| 摘要: | 台灣由於灌排不分的關係，常使得灌溉渠道底泥與其周遭農地土壤容易因偷排而受到重金屬的污染。這些污染對環境所造成的衝擊雖然與所排出的重金屬總濃度有關，但更關鍵的是這些重金屬在特定環境條件下所呈現出的生物有效性，因生物有效性不僅可左右重金屬對於生態系統所帶來的毒性風險程度外，也已被懷疑可能與抗生素抗藥性在環境中的誘發與持續有關。有鑑於此，本研究即以不具czc鎘抗性基因的革蘭氏陰性菌E. coli K-12為模式生物，在調整培養液鎘濃度0.4 ppm中添加三種不同氯離子濃度(1, 50, 200 mM)以改變系統內的優勢鎘物種分佈後，並藉由量化諸如生長抑制、細胞死亡、胞內能量費用(AMP/ADP/ATP比例多寡有關的energy charge)和氧化還原平衡狀態(NAD+/NADH和GSH/GSSG比例有關的redox balance)等生理表徵(phenotypes)。在細胞生長曲線中1 mM [Cl-]有最長的遲滯期。除了使用細胞生長方式探討外，也使用較生物分子層面，如LIVE/DEAD螢光試劑染色細胞內核酸去探討暴露自由鎘離子的死活比，結果如同生長曲線在較多自由鎘離子情況下活菌有較有延遲生長現象，而在死菌部分各個時間點沒有差異(p>0.05)，原因為使用的鎘濃度為亞致死水平(sublethal level)，故不會造成死菌累積情形。並且在16小時細胞能量貨幣(ATP)含量以及計算出的腺苷酸能電荷(Adenylate energy charge, AEC)為1 mM [Cl-]最少，意味著自由鎘離子進入細胞後會影響ATP合成導致菌遲滯期延長現象，也和細胞培養實驗相呼應。量測細胞內氧化還原平衡NADH/NAD+，說明在暴露鎘後1小時內有較高的NADH/NAD+比例，並且1 mM [Cl-]之較多自由鎘離子又較高NADH/NAD+比例，所造成的氧化還原平衡差異更大。穀胱甘肽為細胞內維持細胞內氧化還原平衡方式有兩種(i)自身氧化成GSSG丟失電子去還原自由基；(ii)金屬穩態(homeostatic)方式，在自由鎘離子進入細胞內將會和帶有硫醇官能基的GSH錯合，而不是自身丟失電子氧化方式保護細胞，這使得有無暴露鎘細胞內GSH濃度均會隨時間含量減少，而暴露鎘之GSSG濃度不隨時間點改變，說明了自由鎘離子進入細胞後造成GSH的改變。研究的結果與假說相符：E. coli K-12對於鎘的攝取符合自由離子活性模型(Free ion activity model, FIAM)理論，即以自由型態的Cd2+為最主要被攝入的鎘物種，且當細胞在一定時間內攝取到較多的鎘時，除了造成遲滯期較長，也導致細胞內生物分子受影響，藉由上述種種影響，也假設會與共選擇效應較快誘發/產生對四環素的抗藥性/耐受力，首先發現在有預暴露鎘的實驗組其遲滯期較短和有較高的比生長速率，又在暴露較多自由鎘離子其遲滯期較短和有較高的比生長速率，說明了較多自由鎘離子為主要引起四環黴素耐藥性關鍵因子。推測是暴露重金屬鎘後會提升本菌株帶有的ZntA外排基因，並且減少OmpC孔蛋白的通透性，以產生這種對於四環黴素非特異性的耐藥機制，又在1 mM [Cl-]預暴露鎘0.4 ppm因為生物有效性緣故又有較高的耐藥性表現。本研究僅對一種金屬(Cd)與一種抗生素(Tet)做探討，且尚未觸及基因表徵(genotypes)的量化，因此此現象是否可概括性的擴及其它金屬與抗生素(generalizable)仍待進一步證實。即使如此，本研究結果仍可說明環境污染物彼此間的協同作用，也表明抗藥性問題的複雜度。;Due to the unique policy in Taiwan that water from channels directly receiving wastewater discharges can be used as the irrigation water, irrigation channel sediments and the irrigated farmland often easily get contaminated by heavy metals. While the impact of heavy metal pollution depends on the total concentration of heavy metals, it is more critical resulted from the bioavailability of these heavy metals under specific environmental conditions. This is because bioavailability not only is related to the extent of toxicity that heavy metals can cause to the ecosystem, but also has been suspected to contribute to the induction and persistence of antibiotic resistance in the environment. However, to date this untraditional route of environmental antibiotic resistance development has not been systematically investigated. In this study, the Gram-negative bacterium E. coli K-12 that does not harbor the genes encoded for the czc system was used as a model organism to probe the association between the bioavailability of cadmium and the co-selection of resistance towards tetracycline. Cadmium chemistry in the culture medium was manipulated by adjusting the chloride content (i.e., 1, 50, 200 mM) at a fixed total cadmium concentration (i.e., 0.4 ppm) so that a concentration gradient of free cadmium ion could be produced. The followings were observed after cell exposure to cadmium: (1) lower chloride concentrations resulted in more prolonged lag phase of growth; (2) within the 16-hr exposure, cultures grown at 1 mM chloride had the lowest cellular adenylate energy charge (AEC); (3) higher ratios of cellular NADH/NAD+ were measured in cultures exposed to cadmium for 1 hr than those grown in the absence of cadmium, and of them cells incubated with 1 mM chloride showed a relatively high ratio of NADH/NAD+; (4) while intracellular GSH levels continued to decrease in both Cd-exposed and Cd-unexposed cultures in a 2-hr incubation period, GSSG levels only increased in Cd-exposed cells. These results could be explained with the free ion activity model (FIAM) that the free ion form of cadmium was the most bioavailable cadmium, consistent with our hypothesis. Lastly, pre-exposure of K-12 cells to 0.4 ppm cadmium at 1 mM chloride did exhibit stronger tetracycline (Tet) tolerance than cells that were grown at 200 mM chloride. Given that this study only discusses one metal (Cd) and one antibiotic (Tet), and has not yet touched the resistance at the genetic level (genotypes), whether this phenomenon can be generalized to other metals and antibiotics remains to confirmed (i.e., the generalizable issue). Nonetheless, results of this study can demonstrate the synergy of environmental pollutants, and also indicate the complexity of environmental antibiotic resistance problems. |
