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Susceptability of mature infectious forms (MIFs) of Legionella pneumophila against windscreen wiper fluids (WWFs)
mature infectious forms (MIFs) of Legionella pneumophila,windscreen wiper fluid,antibacterial activity,viable but nonculturable (VBNC),infectivity,resuscitation,
|Publication Year :||2019|
|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。
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.
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