污泥蚯蚓堆肥對染色體和質粒上耐藥基因歸趨的影響

徐俊杰,夏 慧*,魏楓沂,陳 進,謝佳辰,黃 魁,2

污泥蚯蚓堆肥對染色體和質粒上耐藥基因歸趨的影響

徐俊杰1,夏 慧1*,魏楓沂1,陳 進1,謝佳辰1,黃 魁1,2

(1.蘭州交通大學環境與市政工程學院,甘肅 蘭州 730070；2.甘肅省黃河水環境重點實驗室,甘肅 蘭州 730070)

染色體和質粒分別介導污泥中的抗生素抗性基因(Antibiotic resistance genes, ARGs)進行垂直和水平轉移,使ARGs在親代或不同菌種之間傳播,導致污泥蚯蚓堆肥對ARGs的削減有限.為了解決這個問題,本實驗通過研究蚯蚓堆肥過程中染色體與質粒上ARGs和移動遺傳元件(Mobile genetic elements, MGEs)的豐度變化,以無添加蚯蚓為對照,進行20d的蚯蚓堆肥,探究蚯蚓堆肥對污泥中ARGs的垂直和水平轉移的影響.結果顯示:前10d是污泥蚯蚓堆肥中ARGs轉移的高峰期.除了,蚯蚓組其余ARGs豐度在質粒和染色體上均發生了顯著的增加(<0.05).與對照組相比,質粒上的、、、的豐度在蚯蚓組顯著增加了1.02倍、1.97倍、2.43倍、0.75倍(<0.05),而染色體上僅在蚯蚓組顯著增加(<0.05).對于MGEs,質粒上的1豐度在蚯蚓組中比對照組顯著增加了1.63倍(<0.05),而染色體上的卻截然相反,是對照組大于蚯蚓組.堆肥的后10d,兩組染色體和質粒中的MGEs和總ARGs的豐度均降低,且蚯蚓堆肥組降低速度更快.蚯蚓堆肥中,在質粒上MGEs與、、有顯著的正相關性(<0.05),而在染色體上MGEs與所有ARGs均無顯著相關性.冗余分析發現,ARGs的變化與MGEs、蚯蚓堆肥引起的環境變化有關,而且環境因素如電導率、有機質、氨氮和硝酸鹽氮,對質粒上ARGs和MGEs的影響比對染色體的更為強烈.綜上所述,攜帶MGEs的質粒介導的水平轉移是蚯蚓堆肥中ARGs難以削減的的主要原因.

抗生素；抗性基因；遺傳元件；剩余污泥；堆肥；蚯蚓

隨著我國城市污水處理規模的提升,剩余污泥產量也逐年增加[1].然而,由于長期的“重水輕泥”,污泥處理處置形勢非常嚴峻[2].污泥成分極為復雜,既含有碳、氮、磷等可利用物質,也含有重金屬、有機污染物、微塑料、ARGs等污染物[3]. ARGs為環境中新型生物污染物,污泥中已發現有360種ARGs[4].污水中的ARGs積蓄在污泥中,其去除效果甚微[5].因此,控制和減少污泥資源化過程中的ARGs的傳播和污染,成為亟待解決的問題.

蚯蚓堆肥是利用蚯蚓和微生物的協同作用,分解轉化污泥中難降解有機物的一種資源化技術,其成本低,操作簡單,可持續性處理污泥,同時蚯蚓糞富含較多的植物可利用的營養物質以及豐富的農業有益菌群,具有較高的市場價值[6-7].但是由于污泥來源的復雜性,蚯蚓堆肥對其中ARGs的削減并不顯著[8-9].Huang等[10]發現蚯蚓堆肥可以選擇性地清除剩余活性污泥中一些四環素和磺胺類抗性基因的相對豐度.Cui等[11]在蚓堆肥過程中發現喹諾酮類耐藥基因被顯著去除.然而也有報道污泥蚯蚓糞中、和的豐度在堆肥后顯著增加[12-13].對ARGs在蚯蚓污泥堆肥中的傳播認知不足,可能是難以控制ARGs的主要原因,因此研究ARGs在蚯蚓堆肥中的傳播機制,對有效減低污泥蚯蚓糞中ARGs的環境風險尤為重要.

由于抗生素的濫用,導致抗性致病菌甚至超級細菌的滋生,ARGs的產生和傳播擴散也成為一個備受矚目的公共安全問題.ARGs的擴散傳播分為垂直轉移與水平轉移.一方面,通過親代遺傳的垂直轉移對接合子的形成和ARGs的擴散有重要作用[14].另一方面,在不同菌種之間,ARGs通過質粒進行的接合轉移是水平轉移的主要方式[15-16].有研究表明質粒幾乎可以攜帶所有臨床相關的抗生素抗性基因[17-18].雖然染色體和質粒在環境中均可攜帶ARGs進行傳播,但區分染色體與質粒來研究ARGs極為鮮見.

本實驗通過研究污泥蚯蚓堆肥前后染色體與質粒上ARGs的豐度變化,探究蚯蚓堆肥對ARGs的轉移有何影響,為控制控制蚯蚓堆肥中ARGs的傳播提出新思路.

1 材料和方法

1.1 供試材料和實驗設置

實驗所用新鮮脫水污泥(含水率64.54%)取自蘭州市安寧區七里河污水廠,堆肥蚓種為赤子愛勝蚓(),經脫水污泥馴化7d后用于本實驗.實驗選用長方體塑料箱(58cm×38cm×25cm)作為堆肥反應器,供試污泥物理化學性質如表1所示.

表1 供試污泥物理化學性質

注:同列指標后存在相同字母表明兩兩之間不具有顯著差異性(>0.05),同行字母之間無比較意義.

使用5mm×5mm金屬方格網對新鮮脫水污泥進行造粒,然后在各反應器中投加12kg脫水污泥,并接種1200條蚯蚓(均重0.31g)開始堆肥實驗.以無添加蚯蚓為對照組,每組設3個平行,堆肥共進行20d.所有反應器都使用遮陽布覆蓋,并保持室溫(20～25℃).為了保持水分,每3d噴灑一次自來水.為保持有氧條件,每隔一周手動翻堆減少污泥顆粒積壓團聚.實驗第0d、10d、20d各取樣1次,每個反應器取2份樣品,一份自然風干后研磨,過60目篩,置于4℃冰箱中保存,用于理化性質分析;另一份新鮮樣品提取DNA,并置于-20℃冰箱中冷凍保存,用于DNA相關分析.

1.2 測定方法

1.2.1 理化性質分析 有機質含量采用恒重法,使用約2g新鮮樣品在105℃環境下12h以烘干水分得到干樣品,干樣品在650℃的馬弗爐中2h以測量有機物.將風干研磨樣品與去離子水1:50(質量濃度)混勻,磁力攪拌30min后測定pH值(雷磁PHS-3C,上海)和電導率(雷磁DDS-307,上海).硝酸鹽氮采用紫外分光光度法(HJ/T 346-2007),氨氮采用多參數水質分析儀(CNPN-7SII)測定.具體理化測試參照黃魁等[19]方法.

1.2.2 DNA和質粒提取及熒光定量PCR 取約0.25g新鮮污泥樣品用DNeasy?Power Soil?Kit(Qiagen,德國)試劑盒提取DNA,并用1%瓊脂糖凝膠電泳檢測其濃度,所得DNA樣品于-20℃冰箱保存備用.取25μL各樣品DNA,使用SanPrep柱式質粒DNA小量抽提試劑盒(生工,上海)提取質粒.選用6種常見的ARGs和2種MGEs進行定量,其中包括四環素類抗性基因(、)、大環內酯類抗性基因(、)、磺胺類抗性基因(、)以及整合子()和整座子(-)引物序列及條件見文獻[16].其中,和的耐藥機制是靶點替換,和是靶點改變,是抗生素滅活,是靶點保護.定量反應為25μL體系:SYBR Green (艾科瑞,湖南)12.5μL,10μmoL上下游引物各1μL,DNA模板1μL, DNA-free超純水9.5μL.所用引物均購置于生工生物工程(上海)股份有限公司,標準品為攜帶目的基因的質粒,詳細制備過程見文獻[20].

1.3 統計方法

使用Statistica 10.0統計軟件對樣品的理化性質、抗性基因數量在各組之間的差異進行單因素方差分析和相關性分析,顯著性水平為0.05.各處理組堆肥前后ARGs的豐度圖以及ARGs和MGEs之間的相關性熱圖使用OriginPro 2021繪制,用Canoco 4.5軟件對環境因子、MGEs和ARGs之間的關系進行冗余分析.

2 結果與討論

2.1 理化性質變化

蚯蚓堆肥引起的理化性質的變化不僅可以表征污泥穩定化效果,也有可能間接影響ARGs的豐度變化.有機質的降解和礦化作用可以直接反應蚯蚓堆肥過程中污泥的穩定性.由表1可知,堆肥第10d,對照組與蚯蚓組有機質含量比原污泥分別減少了11.40%和16.87%(<0.05).但堆肥的后10d,兩組有機質含量趨于穩定,甚至在蚯蚓組出現小幅上升.電導率是反映有機質礦化程度的重要指標[21].在堆肥的前10d,電導率并沒有因為有機質的降解而增加,反而出現下降趨勢,表明堆肥中有機質降解后并沒有及時轉化為礦物鹽或無機離子等物質.但在堆肥第20d,對照組與蚯蚓組電導率增加了32.58%和109.52%(<0.05).這表明在堆肥前期,有機質可能主要依靠蚯蚓的攝食作用降解為中間代謝產物或者大分子有機物等,而堆肥后期污泥中的微生物進一步降解生成小分子和無機鹽物質.蚯蚓堆肥能夠顯著加快污泥堆肥的礦化進程,與先前研究結果一致[22].

氨氮和硝酸鹽氮的含量是評估蚯蚓堆肥成熟度的重要指標.從表1可知,對照組和蚯蚓組的氨氮含量在前10d堆肥過程中持續下降,可能是堆肥初期的功能細菌AOA和AOB數量少且活性低,導致大量NH4+以NH3的形式損失[23].而堆肥的第20d,蚯蚓組比對照組氨氮顯著增加了75.97%(<0.05),可能是蚯蚓的鉆洞行為增加了污泥內部孔隙率并降低了厭氧率,從而促進了AOB的快速繁殖[19].堆肥前10d兩組的硝酸鹽氮含量持續增加但增幅較小,但后10d增加較快,堆肥前10d兩組的硝酸鹽氮增加較慢可能是由于堆肥前期蚯蚓攝食作用是主導者,微生物的作用并不占優勢.而隨著蚯蚓攝食、鉆洞等行為,為硝化細菌的生長提供了良好的環境并逐漸繁殖[24],從而導致后10d硝酸鹽氮快速增加.整個堆肥結束,對照組和蚯蚓組硝酸鹽氮含量分別從0增加到356.87mg/kg和1253.16mg/kg,蚯蚓組的硝酸鹽氮含量比對照組增加顯著(<0.05),表明由于蚯蚓活動促進硝化反應的進行.所以,蚯蚓堆肥能夠協同微生物促進有益氮循環,提高堆肥產物的利用價值.

2.2 ARGs豐度變化

由圖1(a)染色體中ARGs豐度所示,堆肥至第20d,對于大環內酯類ARGs,蚯蚓組的和分別增加了37.45%和21.94%,而在對照組中,減少了20.16%,但增加了18.90%.堆肥至第20d,四環素類的兩種ARGs呈現出截然不同的變化趨勢.在蚯蚓組和對照組中分別增加了9.36倍和1.58倍,兩組差異極為顯著(<0.001).在蚯蚓組和對照組中分別減少了66.16%和52.48%,兩組并無顯著差異.對于磺胺類的ARGs,堆肥第10d,在蚯蚓組和對照組中分別增加了2.32倍和2.18倍,則分別增加了2.64倍和2.36倍.堆肥的后,10d,緩慢增加,蚯蚓組和對照組分別增加了8.70%和0.76%,而在蚯蚓組和對照組分別顯著(<0.05)減少了20.84%和40.00%.

由圖1(b)質粒中ARGs豐度可知,大環內酯類的兩個ARGs在含量與變化趨勢方面都比較相似.堆肥至第10d,蚯蚓組的和分別顯著增加了94.63%(<0.01)和153.08%(<0.001),而在對照組中卻分別減少了6.85%和14.75%.堆肥至第20d,相比于原始污泥,在蚯蚓組和對照組分別減少了56.23%和81.13%,分別減少了57.91%和68.63%.對于四環素類ARGs,堆肥至第20d,和在對照組中分別減少了53.85%和78.57%,而在蚯蚓組,增加了4.15倍,減少了83.87%.磺胺類ARGs與大環內酯類相似,都呈現出先上升再下降的趨勢,堆肥至第20d,和在蚯蚓組中分別增加了104.27%(<0.01)和61.05%,而在對照組中基本沒變化.

圖1 污泥穩定化過程中各組的染色體(a)和質粒(b)上ARGs的絕對豐度

*<0.05, **<0.01, ***<0.001

結合圖1(a)和圖1(b)可知,同是四環素類ARGs的和呈現出截然不同的變化趨勢,可能是因為兩個抗性基因的抗性機制不同,是抗生素滅活基因,是靶點保護基因.值得注意的是磺胺類的兩種抗性基因和豐度最高,比其他ARGs大了3個數量級左右.Luo等[17]在海河中檢測ARGs也有類似的結果,這可能是由于磺胺類抗生素是人工合成的抑菌藥且成本低、抗菌譜廣、穩定性較高和親水性較強[25].結合耐藥機制看,污泥蚯蚓堆肥可能對靶點改變和靶點保護類ARGs削減效果較好,對靶點替換和抗生素滅活類ARGs削減效果較差.而本實驗ARGs數據量較少,對耐藥機制更深入的分析需要進一步的研究.

*<0.05

堆肥至第10d,蚯蚓組除了,其他ARGs在質粒和染色體中都發生了增加,且大多數ARGs較對照組增加顯著(<0.05).而堆肥的后10d,質粒上的ARGs除了,其余的均處于減少狀態,染色體上的ARGs除了和,其余的也均處于減少狀態.大多數ARGs出現先增加后減少的現象,可能是堆肥前10d蚯蚓作用比較活躍,ARGs在蚯蚓腸道中傳播比較劇烈,Cui等[11]在對剩余污泥進行蚯蚓堆肥時也發現了ARGs在前7d先增加后減少的現象.有研究表明,在蚯蚓胃中聚集的細菌群落會成為ARGs的受體[26].而后期ARGs減少可能是蚯蚓進食活動減少,細菌經過蚯蚓腸道厭氧環境的篩選,蚯蚓糞中多數為厭氧菌[27-28],排出體外后為好氧環境,厭氧菌受到抑制從而減少并影響了ARGs的傳播.由圖3可知,蚯蚓組對比對照組,質粒上的總ARGs和MGEs豐度在第10d和第20d分別顯著(<0.05)增加82.6%和77.9%,而染色體上的增加不顯著,表明蚯蚓堆肥中的ARGs很有可能是通過質粒傳播的.在染色體和質粒上,總ARGs和MGEs的豐度均表現為蚯蚓組大于對照組,并且除了,蚯蚓組其他的ARGs比對照組均有增加.表明蚯蚓堆肥難以對ARGs有效削減,這與Huang等[10]通過宏基因組分析脫水污泥蚯蚓堆肥中ARGs的研究結果并不相同,這可能與堆肥周期長短有關.

*<0.05

2.3 MGEs對ARGs的影響

整合子(Ⅰ1)是一種可移動的DNA分子,具有特殊的結構,可捕獲和整合外源性基因,特別是抗生素抗性基因、重金屬抗性基因等,使之轉變為功能性基因的表達單位[30-31].轉座子(-)也能攜帶其他功能性外源基因在染色體和質粒間轉移,其中以攜帶ARGs最為常見[32].所以MGEs在 ARGs的傳播中發揮著重要作用[33].由圖2可知,在染色體上,蚯蚓組的在堆肥的20d內持續增加,最終增加了1.37倍;而在對照組則是先增加后減少,最后比原污泥增加了2.64倍.-在蚯蚓組和對照組中均是先增加后減少,最終和原污泥相比基本上沒變化,兩組之間也并無差異(>0.05).兩個MGEs在堆肥第10d均發生了顯著增加,表明堆肥前10d可能是ARGs轉移傳播的高峰期.

在質粒上,堆肥至第10d,在蚯蚓組和對照組分別增加了2.61倍和0.37倍,兩組有顯著差異(<0.05);質粒上的-分別增加了43.73%和6.38%,兩組并無顯著差異.而堆肥結束后,相比原污泥,-在蚯蚓組和對照組分別減少了60.62%和70.63%,而在兩組的豐度基本和原污泥相等.

結合圖1和圖2,質粒中的和-先增加后減少的趨勢與大部分ARGs相同,且蚯蚓組豐度高于對照組,而染色體中MGEs的豐度則是對照組高于蚯蚓組.結果表明蚯蚓堆肥中質粒上的MGEs可能對ARGs的轉移貢獻度更大.

由圖4所見,在質粒上,對照組中與、、呈顯著正相關(<0.05),與有較強正相關(<0.01).而蚯蚓組中與、、有極為顯著的正相關(<0.001),與有較強正相關性(<0.01).該結果與Duan等[34]研究的ARGs的增多可能與堆肥產物中的豐度有關相符.對照組中-與、有顯著正相關(<0.05),與、、有極為顯著的正相關(<0.001).蚯蚓組中-與有顯著正相關(<0.05),與有較強正相關(<0.01),與、有極為顯著的正相關(<0.001).以上結果顯示,蚯蚓組的MGEs比對照組,與ARGs的相關性更加密切.蚯蚓組ARGs增加的比對照組更快,進一步表明蚯蚓堆肥體中ARGs的歸趨受MGEs的影響[35-36].在染色體上,蚯蚓組的和-與所有ARGs都無顯著正相關,說明MGEs對ARGs垂直轉移的幾乎無幫助.綜上所述,攜帶著MGEs的質粒所介導的水平轉移是蚯蚓堆肥中ARGs傳播的重要方式.

2.4 環境因子、MGEs和ARGs之間的關系

由圖5可知,堆肥至第10d,蚯蚓堆肥進程和、-在染色體和質粒中均呈現顯著正相關(<0.05),而且蚯蚓堆肥進程與質粒中的、、、,染色體中的、、都呈現顯著正相關(<0.05).而堆肥20d時,蚯蚓堆肥進程與上述指標幾乎無相關性.這個現象與先前的數據結合,ARGs和MGEs在蚯蚓堆肥第10d顯著增加,表明蚯蚓堆肥會增加ARGs傳播的風險.由圖5(b)可知,在ARGs激增的前10d,質粒中的ARGs與MGEs在蚯蚓堆肥組中比對照組中增加顯著,進一步證明蚯蚓堆肥中ARGs的傳播主要依靠的是質粒.質粒中的、、、和-與電導率、氨氮、硝酸鹽氮呈負相關,而染色體中的這些ARGs與環境因子之間相關性很低.這表明環境因素主要影響的是質粒介導的水平轉移[37],且電導率、氨氮和硝酸鹽氮可能會對ARGs和MGEs的豐度有一定的抑制作用.因此,在污泥蚯蚓堆肥中,進一步加快有益氮循環,既可以增加蚯蚓糞的可利用價值,又可能對質粒上ARGs和MGEs的削減有所幫助.

有研究表明,環境因子對ARGs豐度的影響主要取決于它們對其潛在宿主細菌的影響[38-39].同時,質粒和染色體中的和-與、、都呈顯著正相關(<0.05),表明這些ARGs可能在蚯蚓堆肥前10d借助MGEs快速傳播.進一步說明MGEs在蚯蚓堆肥過程中ARGs的傳播中發揮了重要作用[40].綜上所述,環境因子會強烈地影響蚯蚓堆肥時質粒中ARGs和MGEs的變化.因此如何調控蚯蚓堆肥體中環境因子來阻控質粒介導的水平轉移,有待進一步研究.

3 結論

3.1 蚯蚓堆肥的前10d是ARGs轉移的高峰期,而后10d,ARGs的豐度會下降.

3.2 蚯蚓堆肥引起的環境因子變化會影響ARGs和MGEs的豐度,從而影響ARGs的水平轉移.

3.3 質粒上的MGEs與ARGs豐度變化呈顯著正相關,表明攜帶MGEs的質粒在ARGs的水平轉移中起著至關重要的作用.

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Fate of antibiotic resistance genes on chromosomes and plasmids affected by earthworms during vermicomposting of dewatered sludge.

XU Jun-jie1, XIA Hui1*, WEI Feng-yi1, CHEN Jin1, XIE Jia-chen1, HUANG Kui1,2

(1.School of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China；2.Key laboratory of Yellow River Water Environment in Gansu Province, Lanzhou 730070, China)., 2023,43(2)：694～701

Chromosomes and plasmids mediate the vertical and horizontal transfer of antibiotic resistance genes (ARGs) in sludge, respectively, which allows ARGs to spread between parents or different bacteria strains, resulting in limited reduction of ARGs during vermicomposting. To address this issue, the effects of vermicomposting on vertical and horizontal transfer of ARGs in sludge were investigated by detecting the abundance changes of ARGs and mobile genetic elements (MGEs) on chromosomes and plasmids during vermicomposting for 20 days, with no addition of earthworms as the control. The results showed that the first 10d was the peak of ARGs transfer in sludge vermicomposting. Except forgene, a significant increase in the abundance of the remaining ARGs in the vermicomposting occurred on both plasmids and chromosomes (<0.05). Compared with the control, the gene abundances of,,, and1on plasmids significantly increased by 1.02-fold, 1.97-fold, 2.43-fold, and 0.75-fold in the vermicomposting (<0.05), while onlyon chromosomes significantly increased (<0.05). Compared with the control, the MGEs abundance of1 on plasmids significantly enriched by 1.63-fold in the vermicomposting (<0.05), while its abundance on chromosomes was diametrically opposite, its abundance in the control was larger than vermicomposting. In the 10～20 d of composting, the abundance of MGEs and total ARGs on chromosomes and plasmids decreased in both treatments, with a faster decrease in the vermicomposting. In addition, the MGEs had a significant positive correlation (<0.05) with,, and2 on plasmids, while no significant correlation among MGEs and all ARGs on chromosomes was recorded during vermicomposting. The redundancy analysis revealed that the changes of ARGs were related to the MGEs and environmental changes during vermicomposting, and the environmental factors such as conductivity, organic matter, ammonia and nitrate had a stronger effect on ARGs and MGEs on plasmids than those on chromosomes. This study suggests that the plasmids carrying MGEs mediated horizontal transfer is a major reason for hardly reducing ARGs in sludge vermicompost.

antibiotic；resistance gene；mobile genetic element；excesssludge；composting；earthworms

X171.5

A

1000-6923(2023)02-0694-08

徐俊杰(1998-),男,安徽宿州人,蘭州交通大學碩士研究生,主要研究污泥資源化技術.發表論文4篇.

2022-07-01

國家自然科學基金資助項目(51868036;52000095);甘肅省科技計劃資助項目(20JR2RA002);甘肅省優秀研究生“創新之星”項目(2022CXZX-559)

* 責任作者, 副教授, xiahui@mail.lzjtu.cn