmec UT1 (主要分佈於pulsotype L)及UT4 (主要分佈於pulsotype A)與NTUH20085513ΨSCCmec有較相似的基因結構，並發現SCCmec UT1 (主要分佈於pulsotype L)及UT4 (主要分佈於pulsotype A)兩者之SCCmec基因結構差異主要存在於R1的區域，本研究發現溶血葡萄球菌之SCCmec基因結構多樣性高，並由基因序列相似性推斷MRSA形成新型SCCmec type X來源可能為溶血葡萄球菌。

Among coagulase-negative staphylococci (CoNS), Staphylococcus haemolyticus, a commensal organism, is the second next to S. epidermidis in its frequency of isolation from human blood cultures. Within the past few years, CoNS have been increasingly recognized to cause clinically significant infections and are particularly associated with the use of indwelling medical devices. S. haemolyticus is notorious for its multidrug resistance and early aquisition of resistance to methicillin and glycopeptide antibiotics. In this study, we collected 106 S. haemolyticus clinical isolates from National Taiwan University Hospital. These isolates were tested for oxacillin susceptibility testing, and 99 isolates were oxacillin resistant. All these 99 isolates contained mecA gene. These isolates were then characterized for SCCmec elements. In addition to SCCmec type V, we found five unnamed types SCCmec UT1~UT5, which have not seen in MRSA. Comparison between SCCmec type and oxacillin MIC, we found that the SCCmec UT1~UT4 were more resistant to oxacillin than SCCmec type V. Analysis of genetic relatedness using PFGE, 99 isolates can be separated into 21 groups. The pulsotype A was the most predominant (42/99). Comparison between SCCmec type and PFGE type, we found that the predominant pulsotype A almost belong to SCCmec type UT4 (39/42), indicating that these isolates might have epidemiology significance. Using LA-PCR and Inverse PCR to analyse the SCCmec structure of a isolate belonging to pulsotype A and carrying SCCmec UT4 (NTUH20085513). The results indicated that NTUH20085513ΨSCCmec structure has high similarity with MRSA JCSC6945 SCCmec type X and S. haemolyticus WCH1 ΨSCCmec, but no ccr gene was found in this gene structure. Using PCR mapping and sourthern blot to compare other clinical isolates' SCCmec gene structures with NTUH20085513 ΨSCCmec, we found that SCCmec UT1(pulsotype L) and UT4(pulsotype A) have the highest similarity with NTUH20085513 ΨSCCmec gene structure, and the gene structures of these two groups are mainly different at the Region 1 of SCCmec gene structures. This study reinforce the hypothesis that it is possible that MRSA SCCmec type X may aquire SCCmec from MR-S. haemolyticus by horizontal gene transfer.