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標題: | 利用多位址重複序列分析ST59型非抗藥性及抗藥性金黃色葡萄球菌SCCmec IV和VT之親緣演化關係 The Evolutionary Relationship between Methicillin-susceptible Staphylococcus aureus、Methicillin-resistant Staphylococcus aureus SCCmec IV and VT of the ST59 by MLVA |
作者: | Yu-Chia Kuo 郭昱嘉 |
指導教授: | 鄧麗珍 |
關鍵字: | 多位址重複序列,金黃色葡萄球菌, MLVA,Staphylococcus aureus, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 金黃色葡萄球菌是人類重要的致病菌之一,而隨著抗生素的廣泛使用,金黃色葡萄球菌也逐漸產生了抗藥性,像是藉由得到了SCCmec DNA片段而對β-lactum類藥物有抗藥性的MRSA (methicillin-resistant Staphylococcus aureus)。MRSA又可根據病人的的醫療歷史分成hospital-associated MRSA (HA-MRSA)和community-asociated MRSA (CA-MRSA),其中CA-MRSA是從1990年代後期才開始出現,但近期快速散播的致病株。亞太地區之CA-MRSA主要是ST59,其中包括了台灣。在台灣ST59 MRSA又有兩種clones,“Taiwan”和“Asian-Pacific”clones,分別屬於SCCmec IV和SCCmec VT,而台灣地區的MSSA雖然ST188占了多數,但仍有約10%是ST59,有趣的是在台灣地區,只有ST59在MRSA和MSSA都可以被發現,根據MRSA是從MSSA演化過來的假說,期望以高解析度的多位址重複序列分析(Multiple Loci Variable Number Tandem Repeat Analysis, MLVA)分型方法找出台灣ST59 MRSA SCCmec IV、VT和MSSA的親緣演化關係。
蒐集臺大醫院2000、2005、2010和2011年MRSA和MSSA臨床菌株,SCCmec typing結果,type IV有128株,type VT有85株,MSSA有31株,總共244株再做spa typing,主要都是t437,分別有57.0%、77.6%和80.6%。244株以MLVA 10個VNTR locus分析,發現雖然可以將各個clonal complex (CC)區分開,但在CC59 (包含ST59)中無法看出SCCmec IV和VT和MSSA有明顯的演化關係,因此針對CC59的194株菌株以MLVA 16個VNTR locus分析,發現SCCmec IV在Sa0964 locus有81.3%是10,VT有94.5%是7,而MSSA是7或10的比率大約各占一半,或許就暗示著MSSA先演化成兩個clones,再個別獲取SCCmec而演化成SCCmec IV或VT,而PFGE的結果也部份地支持了這樣的推論。最後針對Sa0964這個locus與其下游基因SAV0921進行研究:既然此locus的repeat個數在SCCmec IV和VT之間有明顯的差異,則可以合理推論可能會影響下游基的轉錄。但是根據RT-PCR半定量的結果發現,無論repeat個數是7或10,甚至只有1個repeat,下游基因的轉錄量仍然相同,因此推測VNTR locus Sa0964可能只是一個演化上的痕跡,並不會對下游基因轉錄有所影響而造成SCCmec IV或VT有所差異。 Staphylococcus aureus is one of the important human’s bacterial pathogen. With broad usage of antibiotics, S. aureus gradually emerged antibiotic-resistant strain, for instance, methicillin-resistant Staphylococcus aureus (MRSA), by gaining staphylococcal chromosome cassette mec (SCCmec). According to patients’ medical history, MRSA can be separated into two types: hospital-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA), and the later immerged in late 1990s and spread rapidly. In Taiwan, there are two major clones in ST59 CA-MRSA, “Taiwan” and “Asian-Pacific” clone. Generally, the former carried the type VT SCCmec cassette, and the later carried the type IV SCCmec cassette. For methicillin- susceptible Staphylococcus aureus (MSSA), although ST188 clones were most commonly found, some of the MSSA belonged to ST59, roughly 10%. Interestingly, only ST59 was found among both MSSA and MRSA isolates. MRSA was recognized as emerging from MSSA by acquiring SCCmec chromosome cassette. However, the evolutionary relationship between SCCmec type IV and VT of ST59 remains unclear. Since Multiple Loci Variable Number Tandem Repeat Analysis (MLVA) may provides higher discriminatory power, it is expected to gain more understanding for the evolutionary relationship between MSSA and SCCmec IV, VT . In this study, we collected clinical isolates of MRSA and MSSA recovered from NTUH in 2000, 2005, 2010, and 2011. A total of 244 clinical isolates were collected, including 128 SCCmec type IV, 85 type VT, and 31 MSSA. By spa typing, the major type, t437, was found in SCCmec IV (57.0%), VT (77.6%), and MSSA (80.6%), respectively. By MLVA-10-variable number tandem repeat (VNTR)-locus typing, each clonal complex (CC) can be separated clearly, but there was no obvious difference in evolutionary relationship among SCCmec IV, VT, and MSSA in CC59. Therefore, we used MLVA-16-VNTR-locus, expecting higher discriminatory power, to analyze 194 clinical isolates, belonging to CC59. Finally, according to the MLVA code of VNTR locus, Sa0964, 81.3% SCCmec type IV was 10, and 94.5% SCCmec type VT was 7. Interestingly, in MSSA, the ratio of 7 or 10 is about half to half. Thus, it is possible that MSSA initially separated into two lineages long time ago, and then acquired type IV and type VT SCCmec cassette, respectively. The PFGE data also partially support this speculation. After that, we studied this VNTR locus, Sa0964, and the downstream gene, SAV0921. Given the obviously different repeat copy number between SCCmec IV and VT, it is possible that the transcription of SAV0921 might be different, too. According to reverse transcription PCR data, the transcription of SAV0921 are the same when the repeat copy number is 1, 7, and 10. That is, the VNTR locus, Sa0964, won’t affect the transcription of downsteam gene, and play the role much like an evolutional marker among the genome of CC59 MRSA and MSSA. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57327 |
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