紫外光波長及自由基對於降解細胞外抗生素抗性基因之影響

Impact of wavelength and free radical on degradation of a plasmid-encoded extracellular antibiotic resistance gene by UV/Chlorine processes

王思云(Szu-Yun Wang)

指導教授 ： 童心欣

國立臺灣大學/工學院/環境工程學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101529

摘要

抗生素抗藥性基因(antibiotic resistance genes, ARGs)於近來年開始被視為一種新興汙染物(emerging contaminants)，由於人類的大量使用，導致抗生素抗性基因已於各種環境被檢測到，特別是水生環境中。抗生素抗藥性基因可能會透過水平基因轉移增強抗生素抗性於非抗藥性細菌菌群中的傳播，因此如何去除水中的抗藥性抗性基因便成為一大研究課題。本研究將使用紫外光結合自由餘氯之高級氧化處理程序，評估其對於所選定之目標基因–帶有四環黴素及氨芐青黴素抗性的質體pWH1266的降解效果，以探討使用不同紫外光波長做為高級氧化處理之紫外光光源下對於細胞外抗生素抗性基因之降解效果，以及自由基在降解抗生素抗性基因中的貢獻度，並透過即時聚合酶連鎖反應偵測抗生素抗性基因在經過高級氧化處理後基因之完整性以定量抗生素抗性基因以及使用塗盤培養法測試基因在受到損傷後的自然轉形能力是否受到抑制。透過批次式實驗的結果可以發現不論是以處理程序最終可達成之log reduction還是一階反應動力常數k，LPUV (254 nm)/Chlorine對於抗生素抗性基因降解之效果優於UV-C LED (275 nm)/Chlorine，表示在本研究中波長254 nm之紫外光更適合做為UV/Chlorine AOP的紫外光光源；當在UV/Chlorine AOP中添加scavenger以去除系統中的ROS或RCS後可發現LPUV/Chlorine和UV-C/Chlorine對於降解抗生素抗性基因的效果在短擴增子中兩者皆下降了20%，在長擴增子則分別下降了20%及25~30%，且ROS對基因降解佔了大部分的貢獻度，RCS則只佔了小部分的貢獻度；而於自然轉形抑制實驗中則可發現抗生素抗性基因完整性的破壞確實可以抑制自然轉形的發生。

關鍵字

抗生素抗性基因 ； 高級氧化處理 ； 紫外光波長 ； 水平基因轉移 ； 自然轉形 ； 自由基

並列摘要

Antibiotic resistance genes (ARGs) is considered to be a kind of emerging contaminants in recent years. Owing to the massive use of humans, antibiotic resistance genes have been detected in various environments, especially in aquatic environments. The spread of antibiotic resistance amongst non-resistant bacterial communities may enhanced by horizontal gene transfer. Therefore, it is important to remove antibiotic resistance genes in aquatic environments. This research was aimed to evaluate the degradation of gene pWH1266 which contains ampicillin and tetracycline resistance genes by UV/Chlorine AOP with different ultraviolet wavelengths. In addition, the effects of different free radicals such as reactive oxygen species (ROS) and reactive chlorine species (RCS) on antibiotic resistance genes degradation were also explored. Real-time quantitative PCR (qPCR) and culture-base method were used to detect the damage of antibiotic resistance genes from UV/Chlorine AOP. Furthermore, the impact on nature transformation from UV/Chlorine AOP was verified by culture-based method with Acinetobacter baylyi strain AC811. The results showed that the log reductions and the first-order reaction kinetic constants for LPUV (254 nm)/Chlorine were better than that for UV-C LED (275 nm)/Chlorine. Therefore, ultraviolet light with a wavelength of 254 nm may be more suitable for ARGs degradation by UV/Chlorine AOP. When the radical scavengers were added to the UV/Chlorine AOP experiments, the degradation efficiencies of ARGs short amplicons were reduced by 20% for both LPUV/Chlorine and UV-C LED/Chlorine process. For long amplicons, the degradation efficiencies were reduced by 20% and 25~30% from LPUV/Chlorine and UV-C LED/Chlorine process, respectively. In addition, the ROS was accounted for the highest contribution to ARGs degradation. And, the RCS only played a minor role in the UV/Chlorine AOP. From the nature transformation assays, the UV/Chlorine AOP reduced that natural transformation efficiencies successfully.

並列關鍵字

Antibiotic resistance genes (ARGs) ； Advanced oxidation process (AOPs) ； Ultraviolet wavelengths ； Horizontal gene transfer ； nature transformation ； free radicals

參考文獻

Ahmed, Y., Lu, J., Yuan, Z., Bond, P. L., Guo, J. (2020). Efficient inactivation of antibiotic resistant bacteria and antibiotic resistance genes by photo-Fenton process under visible LED light and neutral pH. Water research, 179, 115878.

Albrich, J. M., McCarthy, C. A., Hurst, J. K. (1981). Biological reactivity of hypochlorous acid: implications for microbicidal mechanisms of leukocyte myeloperoxidase. Proceedings of the National Academy of Sciences, 78(1), 210-214.

Aslam, B., Wang, W., Arshad, M. I., Khurshid, M., Muzammil, S., Rasool, M. H., Nisar, M. A., Alvi, R. F., Aslam, M. A., Qamar, M. U. (2018). Antibiotic resistance: a rundown of a global crisis. Infection and drug resistance, 11, 1645.

Augsburger, N., Mantilla-Calderon, D., Daffonchio, D., Hong, P.-Y. (2019). Acquisition of Extracellular DNA by Acinetobacter baylyi ADP1 in response to Solar and UV-C254nm Disinfection. Environmental science technology, 53(17), 10312-10319.

Barbe, V., Vallenet, D., Fonknechten, N., Kreimeyer, A., Oztas, S., Labarre, L., Cruveiller, S., Robert, C., Duprat, S., Wincker, P. (2004). Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic acids research, 32(19), 5766-5779.