徐俊杰,夏 慧*,魏楓沂,陳 進(jìn),謝佳辰,黃 魁,2

污泥蚯蚓堆肥對(duì)染色體和質(zhì)粒上耐藥基因歸趨的影響

徐俊杰1,夏 慧1*,魏楓沂1,陳 進(jìn)1,謝佳辰1,黃 魁1,2

(1.蘭州交通大學(xué)環(huán)境與市政工程學(xué)院,甘肅 蘭州 730070；2.甘肅省黃河水環(huán)境重點(diǎn)實(shí)驗(yàn)室,甘肅 蘭州 730070)

染色體和質(zhì)粒分別介導(dǎo)污泥中的抗生素抗性基因(Antibiotic resistance genes, ARGs)進(jìn)行垂直和水平轉(zhuǎn)移,使ARGs在親代或不同菌種之間傳播,導(dǎo)致污泥蚯蚓堆肥對(duì)ARGs的削減有限.為了解決這個(gè)問題,本實(shí)驗(yàn)通過研究蚯蚓堆肥過程中染色體與質(zhì)粒上ARGs和移動(dòng)遺傳元件(Mobile genetic elements, MGEs)的豐度變化,以無(wú)添加蚯蚓為對(duì)照,進(jìn)行20d的蚯蚓堆肥,探究蚯蚓堆肥對(duì)污泥中ARGs的垂直和水平轉(zhuǎn)移的影響.結(jié)果顯示:前10d是污泥蚯蚓堆肥中ARGs轉(zhuǎn)移的高峰期.除了,蚯蚓組其余ARGs豐度在質(zhì)粒和染色體上均發(fā)生了顯著的增加(<0.05).與對(duì)照組相比,質(zhì)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(duì)于MGEs,質(zhì)粒上的1豐度在蚯蚓組中比對(duì)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(duì)照組大于蚯蚓組.堆肥的后10d,兩組染色體和質(zhì)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(zhì)粒上MGEs與、、有顯著的正相關(guān)性(<0.05),而在染色體上MGEs與所有ARGs均無(wú)顯著相關(guān)性.冗余分析發(fā)現(xiàn),ARGs的變化與MGEs、蚯蚓堆肥引起的環(huán)境變化有關(guān),而且環(huán)境因素如電導(dǎo)率、有機(jī)質(zhì)、氨氮和硝酸鹽氮,對(duì)質(zhì)粒上ARGs和MGEs的影響比對(duì)染色體的更為強(qiáng)烈.綜上所述,攜帶MGEs的質(zhì)粒介導(dǎo)的水平轉(zhuǎn)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(guó)城市污水處理規(guī)模的提升,剩余污泥產(chǎn)量也逐年增加[1].然而,由于長(zhǎng)期的“重水輕泥”,污泥處理處置形勢(shì)非常嚴(yán)峻[2].污泥成分極為復(fù)雜,既含有碳、氮、磷等可利用物質(zhì),也含有重金屬、有機(jī)污染物、微塑料、ARGs等污染物[3]. ARGs為環(huán)境中新型生物污染物,污泥中已發(fā)現(xiàn)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(xié)同作用,分解轉(zhuǎn)化污泥中難降解有機(jī)物的一種資源化技術(shù),其成本低,操作簡(jiǎn)單,可持續(xù)性處理污泥,同時(shí)蚯蚓糞富含較多的植物可利用的營(yíng)養(yǎng)物質(zhì)以及豐富的農(nóng)業(yè)有益菌群,具有較高的市場(chǎng)價(jià)值[6-7].但是由于污泥來(lái)源的復(fù)雜性,蚯蚓堆肥對(duì)其中ARGs的削減并不顯著[8-9].Huang等[10]發(fā)現(xiàn)蚯蚓堆肥可以選擇性地清除剩余活性污泥中一些四環(huán)素和磺胺類抗性基因的相對(duì)豐度.Cui等[11]在蚓堆肥過程中發(fā)現(xiàn)喹諾酮類耐藥基因被顯著去除.然而也有報(bào)道污泥蚯蚓糞中、和的豐度在堆肥后顯著增加[12-13].對(duì)ARGs在蚯蚓污泥堆肥中的傳播認(rèn)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(jī)制,對(duì)有效減低污泥蚯蚓糞中ARGs的環(huán)境風(fēng)險(xiǎn)尤為重要.

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

本實(shí)驗(yàn)通過研究污泥蚯蚓堆肥前后染色體與質(zhì)粒上ARGs的豐度變化,探究蚯蚓堆肥對(duì)ARGs的轉(zhuǎn)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(shí)驗(yàn)設(shè)置

實(shí)驗(yàn)所用新鮮脫水污泥(含水率64.54%)取自蘭州市安寧區(qū)七里河污水廠,堆肥蚓種為赤子愛勝蚓(),經(jīng)脫水污泥馴化7d后用于本實(shí)驗(yàn).實(shí)驗(yàn)選用長(zhǎng)方體塑料箱(58cm×38cm×25cm)作為堆肥反應(yīng)器,供試污泥物理化學(xué)性質(zhì)如表1所示.

表1 供試污泥物理化學(xué)性質(zhì)

注:同列指標(biāo)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(wú)比較意義.

使用5mm×5mm金屬方格網(wǎng)對(duì)新鮮脫水污泥進(jìn)行造粒,然后在各反應(yīng)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(shí)驗(yàn).以無(wú)添加蚯蚓為對(duì)照組,每組設(shè)3個(gè)平行,堆肥共進(jìn)行20d.所有反應(yīng)器都使用遮陽(yáng)布覆蓋,并保持室溫(20～25℃).為了保持水分,每3d噴灑一次自來(lái)水.為保持有氧條件,每隔一周手動(dòng)翻堆減少污泥顆粒積壓團(tuán)聚.實(shí)驗(yàn)第0d、10d、20d各取樣1次,每個(gè)反應(yīng)器取2份樣品,一份自然風(fēng)干后研磨,過60目篩,置于4℃冰箱中保存,用于理化性質(zhì)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(guān)分析.

1.2 測(cè)定方法

1.2.1 理化性質(zhì)分析 有機(jī)質(zhì)含量采用恒重法,使用約2g新鮮樣品在105℃環(huán)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(cè)量有機(jī)物.將風(fēng)干研磨樣品與去離子水1:50(質(zhì)量濃度)混勻,磁力攪拌30min后測(cè)定pH值(雷磁PHS-3C,上海)和電導(dǎo)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(shù)水質(zhì)分析儀(CNPN-7SII)測(cè)定.具體理化測(cè)試參照黃魁等[19]方法.

1.2.2 DNA和質(zhì)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(guó))試劑盒提取DNA,并用1%瓊脂糖凝膠電泳檢測(cè)其濃度,所得DNA樣品于-20℃冰箱保存?zhèn)溆?取25μL各樣品DNA,使用SanPrep柱式質(zhì)粒DNA小量抽提試劑盒(生工,上海)提取質(zhì)粒.選用6種常見的ARGs和2種MGEs進(jìn)行定量,其中包括四環(huán)素類抗性基因(、)、大環(huán)內(nèi)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(xiàn)[16].其中,和的耐藥機(jī)制是靶點(diǎn)替換,和是靶點(diǎn)改變,是抗生素滅活,是靶點(diǎn)保護(hù).定量反應(yīng)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(gòu)置于生工生物工程(上海)股份有限公司,標(biāo)準(zhǔn)品為攜帶目的基因的質(zhì)粒,詳細(xì)制備過程見文獻(xiàn)[20].

1.3 統(tǒng)計(jì)方法

使用Statistica 10.0統(tǒng)計(jì)軟件對(duì)樣品的理化性質(zhì)、抗性基因數(shù)量在各組之間的差異進(jìn)行單因素方差分析和相關(guān)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(guān)性熱圖使用OriginPro 2021繪制,用Canoco 4.5軟件對(duì)環(huán)境因子、MGEs和ARGs之間的關(guān)系進(jìn)行冗余分析.

2 結(jié)果與討論

2.1 理化性質(zhì)變化

蚯蚓堆肥引起的理化性質(zhì)的變化不僅可以表征污泥穩(wěn)定化效果,也有可能間接影響ARGs的豐度變化.有機(jī)質(zhì)的降解和礦化作用可以直接反應(yīng)蚯蚓堆肥過程中污泥的穩(wěn)定性.由表1可知,堆肥第10d,對(duì)照組與蚯蚓組有機(jī)質(zhì)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(jī)質(zhì)含量趨于穩(wěn)定,甚至在蚯蚓組出現(xiàn)小幅上升.電導(dǎo)率是反映有機(jī)質(zhì)礦化程度的重要指標(biāo)[21].在堆肥的前10d,電導(dǎo)率并沒有因?yàn)橛袡C(jī)質(zhì)的降解而增加,反而出現(xiàn)下降趨勢(shì),表明堆肥中有機(jī)質(zhì)降解后并沒有及時(shí)轉(zhuǎn)化為礦物鹽或無(wú)機(jī)離子等物質(zhì).但在堆肥第20d,對(duì)照組與蚯蚓組電導(dǎo)率增加了32.58%和109.52%(<0.05).這表明在堆肥前期,有機(jī)質(zhì)可能主要依靠蚯蚓的攝食作用降解為中間代謝產(chǎn)物或者大分子有機(jī)物等,而堆肥后期污泥中的微生物進(jìn)一步降解生成小分子和無(wú)機(jī)鹽物質(zhì).蚯蚓堆肥能夠顯著加快污泥堆肥的礦化進(jìn)程,與先前研究結(jié)果一致[22].

氨氮和硝酸鹽氮的含量是評(píng)估蚯蚓堆肥成熟度的重要指標(biāo).從表1可知,對(duì)照組和蚯蚓組的氨氮含量在前10d堆肥過程中持續(xù)下降,可能是堆肥初期的功能細(xì)菌AOA和AOB數(shù)量少且活性低,導(dǎo)致大量NH4+以NH3的形式損失[23].而堆肥的第20d,蚯蚓組比對(duì)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(nèi)部孔隙率并降低了厭氧率,從而促進(jìn)了AOB的快速繁殖[19].堆肥前10d兩組的硝酸鹽氮含量持續(xù)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(dǎo)者,微生物的作用并不占優(yōu)勢(shì).而隨著蚯蚓攝食、鉆洞等行為,為硝化細(xì)菌的生長(zhǎng)提供了良好的環(huán)境并逐漸繁殖[24],從而導(dǎo)致后10d硝酸鹽氮快速增加.整個(gè)堆肥結(jié)束,對(duì)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(duì)照組增加顯著(<0.05),表明由于蚯蚓活動(dòng)促進(jìn)硝化反應(yīng)的進(jìn)行.所以,蚯蚓堆肥能夠協(xié)同微生物促進(jìn)有益氮循環(huán),提高堆肥產(chǎn)物的利用價(jià)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(duì)于大環(huán)內(nèi)酯類ARGs,蚯蚓組的和分別增加了37.45%和21.94%,而在對(duì)照組中,減少了20.16%,但增加了18.90%.堆肥至第20d,四環(huán)素類的兩種ARGs呈現(xiàn)出截然不同的變化趨勢(shì).在蚯蚓組和對(duì)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(duì)照組中分別減少了66.16%和52.48%,兩組并無(wú)顯著差異.對(duì)于磺胺類的ARGs,堆肥第10d,在蚯蚓組和對(duì)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(duì)照組分別增加了8.70%和0.76%,而在蚯蚓組和對(duì)照組分別顯著(<0.05)減少了20.84%和40.00%.

由圖1(b)質(zhì)粒中ARGs豐度可知,大環(huán)內(nèi)酯類的兩個(gè)ARGs在含量與變化趨勢(shì)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(duì)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(duì)照組分別減少了56.23%和81.13%,分別減少了57.91%和68.63%.對(duì)于四環(huán)素類ARGs,堆肥至第20d,和在對(duì)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(huán)內(nèi)酯類相似,都呈現(xiàn)出先上升再下降的趨勢(shì),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(duì)照組中基本沒變化.

圖1 污泥穩(wěn)定化過程中各組的染色體(a)和質(zhì)粒(b)上ARGs的絕對(duì)豐度

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

結(jié)合圖1(a)和圖1(b)可知,同是四環(huán)素類ARGs的和呈現(xiàn)出截然不同的變化趨勢(shì),可能是因?yàn)閮蓚€(gè)抗性基因的抗性機(jī)制不同,是抗生素滅活基因,是靶點(diǎn)保護(hù)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(gè)數(shù)量級(jí)左右.Luo等[17]在海河中檢測(cè)ARGs也有類似的結(jié)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(wěn)定性較高和親水性較強(qiáng)[25].結(jié)合耐藥機(jī)制看,污泥蚯蚓堆肥可能對(duì)靶點(diǎn)改變和靶點(diǎn)保護(hù)類ARGs削減效果較好,對(duì)靶點(diǎn)替換和抗生素滅活類ARGs削減效果較差.而本實(shí)驗(yàn)ARGs數(shù)據(jù)量較少,對(duì)耐藥機(jī)制更深入的分析需要進(jìn)一步的研究.

*<0.05

堆肥至第10d,蚯蚓組除了,其他ARGs在質(zhì)粒和染色體中都發(fā)生了增加,且大多數(shù)ARGs較對(duì)照組增加顯著(<0.05).而堆肥的后10d,質(zhì)粒上的ARGs除了,其余的均處于減少狀態(tài),染色體上的ARGs除了和,其余的也均處于減少狀態(tài).大多數(shù)ARGs出現(xiàn)先增加后減少的現(xiàn)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(duì)剩余污泥進(jìn)行蚯蚓堆肥時(shí)也發(fā)現(xiàn)了ARGs在前7d先增加后減少的現(xiàn)象.有研究表明,在蚯蚓胃中聚集的細(xì)菌群落會(huì)成為ARGs的受體[26].而后期ARGs減少可能是蚯蚓進(jìn)食活動(dòng)減少,細(xì)菌經(jīng)過蚯蚓腸道厭氧環(huán)境的篩選,蚯蚓糞中多數(shù)為厭氧菌[27-28],排出體外后為好氧環(huán)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(duì)比對(duì)照組,質(zhì)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(zhì)粒傳播的.在染色體和質(zhì)粒上,總ARGs和MGEs的豐度均表現(xiàn)為蚯蚓組大于對(duì)照組,并且除了,蚯蚓組其他的ARGs比對(duì)照組均有增加.表明蚯蚓堆肥難以對(duì)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(jié)果并不相同,這可能與堆肥周期長(zhǎng)短有關(guān).

*<0.05

2.3 MGEs對(duì)ARGs的影響

整合子(Ⅰ1)是一種可移動(dòng)的DNA分子,具有特殊的結(jié)構(gòu),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(zhuǎn)變?yōu)楣δ苄曰虻谋磉_(dá)單位[30-31].轉(zhuǎn)座子(-)也能攜帶其他功能性外源基因在染色體和質(zhì)粒間轉(zhuǎn)移,其中以攜帶ARGs最為常見[32].所以MGEs在 ARGs的傳播中發(fā)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(nèi)持續(xù)增加,最終增加了1.37倍;而在對(duì)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(duì)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(wú)差異(>0.05).兩個(gè)MGEs在堆肥第10d均發(fā)生了顯著增加,表明堆肥前10d可能是ARGs轉(zhuǎn)移傳播的高峰期.

在質(zhì)粒上,堆肥至第10d,在蚯蚓組和對(duì)照組分別增加了2.61倍和0.37倍,兩組有顯著差異(<0.05);質(zhì)粒上的-分別增加了43.73%和6.38%,兩組并無(wú)顯著差異.而堆肥結(jié)束后,相比原污泥,-在蚯蚓組和對(duì)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(jié)合圖1和圖2,質(zhì)粒中的和-先增加后減少的趨勢(shì)與大部分ARGs相同,且蚯蚓組豐度高于對(duì)照組,而染色體中MGEs的豐度則是對(duì)照組高于蚯蚓組.結(jié)果表明蚯蚓堆肥中質(zhì)粒上的MGEs可能對(duì)ARGs的轉(zhuǎn)移貢獻(xiàn)度更大.

由圖4所見,在質(zhì)粒上,對(duì)照組中與、、呈顯著正相關(guān)(<0.05),與有較強(qiáng)正相關(guān)(<0.01).而蚯蚓組中與、、有極為顯著的正相關(guān)(<0.001),與有較強(qiáng)正相關(guān)性(<0.01).該結(jié)果與Duan等[34]研究的ARGs的增多可能與堆肥產(chǎn)物中的豐度有關(guān)相符.對(duì)照組中-與、有顯著正相關(guān)(<0.05),與、、有極為顯著的正相關(guān)(<0.001).蚯蚓組中-與有顯著正相關(guān)(<0.05),與有較強(qiáng)正相關(guān)(<0.01),與、有極為顯著的正相關(guān)(<0.001).以上結(jié)果顯示,蚯蚓組的MGEs比對(duì)照組,與ARGs的相關(guān)性更加密切.蚯蚓組ARGs增加的比對(duì)照組更快,進(jìn)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(wú)顯著正相關(guān),說(shuō)明MGEs對(duì)ARGs垂直轉(zhuǎn)移的幾乎無(wú)幫助.綜上所述,攜帶著MGEs的質(zhì)粒所介導(dǎo)的水平轉(zhuǎn)移是蚯蚓堆肥中ARGs傳播的重要方式.

2.4 環(huán)境因子、MGEs和ARGs之間的關(guān)系

由圖5可知,堆肥至第10d,蚯蚓堆肥進(jìn)程和、-在染色體和質(zhì)粒中均呈現(xiàn)顯著正相關(guān)(<0.05),而且蚯蚓堆肥進(jìn)程與質(zhì)粒中的、、、,染色體中的、、都呈現(xiàn)顯著正相關(guān)(<0.05).而堆肥20d時(shí),蚯蚓堆肥進(jìn)程與上述指標(biāo)幾乎無(wú)相關(guān)性.這個(gè)現(xiàn)象與先前的數(shù)據(jù)結(jié)合,ARGs和MGEs在蚯蚓堆肥第10d顯著增加,表明蚯蚓堆肥會(huì)增加ARGs傳播的風(fēng)險(xiǎn).由圖5(b)可知,在ARGs激增的前10d,質(zhì)粒中的ARGs與MGEs在蚯蚓堆肥組中比對(duì)照組中增加顯著,進(jìn)一步證明蚯蚓堆肥中ARGs的傳播主要依靠的是質(zhì)粒.質(zhì)粒中的、、、和-與電導(dǎo)率、氨氮、硝酸鹽氮呈負(fù)相關(guān),而染色體中的這些ARGs與環(huán)境因子之間相關(guān)性很低.這表明環(huán)境因素主要影響的是質(zhì)粒介導(dǎo)的水平轉(zhuǎn)移[37],且電導(dǎo)率、氨氮和硝酸鹽氮可能會(huì)對(duì)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(jìn)一步加快有益氮循環(huán),既可以增加蚯蚓糞的可利用價(jià)值,又可能對(duì)質(zhì)粒上ARGs和MGEs的削減有所幫助.

有研究表明,環(huán)境因子對(duì)ARGs豐度的影響主要取決于它們對(duì)其潛在宿主細(xì)菌的影響[38-39].同時(shí),質(zhì)粒和染色體中的和-與、、都呈顯著正相關(guān)(<0.05),表明這些ARGs可能在蚯蚓堆肥前10d借助MGEs快速傳播.進(jìn)一步說(shuō)明MGEs在蚯蚓堆肥過程中ARGs的傳播中發(fā)揮了重要作用[40].綜上所述,環(huán)境因子會(huì)強(qiáng)烈地影響蚯蚓堆肥時(shí)質(zhì)粒中ARGs和MGEs的變化.因此如何調(diào)控蚯蚓堆肥體中環(huán)境因子來(lái)阻控質(zhì)粒介導(dǎo)的水平轉(zhuǎn)移,有待進(jìn)一步研究.

3 結(jié)論

3.1 蚯蚓堆肥的前10d是ARGs轉(zhuǎn)移的高峰期,而后10d,ARGs的豐度會(huì)下降.

3.2 蚯蚓堆肥引起的環(huán)境因子變化會(huì)影響ARGs和MGEs的豐度,從而影響ARGs的水平轉(zhuǎn)移.

3.3 質(zhì)粒上的MGEs與ARGs豐度變化呈顯著正相關(guān),表明攜帶MGEs的質(zhì)粒在ARGs的水平轉(zhuǎn)移中起著至關(guān)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(xué)碩士研究生,主要研究污泥資源化技術(shù).發(fā)表論文4篇.

2022-07-01

國(guó)家自然科學(xué)基金資助項(xiàng)目(51868036;52000095);甘肅省科技計(jì)劃資助項(xiàng)目(20JR2RA002);甘肅省優(yōu)秀研究生“創(chuàng)新之星”項(xiàng)目(2022CXZX-559)

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