近年來，在歐美地區困難梭狀桿菌被認為是目前造成院內感染性腹瀉的主要致病菌。然而，台灣對於困難梭狀桿菌的流行病學調查是缺乏的。為了瞭解困難梭狀桿菌在台灣的分佈與基因型多樣性，我們收集了從2011年到2012年兩年來自台南醫院的251個不重複的臨床病人分離菌株。針對臨床菌株的毒素基因分型，我建立一個快速篩檢的多重聚合酶鏈鎖反應，同時搭配一個針對tcdA基因3端截斷突變的聚合酶鏈鎖反應來偵測許多困難梭狀桿菌毒力相關的基因，包含毒素A（tcdA）、毒素B（tcdB）、二元毒素CDT（cdtA and cdtB）以及毒素負調控子TcdC（tcdC）的截斷突變。分型結果顯示臨床檢體有45.8%（115/251）同時帶有tcdA 與 tcdB 基因（A+B+），而32.7%（54/251）的臨床菌株是不具有毒性基因存在（A-B-）。值得我們注意的是這些臨床菌株中有21.5%（54/251）的菌株只具有tcdB基因（A-B+）。然而，在A+B+基因型的菌株中只有十八株菌株（7.2%）是二元毒素基因cdtA與cdtB陽性並同時帶有毒素負調控基因tcdC截斷突變，包含 22%的18個鹼基缺失與78%的39個鹼基缺失。進一步，這十八株高毒力基因型的菌株（A+B+CDT+）利用聚合酶鏈鎖反應進行核醣型分型（Ribotying）。菌株核醣型的鑑定主要是透過英國的困難梭狀桿菌核醣型網絡中心（CDRN）協助，結果顯示在本研究中並沒有核醣型027的存在，其中高毒力基因型的菌株中佔大多數的核醣型屬於核醣型078家族（77.8%, 14/18），包含了核醣型078（5.5%）、核醣型127（33.3%）、核醣型126（22.2%）以及一個具有RT078 marker的未知核醣型（16.7%）。對應臨床資料顯示這十八株高毒力菌株中有七株（38.9%）造成病人產生典型腹瀉症狀，其中核醣型078家族佔了五株。值得注意的是一名被核醣型126菌株感染的病人甚至發展成偽膜性結腸炎。此外，細胞毒性實驗的結果顯示核醣型078家族與其他核醣型的菌株比較下具有較高的細胞毒性。過去的研究曾指出核醣型078家族普遍存在於全世界的動物，特別是豬隻。因此，我分析從2011年八月到2015年三月收集來自台灣十六個不同豬場的134個檢體。結果顯示在豬隻身上有85.1%（114/134）的陽性分離率而且所有的分離菌株也都屬於核醣型078-家族，包含核醣型078（32.5%）、核醣型126（28.9%）、 核醣型127（37.7%）以及一個未知的核醣型（0.9%）。其中豬隻分離的三個核醣型078與病人分離的核醣型078菌株具有相同的重複序列分型圖譜（Rep-PCR）與相同的tcdB基因次分型，這表示人與豬之間的核醣型078菌株在親緣關係上相近，具有潛在人畜共通的特性。同時我也發現核醣型078和126在血盤培養上都展現獨特的菌株型態，同時在液體培養中也表現特殊的高聚集性（aggregation），但是這些特性卻與菌株生長快速及生物膜的形成無關。總結，所有的實驗結果都指出台灣地區所存在的核醣型078家族菌株是需要持續監控與調查，以防未來菌株有擴散與群突發的風險。

Clostridium difficile is the major cause of nosocomial diarrhea. Epidemiological study on C. difficile in Taiwan is limited. A total of 251 C. difficile isolates from patients were collected between January 2011 and December 2012 in southern Taiwan. I analyzed the distribution of toxin A (tcdA), toxin B (tcdB), and binary toxin genes (both cdtA and cdtB) and tcdC truncated mutations in all isolates by a newly established multiplex-PCR with additional truncated-tcdA PCR screening. A total of 45.8% (115/251) isolates harbored tcdA and tcdB (A+B+), and 32.7% (54/251) isolates did not have tcdA and tcdB (A-B-). Notably, 21.5% (54/251) of isolates were tcdA-nagative and tcdB-positive (A-B+). Among these A+B+ strains, eighteen isolates (7.2%) were also positive for cdtA/B, which carry a tcdC gene deletion, including an 18-bp (22%) or 39-bp deletion (78%). These eighteen hypertoxigenic (A+B+CDT+) strains were further typed by PCR-ribotyping. There was no ribotype 027 in this study and the predominant PCR-ribotypes were defined as RT078-family (77.8%, 14/18) including RT078 (5.5%), RT127 (33.3%), RT126 (22.2%) and unknown RT-type (16.7%) with a RT078 marker, which were confirmed by the C. difficile Ribotyping Network (CDRN) in England. Retrospective review of clinical data demonstrated 7 hypertoxigenic strains caused C. difficile associated diarrhea (CDAD) in patients and 5 of 7 belonged to RT078-family. Notably, one patient infected by RT126 even developed the severe pseudomembranous colitis (PMC). In addition, RT078-family is more cytotoxic than other ribotypes. Previous studies demonstrated that C. difficile RT078-family is predominant in animals worldwide, particularly in swine. Therefore, I surveyed C. difficile strains isolated from swine in sixteen farms during August 2011 and March 2015 in Taiwan. I found the positive culture rate is 85.1% (114/134) and all strains from swine belonged to RT078-family, including RT078 (32.5%), RT126 (28.9%), RT127 (37.7%) and one unknown-RT (0.9%) with RT078 marker. Among RT078 isolates, I found three swine isolates might that be identical to the strains isolated from patient by Rep-PCR and tcdB sequencing, suggesting these is potential zoonosis between human and swine in Taiwan. Meanwhile, we observed that RT078 and RT126 strains presented distinctive colony morphology on agar plate and aggregation phenotype in broth culture, which were not associated with rapid growth or biofilm formation. In conclusion, these results highlighted that toxigenic RT078 family strains in Taiwan is predominant and more attention should be given in order to minimize risk of further spreading.

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