2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647495摘要：金黃色葡萄球菌 (Staphylococcus aureus) 是重要的院內感染或社區感染病原菌。表皮葡萄球菌(Staphylococcus epidermidis) 造成的感染主要是在免疫低下或與身上裝有人造醫療置入物有密切關連。夫西地酸(Fusidic acid)為類固醇類(steroid-like)的抗生素，自Fusidium coccineum被分離出，臨床上主要用於皮膚感染，及少數的全身性感染，常用於治療骨頭關節葡萄球菌的感染。此外也常用於對兒童異位性皮膚炎的預防感染。夫西地酸抗藥菌株雖不是很多，但近年來已有增加趨勢，因此了解抗藥機轉為重要課題。夫西地酸主要作用於細菌在轉譯 (translation)合成蛋白質的過程當中，與elongation factor G (EF-G)結合而抑制細菌生長。若細菌EF-G (fusA)有變異或得到另外的因子(FusB-family proteins, 主要是FusB, FusC)保護EF-G的作用，則細菌會對夫西地酸產生抗藥性。最近幾年，本實驗室團隊已先針對台大醫院收集的金黃色葡萄球菌及coagulase-negative較常見的葡萄球菌(CoNS)臨床菌株，分析夫西地酸抗藥基因(fusA point mutation、fusB、fusC與fusD)的盛行率及抗藥程度的差異，發現methicillin-resistant Staphylococcus aureus (MRSA)與methicillin-susceptible S. aureus (MSSA)之夫西地酸抗藥基因分佈有明顯差異。在 MRSA中，大多(22/38, 58%)是因為fusA點突變，而大部分的MSSA (15/26，58%)及S. epidermidis (34/36，94%)則主要是帶有fusB或fusC抗藥基因。在fusA點突變中，我們發現有六個新的點突變位置，其中E444K、E444V、C473S、P478S位於domain III，M651I位於domain V，R76C位於domain I。為證實這些變異點的確是造成抗藥的原因或是與生長fitness有關，將於第一年進行；探討金黃色葡萄球菌fusA點突變位置與抗藥的關係。而由於我們初步以PCR或南方墨點法，顯示表皮葡萄球菌之fusB基因結構與金黃葡萄球菌不太相同，因此擬於第二年︰探討夫西地酸抗藥金黃色葡萄球菌與表皮葡萄球菌菌株之fusB基因結構分析。另外針對fusC的部份，雖然在我們之前的分析中，MRSA中以fusA變異為主，但2006年後MRSA帶有fusC的比率似乎有增加。有文獻報導fusC在一MSSA的菌株是位在SCC island上，由於MRSA均有SCCmec，為了解MRSA之fusC與SCCmec的關聯，及帶有fusC菌株增加的原因，因此擬於第三年︰探討夫西地酸抗藥金黃色葡萄球菌菌株之fusC基因結構分析，以進一步了解fusC與MRSA的關聯性。<br> Abstract: Staphylococcus aureus is an important pathogen causing nosocomial or community-acquired infections. Staphylococcus epidermidis, one of the coagulase-negative staphylococci, is associated with infections with indwelling medical devices. Fusidic acid, a steroid-like antibiotic, has been used as a topical agent for skin infection and for some systemic infections caused by S. aureus. Fusidic acid inhibits bacteria protein synthesis by binding to a complex of elongation factor G (EF-G)-GTP/GDP and the ribosome, and then inhibiting the release of EF-G-GDP complex. Two major fusidic acid–resistance mechanisms have been reported in S. aureus: the alteration of the drug target site, which is due to mutations in fusA (encoding elongation factor G, EF-G) or rplF (FusE, encoding ribosome protein L6), and the protection of the drug target site by FusB-family proteins, including fusB, fusC, and fusD. Recently we have analyzed the distribution of fusA mutation and the presence of fusB, fusC, and fusD in S. aureus and several coagulase-negative Staphylococcus species. We found that the distribution of fusA mutations, fusB, and fusC was significantly different between the methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) groups. Among 45 fusidic acid-resistant MRSA, 38 (84%) had fusA mutations conferring high-level resistance to fusidic acid (22/38 with MIC ≥128 μg/ml), none had fusB, and 7 (16%) had fusC. For 26 fusidic acid-resistant MSSA, only 3 possessed fusA mutations, but 15 (58%) had fusB, and 8 (31%) had fusC. For S. epidermidis, most (34/36 [94%]) were due to fusB. For 41 isolates (38 MRSA and 3 MSSA) with fusA mutations, a total of 21 amino-acid substitutions in EF-G (fusA gene) were detected. Of which, R76C (domain I), E444K, E444V, C473S, P478S (domain III), and M651I (domain V), were identified for the first time. Thus in the first year of this project, we will construct the clones with new-found fusA mutation site-carrying plasmids to test the role of mutation. In the second year, we will focus on fusB element in S. epidermidis. This is because the majority of fusidic acid resistant S. epidermidis contained fusB. And our preliminary data indicated that the fusB in S. epidermidis was on chromosome, not on plasmids. The upstream and downstream sequences of fusB in S. epidermidis will be analyzed by Southern blot and subsequently cloning and sequencing. In the third year, we will analyze the genetic structure of fusC in methicillin-resistant S. aureus isolates. The reason is fusC-positive MRSA has been increasing in our hospital since 2006. Since fusC has been reported on SCC element, and our fusC-positive MRSA were SCCmec type III, the correlation of fusC and SCCmec needs further investigation.夫西地酸fusA點突變金黃葡萄球菌fusidic acidfusA point mutationStaphylococcus aureus