以重組KPC-2碳青黴烯抗生素分解酶蛋白製備抗體用 於偵測攜帶KPC基因之微生物

Preparation of a polyclonal antiserum against recombinant KPC-2, a carbapenemase, for screening KPC-2-harboring microorganisms

陳威廷(Wei-Ting Chen)

指導教授 ： 曾銘仁

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2015年)

摘要

抗藥感染性微生物劇增是日益嚴重的世界問題，針對最後一線抗生素「碳青黴烯類 (carbapenem)」藥物，在濫用下導致許多抗藥性細菌的出現。臺灣2010年的調查，發現有6%的臨床克雷伯氏肺炎菌被偵測出帶有碳青黴烯分解酶基因，但不含克雷伯氏肺炎菌碳青黴烯分解酶 (Klebsiella pneumonia carbapenemase, KPC)。但在2012年時，帶有碳青黴稀分解酶的臨床克雷伯氏肺炎菌上升至22.3%，其中有75%為KPC-2。KPC-2基因坐落於Tn3型轉因子 (Tn4401) 上，可轉移至革蘭氏陰性細菌的質體中，作為傳播來源。且由於克雷伯氏肺炎菌存在於腸道中，所以KPC-2基因會轉移至其他菌種，導致KPC-2基因的擴散。目前在臨床微生物實驗室中尚未有有效檢測KPCs基因的方法，為了有效提升檢測方式，本研究目標是以高效率抗體為平台，用來檢測臨床菌株是否表現KPC碳青黴烯分解酶，作為臨床投藥參考指標。
首先自臨床對碳青黴烯抗生素有抗藥性的克雷伯氏肺炎菌 (Carbapenem-resistant K. pneumonia , CRKP)，利用菌落PCR取得KPC-2基因片段，接合至pQE30表現載體，並轉形至E. coli M15中，以IPTG誘導大量KPC-2蛋白表現，經Ni-column來純化，並進一步利用SDS-PAGE將蛋白完全純化，再以免疫兔子產生抗血清。
取得的KPC-2抗血清利用西方墨點法及斑點墨點法偵測純化的KPC-2蛋白，顯示具高度敏感性。再用此抗血清檢測五十株臨床的CRKP，發現表現KPC蛋白的共有二十六株 (52%)。為確定西方墨點法檢測的正確性，利用菌落PCR檢測KPC基因，也發現西方墨點法為陽性的二十六株菌皆攜帶有KPC基因，而西方墨點法陰性的菌株則沒有PCR的產物。這些西方墨點法陰性菌株的抗碳青黴烯抗生素的機制正在調查中。本研究數據顯示,以抗體檢測CRKP是否帶有KPC是有效且快速的方法。

關鍵字

克雷伯氏肺炎菌碳青黴烯分解酶

並列摘要

Antibiotic resistance among infectious microorganisms is a serious and growing crisis worldwide. The carbapenem antibiotics are drugs of last resort, however, their misuse has already led to carbapenem-resistant bacteria. In Taiwan carbapenemase genes were detected in only 6.0% of Klebsiella pneumoniae isolates but no Klebsiella pneumoniae carbapenemase (KPC)-producing isolates detected in 2010. However, in 2012 the carbapenemase genes were detected in 22.3% of K. pneumoniae isolates and 75% of them are KPC-2. The KPC carbapenemase gene has been identified within Tn3-type transposon, Tn4401, and it can insert into diverse plasmids of Gram-negative bacteria and functions as origin of blaKPC-like gene dissemination. Identification of isolates harboring KPCs has proved to be especially challenging in clinical microbiology laboratories. The aim of this study is to establish a quick antibody-based detecting platform to screen KPC-2 harboring clinical isolates. The outcome can serve as a reference for clinical antibiotic administration.
The KPC-2 gene from a carbapenem-resistant K. pneumonia (CRKP) isolate was obtained by colony PCR, subcloned into pQE30 expression vector and KPC-2 protein was overproduced as insoluble inclusion bodies upon IPTG induction in E. coli M15. The urea-solubilized KPC-2 proteins were purified with Ni-column and further resolved by SDS-PAGE to exclude trace contaminates and used to immunize rabbit to produce antiserum against KPC-2 protein by a commercial biotech company.
The KPC-2 antiserum showed high sensitivity toward purified KPC-2 protein by Western blot and Dot blot. Screening 50 clinical CRKP isolates showed that 26 (52%) of them expressing KPCs proteins by Western blot using this KPC-2 antiserum. Colony PCR also confirmed presence of KPCs genes in these 26 corresponding immunoblot-positive isolates, but not in other immunoblot-negative isolates. The mechanisms of carbapenem resistance of those KPC-negative isolates are being under investigation. Nevertheless, our data suggested that this antibody-based method can serve as a quick and efficient screening for KPC-harboring CRKP.

並列關鍵字

Klebsiella pneumoniae carbapenemase ； KPC

參考文獻

3. Report S.o.C.D.a.S (2012) Annual Report of Nosocomial Infections Surveillance System(2012)-Taiwan.

4. Report S.o.C.D.a.S. (2011) Annual Report of Nosocomial Infections Surveillance System(2011)-Taiwan.

2. Wright G D (2013) Q&A: antibiotic resistance: what more do we know and what more can we do? BMC Biol. 11:51.

5. Eugenie Bergogne-Berezin, MD (1995) Treatment and Prevention of Nosocomial Pneumonia. Chest 108:26-34

8. Spierings G, van Silfhout A, Hofstra H, Tommassen J (1992) Identification of Klebsiella pneumoniae by DNA Hybridization and Fatty Acid Analysis. Int J Syst Bacteriol 42: 252-6.