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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張靜文(Ching-Wen Chang) | |
dc.contributor.author | Pei-Husan Lu | en |
dc.contributor.author | 呂珮萱 | zh_TW |
dc.date.accessioned | 2021-06-17T08:07:10Z | - |
dc.date.available | 2024-08-27 | |
dc.date.copyright | 2019-08-27 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-18 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73635 | - |
dc.description.abstract | 具兼性共生特性之嗜肺性退伍軍人菌 (Legionella pneumophila),於自然環境下,可利用原蟲躲避不利生長之環境壓力,並以原蟲作為營養源,自原蟲體內大量複製增生,進而釋出高移動性及高毒性之成熟感染型態 (mature infectious form, MIF),且於相同環境壓力下,MIF-L. pneumophila比高營養環境達生長穩定期 (stationary phase, SP)之L. pneumophila (SP-L. pneumophila)更具耐受性。另外,處於環境壓力下之L. pneumophila,亦可形成具活性但不具可培養性 (viable but non culturable, VBNC)之生理狀態,以適應並存活於不利生長之環境中,待接觸原蟲後,即有感染原蟲,或利用原蟲恢復自身可培養性 (復生,resuscitation)之可能,進而再次造成水體汙染。而於過往研究退伍軍人病零星個案中,職業駕駛屬高風險族群,並證實於汽車雨刷水中可同時檢出L. pneumophila與原蟲,且不同品牌及種類之雨刷精對L. pneumophila抑制程度不一之情況下,本研究以MIF-L. pneumophila為研究對象,探討MIF-L. pneumophila與0.1 g/mL之10種市售雨刷精 (Commercial screenwash, CS)及10種家用清潔劑 (Household detergent, HD),於37℃下接觸7、18及30日後,MIF-L. pneumophila可培養力及活性變化;另外亦延伸觀察接觸雨刷水清潔劑 (windscreen wiper fluids, WWFs)後呈VBNC型態之MIF-L. pneumophila,與原蟲於不同感染比 (multiplicity of infection, MOI)及共培養時日 (2、4、7及10日)下之感染力及復生情形。
本研究實驗分為三階段:首先利用L. pneumophila (ATCC 33152)與Acanthamoeba castellanii (ATCC 30234)共培養後,以自原蟲體內大量增生釋出之MIF-L. pneumophila作為研究對象;再以第一階段獲得之MIF-L. pneumophila與20種雨刷水清潔劑接觸7、18及30日,利用推盤培養法及EMA-qPCR分析接觸雨刷水清潔劑前與接觸後,MIF-L. pneumophila之可培養及活性細菌濃度,並以接觸雨刷水清潔劑前後之MIF-L. pneumophila可培養及活性細菌log濃度差值 (log reduction)作為抑菌效能評估之指標;最後參考抑菌實驗結果,挑選於30日中可使MIF-L. pneumophila呈VBNC型態之雨刷水清潔劑,再次與MIF-L. pneumophila接觸特定時日後,與原蟲進行共培養,並以LIVE/DEAD® BacLightTM Bacterial Viability Kits結合螢光顯微鏡法及推盤培養法,分別觀察MIF-L. pneumophila之感染力及復生。 就不同雨刷水清潔劑品項而言,以廣義估計方程式多變項模式分析發現,10種CS中,AA、CD及CAP相較CP (至接觸30日可培養濃度僅下降0.43 log)可顯著減少4.91、4.94及4.92 log之MIF-L. pneumophila可培養濃度 (p-value均<0.0001),且AA及CD相較CP亦可顯著減少2.82及1.22 log之MIF-L. pneumophila活性濃度 (p-value均<0.0001)。10種HD中,MB2017、BL、MG、MA及AM相較WD (至接觸30日可培養濃度僅下降0.59 log)可顯著減少4.86-5.04 log之MIF-L. pneumophila可培養濃度 (p-value均<0.0001),且MB2017相較WD亦可顯著減少3.27 log之MIF-L. pneumophila活性濃度 (p-value<0.0001)。 廣義估計方程式多變項模式分析亦顯示,與20種WWFs接觸18及30日後均可較接觸7日顯著減少0.34及0.66 log之MIF-L. pneumophila可培養濃度 (p-value≦0.0001);相較於7日,與20種WWFs接觸18日可增加0.06 log之MIF-L. pneumophila活性濃度 (p-value=0.10),接觸30日則較接觸7日顯著減少1.06 log之MIF-L. pneumophila活性濃度 (p-value<0.0001)。 評估是否出現VBNC型態之MIF-L. pneumophila之結果顯示,20種WWFs中,接觸1日即失去可培養性 (<1 CFU/mL)且於7日失去活性 (<527 cells/mL)者有AA及MB2017;於7日失去可培養性但活性細菌濃度介於103-105 cells/mL者共計有CD、CAP、BL、MG、MA及AM;於18日失去可培養性然活性細菌濃度>105 cells/mL者則有WB及SO;於30日失去可培養性且活性細菌濃度>104 cells/mL者為TM2018及LT;於30日仍未失去可培養性者則有BP、BS、GY、TY、CP、DW及WD。 VBNC型態之MIF-L. pneumophila與原蟲共培養之結果顯示,10種受測之WWFs中,與原蟲以感染比為5、50及100共培養2日後,於螢光顯微鏡下觀察,可自原蟲體內清楚發現具≧5隻具綠色螢光 (活性)之L. pneumophila者為CAP、MG、AA及MB2017;原蟲體內雖無法辨識L. pneumophila個體但呈現大片綠色螢光者為AM、CD、BL及LT;無法觀察L. pneumophila及原蟲者為WB及TM2018。培養法分析則發現,接觸CD、AM與BL7日呈VBNC型態之MIF-L. pneumophila,與原蟲分別於感染比為5並共培養7日與10日以及以感染比為100並共培養10日時可達復生。 本研究證實,不同成分雨刷水清潔劑對抑制MIF-L. pneumophila之培養力及活性具差異性,且接觸雨刷水清潔劑之MIF-L. pneumophila於30日內仍具高可培養力,而呈VBNC型態之MIF-L. pneumophila與原蟲接觸不同時日下,尚有感染原蟲甚至利用原蟲恢復繁殖增生之風險,反應出培養法對偵測環境中L. pneumophila之限制,以及現今環境消毒或清潔手段無法完全根除L. pneumophila之潛在原因。 | zh_TW |
dc.description.abstract | Legionella pneumophila, a facultative intracellular bacillus, can use the protozoa to avoid the environmental pressure and use protozoa as a nutrient source to replicate and proliferate.This process will release the mature infectious forms (MIFs) of L. pneumophila, which characterized as hyper-mobility and virulent. Besides, Literature showed that MIF-L. pneumophila was more susceptibility than stationary phase (SP), which growth in high nutrient environment, in same environmental pressure. Moreover, L. pneumophila are capable of developing a viable but nonculturable (VBNC) state to stay in environmental pressure and infect protozoa or be resuscitated after culture on protozoa. Literature showed that drivers possess greater risk of legionellosis, and windscreen wiper systems of vehicles are considered as the potential contamination source of L. pneumophila and amoeba. Otherwise, different brands and types of windscreen wiper fluids (WWFs) have different degrees of inhibition on L. pneumophila. In this study, MIF-L. pneumophila was used as a research object to contact 10 kinds of commercial screenwash (CS) and 10 kinds of household detergent (HD) in 0.1 g/mL for 7, 18 and 30 days and to assess the influence on the culturable and viable MIF-L. pneumophila. In addition, legionella, which was VBNC after exposure to WWFs, was also observed the infectivity and resuscitation on the amoeba in the different multiplicity of infection (MOI) and co-culture time.
There were three experiments in this study. First, preparation of MIF-L. pneumophila by coculturing the L. pneumophila (ATCC 33152) and Acanthamoeba castellanii (ATCC 30234). Second, using MIF-L. pneumophila to contact the 20 kinds of WWFs for 7, 18 and 30 days. Culture method and EMA-qPCR analysis were used for analyzing the concentration of culturable and viable MIF-L. pneumophila. The antibacterial activities of WWFs were further calculated as a logarithmic reduction of (Ca/Cb), whereby Ca and Cb referred to the concentration of MIF-L. pneumophila after and before adding to WWFs, respectively. Finally, selecting the WWFs, which could get VBNC MIF-L. pneumophila after contact within 30 days, to contact the MIF-L. pneumophila on specific time, and co-culture with A. castellanii. LIVE/DEAD® BacLightTM Bacterial Viability Kits with fluorescence microscopy and culture method were used for monitoring of Legionella infections and resuscitation. For different kinds of WWFs, the analysis of the generalized estimation equation multivariate model found that AA, CD and CAP in CS could significantly reduce the culturable concentrations of 4.91, 4.94 and 4.92 log, further AA and CD could significantly reduce the viable concentrations of 2.82 and 1.22 log compared with CP (p-value<0.0001). MB2017, BL, MG, MA and AM in HD could significantly reduce the culturable concentrations of 4.86-5.04 log, further MB2017 could significantly reduce the viable concentrations of 3.27 log compared with WD (p-value<0.0001). Moreover, compared with 7 days, 18 and 30 days of contact with 20 WWFs could significantly reduce the culturable concentrations of 0.34 and 0.66 log (p-value≦0.0001), further 18 days could increase the viable concentration of 0.06 log (p-value=0.10), then 30 days could significantly reduce the viable concentration of 1.06 log (p-value<0.0001). VBNC MIF-L. pneumophila results showed that AA and MB2017 were no culturability (<1 CFU/mL) on the 1st day and no viability (<527 cells/mL) on the 7th day; CD, CAP, BL, MG, MA and AM were no culturability (<1 CFU/mL) on the 7th day but 103-105 cells/mL of viable concentration; WB and SO were no culturability (<1 CFU/mL) on the 18st day but >105 cells/mL of viable concentration; TM2018 and LT were no culturability (<1 CFU/mL) on the 30st day but >104 cells/mL of viable concentration. MIF-L. pneumophila was still culturable within 30 days in BP, BS, GY, TY, CP, DW and WD. The results of VBNC MIF-L. pneumophila co-culture with protozoa showed that≧5 bright L. pneumophila within A. castellanii were observed in CAP, MG, AA and MB2017; L. pneumophila was not recognized but large green fluorescence within A. castellanii were observed in AM, CD, BL and LT; unable to observe L. pneumophila and A. castellanii for WB and TM2018. Furthermore, MIF-L. pneumophila, which was VBNC by contacting with CD, AM and BL for 7 days, was co-cultured with A. castellanii at an MOI of 5 and 100 and co-culture for 7 and 10 days could observe resuscitation. Above all, this study confirmed that different ingredient of WWF have different effects on the culture and viable MIF-L. pneumophila. There was high culturability of MIF-L. pneumophila in WWF within 30 days, and VBNC MIF-L. pneumophila could infect amoeba and even resuscitation by contacting with amoeba in different MOI and contact time. It shown the limitations of the culture method on the detection of L. pneumophila in the environment, and the potential reasons for the current environmental disinfection or cleaning methods to completely eradicate L. pneumophila. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:07:10Z (GMT). No. of bitstreams: 1 ntu-108-R06844005-1.pdf: 23890792 bytes, checksum: 536cb66f33114fb06427f3e8ae72ebfe (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 摘要 i
Abstract iii 圖目錄 xi 表目錄 xv 第一章 前言 1 第二章 文獻回顧 2 2.1 嗜肺性退伍軍人菌 (Legionella pneumophila)與退伍軍人病 (Legionellosis) 2 2.1.1 成熟感染型嗜肺性退伍軍人菌(Mature infectious form L. pneumophila, MIF-L. pneumophila) 4 2.1.2 生長穩定期嗜肺性退伍軍人菌 (Stationary phase L. pneumophila, SP-L. pneumophila) 4 2.1.3 MIF-L. pneumophila於環境壓力下之優勢 5 2.2 棘阿米巴原蟲 (Acanthamoeba castellanii) 8 2.2.1 棘阿米巴原蟲與嗜肺性退伍軍人菌之互動關係 8 2.3 L. pneumophila與原蟲共培養之實驗室備置 9 2.4 退伍軍人菌與棘阿米巴原蟲於車體檢出情形 13 2.5 雨刷水清潔劑對車輛雨刷水中L. pneumophila之檢出影響 15 2.6 雨刷水清潔劑對SP-L. pneumophila之抑菌研究 17 2.7 MIF-L. pneumophila之抑菌研究 20 2.8 具活性但不具培養性 (Viable but nonculturable, VBNC)嗜肺性退伍軍人菌之復生研究 (resuscitation) 23 2.8.1 低營養環境 23 2.8.2 環境消毒處置 27 2.9 VBNC型態嗜肺性退伍軍人菌感染力 (Infectivity)研究 31 第三章 研究目的 35 第四章 研究架構 36 第五章 材料與方法 38 5.1 培養基、緩衝液及無菌自來水之備置 38 5.1.1 Buffered charcoal yeast extract agar (BCYEα agar) 38 5.1.2 ATCC medium 712 38 5.1.3 Page’s Amoeba Saline (PAS) 39 5.1.4 Tryptic soy agar (TSA agar) 39 5.1.5 Non-Nutrient agar (NNA agar) 40 5.1.6 TE緩衝溶液 40 5.1.7 無菌自來水 40 5.2 儀器設備 41 5.3 微生物菌株備置與培養 42 5.3.1 生長穩定期嗜肺性退伍軍人菌 (SP-L. pneumophila)備置 42 5.3.1.1 SP-L. pneumophila可培養 (CFU/mL)及細菌濃度 (cells/mL)檢量線建立 42 5.3.2 棘阿米巴原蟲 (Acanthamoeba castellanii ATCC 30234) 44 5.3.3 大腸桿菌(Escherichia coli ATCC 25922) 45 5.3.4 Heat-killed Escherichia coli/NNA agar 45 5.3.5 成熟感染型嗜肺性退伍軍人菌 (MIF-L. pneumophila)備置 45 5.3.5.1 移除原蟲體外SP-L. pneumophila 49 5.3.5.2 觀察SP-L. pneumophila與A. castellanii共培養時日 49 5.3.5.3 測試收取MIF-L. pneumophila之離心條件 50 5.3.5.4 MIF-L. pneumophila可培養濃度 (CFU/mL)檢量線建立 52 5.4 雨刷水清潔用品 (Windscreen Wiper fluids, WWFs) 52 5.4.1 雨刷水清潔用品受測品項 52 5.4.2 雨刷水清潔用品化學成分分析 53 5.5 MIF-L. pneumophila抑菌實驗 56 5.5.1 MIF-L. pneumophila與WWFs接觸及取樣時間 57 5.5.2 MIF-L. pneumophila抑菌實驗之樣本備置與研究設計 57 5.5.3 MIF-L. pneumophila抑菌實驗之樣本處置 59 5.6 MIF-L. pneumophila與原蟲之共培養實驗 60 5.6.1 選擇共培養實驗用之雨刷水清潔劑種類 60 5.6.2 MIF-L. pneumophila與A. castellanii共培養之研究設計 60 5.6.3 MIF-L. pneumophila與A. castellanii共培養之樣本備置及處理 61 5.7 樣本分析 65 5.7.1 BacLight結合螢光顯微鏡分析 65 5.7.2 EMA結合Real-time PCR分析 66 5.7.2.1 Ethidium monoazide (EMA)核酸染劑處置 66 5.7.2.2 DNA萃取 66 5.7.2.3 L. pneumophila EMA Real-time PCR分析 66 5.7.2.4 DNA標準品檢量線建立 67 5.7.3 共培養樣本鑑定分析 69 5.7.3.1 革蘭氏染色法 69 5.8 資料處理 70 5.8.1 血球計數器細胞濃度計算 70 5.8.1.1 L. pneumophila細胞濃度 (cells/mL)計算 70 5.8.1.2 A. castellanii細胞濃度 (cells/mL)計算 71 5.8.2 L. pneumophila可培養濃度與偵測下限值之計算 71 5.8.3 L. pneumophila活性細菌濃度與偵測下限值之計算 72 5.8.4 雨刷水清潔劑對MIF-L. pneumophila之影響 (Log reduction計算) 73 5.8.5 易感性因子K值計算 73 5.9 統計方法 74 第六章 結果 76 6.1 MIF-L. pneumophila之配置 76 6.1.1 原蟲體外之SP-L. pneumophila移除率 (%) 76 6.1.2 SP-L. pneumophila與A. castellanii於不同共培養時日下之互動 76 6.1.3 獲得MIF-L. pneumophila之離心條件 78 6.2 MIF-L. pneumophila抑菌實驗結果 79 6.2.1 市售雨刷精 (Commercial screenwash, CS)對MIF-L. pneumophila之抑菌效果 79 6.2.1.1 可培養性變化 79 6.2.1.1.1 30日可培養濃度 (CFU/mL)及不同接觸時日可培養濃度之統計檢定 79 6.2.1.1.2 1-6日可培養濃度 (CFU/mL) 83 6.2.1.1.3 Log reduction及抑菌效能統計檢定 85 6.2.1.1.4 易感性因子 (K值) 91 6.2.1.2 活性變化 93 6.2.1.2.1 30日活性細菌濃度 (cells/mL)及不同接觸時日活性細菌濃度之統計檢定 93 6.2.1.2.2 Log reduction及抑菌效能統計檢定 96 6.2.1.2.3 易感性因子(K值) 102 6.2.2 家用清潔劑 (Household detergent, HD)對MIF-L. pneumophila之抑菌效果 104 6.2.2.1 MB2015及MB2017之比較 104 6.2.2.2 可培養性變化 105 6.2.2.2.1 30日可培養濃度 (CFU/mL)及不同接觸時日可培養濃度之統計檢定 105 6.2.2.2.2 1-6日可培養濃度 (CFU/mL) 108 6.2.2.2.3 Log reduction及抑菌效能統計檢定 110 6.2.2.2.4 易感性因子 (K值) 116 6.2.2.3 活性變化 118 6.2.2.3.1 30日活性細菌濃度 (cells/mL)及不同接觸時日活性細菌濃度之統計檢定 118 6.2.2.3.2 Log reduction及抑菌效能統計檢定 121 6.2.2.3.3 易感性因子 (K值) 127 6.2.3 20種雨刷水清潔劑抑菌效能統計檢定 129 6.2.3.1 可培養性變化 129 6.2.3.2 活性變化 130 6.2.4 具活性但不具可培養性 (VBNC)之MIF-L. pneumophila 131 6.3 MIF-L. pneumophila與原蟲共培養之實驗結果 133 6.3.1 陰性控制組 (原蟲攝食觀察結果) 133 6.3.2 陽性控制組 (未接觸清潔劑之MIF-L. pneumophila與原蟲共培養之觀察結果) 135 6.3.3 實驗組 (接觸清潔劑之MIF-L. pneumophila與原蟲共培養之觀察結果) 137 6.3.3.1 與原蟲共培養前可培養力之確認 137 6.3.3.2 感染力及復生 141 第七章 討論 171 7.1 不同製造年分雨刷水清潔劑之比較 171 7.2 MIF-L. pneumophila與SP-L. pneumophila之比較 176 7.2.1 可培養性 177 7.2.2 活性 187 7.2.3 易感因子 (K值) 195 7.3 抑菌影響因子 198 7.3.1 清潔劑抑菌研究 198 7.3.2 雨刷水清潔劑種類及成分 200 7.3.3 接觸時日 211 7.4 共培養實驗 212 7.4.1 感染力 213 7.4.2 復生 214 7.5 研究限制 216 7.5.1 抑菌實驗 216 7.5.2 共培養實驗 216 7.6 未來建議 217 第八章 結論 218 8.1 抑菌效能評估 218 8.1.1 雨刷水清潔劑品項 218 8.1.2 雨刷水清潔劑接觸時日 218 8.1.2.1 Kruskal-Walles test結合Dunn’s test事後檢定分析 218 8.1.2.2 廣義估計方程式多變項模式分析 219 8.1.3 具活性但不具可培養性 219 8.2 共培養 220 8.3 MIF-L. pneumophila與SP-L. pneumophila比較 220 8.3.1 可培養性 220 8.3.2 活性 221 8.3.3 K值 221 參考文獻 222 附錄 230 附件一、SP-L. pneumophila可培養濃度 (CFU/mL)與細胞濃度 (cells/mL)檢量線結果 230 附件二、MIF-L. pneumophila可培養濃度 (CFU/mL)檢量線結果 231 附件三、雨刷水清潔用品化學成分分析 232 附件四、統計檢定表 249 附件五、TM2018對SP-L. pneumophila之抑菌實驗 256 附件六、SP-L. pneumophila及MIF-L. pneumophila於市售雨刷精 (Commercial screenwash, CS)之可培養濃度彙整表 259 附件七、SP-L. pneumophila及MIF-L. pneumophila於市售雨刷精 (Commercial screenwash, CS)之活性細菌濃度彙整表 260 附件八、SP-L. pneumophila及MIF-L. pneumophila於家用清潔劑 (Household detergent, HD)之可培養濃度彙整表 261 附件九、SP-L. pneumophila及MIF-L. pneumophila於家用清潔劑 (Household detergent, HD)之活性細菌濃度彙整表 262 | |
dc.language.iso | zh-TW | |
dc.title | 成熟感染型嗜肺性退伍軍人菌對雨刷水清潔劑之感受性評估 | zh_TW |
dc.title | Susceptability of mature infectious forms (MIFs) of Legionella pneumophila against windscreen wiper fluids (WWFs) | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 曾俊傑(Chun-Chieh Tseng),林菀俞(Wan-Yu Lin) | |
dc.subject.keyword | 成熟感染型嗜肺性退伍軍人菌,雨刷水清潔劑,抑菌實驗,具活性但不具可培養性,感染力,復生, | zh_TW |
dc.subject.keyword | mature infectious forms (MIFs) of Legionella pneumophila,windscreen wiper fluid,antibacterial activity,viable but nonculturable (VBNC),infectivity,resuscitation, | en |
dc.relation.page | 262 | |
dc.identifier.doi | 10.6342/NTU201903955 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-19 | |
dc.contributor.author-college | 公共衛生學院 | zh_TW |
dc.contributor.author-dept | 環境衛生研究所 | zh_TW |
顯示於系所單位: | 環境衛生研究所 |
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ntu-108-1.pdf 目前未授權公開取用 | 23.33 MB | Adobe PDF |
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