氯和紫外消毒過(guò)程中胞外抗性基因的產(chǎn)生特征

付樹(shù)森,王 藝,王肖霖,王尚杰,程 遠(yuǎn),卞 博,張生博,袁青彬

氯和紫外消毒過(guò)程中胞外抗性基因的產(chǎn)生特征

付樹(shù)森,王 藝,王肖霖,王尚杰,程 遠(yuǎn),卞 博,張生博,袁青彬*

(南京工業(yè)大學(xué)環(huán)境科學(xué)與工程學(xué)院,江蘇 南京 211816)

考察了城市污水氯和紫外消毒過(guò)程中不同物理形態(tài)的胞外抗性基因的產(chǎn)生行為與及微生物群落的關(guān)聯(lián)特征.結(jié)果表明,氯消毒盡管使胞內(nèi)抗性基因豐度下降,但使結(jié)合型胞外抗性基因豐度明顯上升(0.7±0.1) log,而游離型胞外抗性基因豐度下降(0.2±0.1)log.紫外消毒也使胞內(nèi)抗性基因下降,但使游離型胞外抗性基因顯著上升(0.4±0.2) log,而結(jié)合型胞外抗性基因豐度下降(0.3±0.1) log.氯消毒后, 結(jié)合型胞外DNA(a-eDNA)中變形菌門(mén)豐度下降而其他菌門(mén)的豐度上升,細(xì)菌多樣性指數(shù)由4.2上升到4.7;而游離型胞外DNA(f-eDNA)中變形菌門(mén)上升了6.6%,多樣性指數(shù)則從3.5降低到2.8.紫外消毒后,a-eDNA中變形菌門(mén)豐度下降了36.6%,多樣性則上升到4.8,而f-eDNA中細(xì)菌豐度變化較小.分子生態(tài)網(wǎng)絡(luò)分析揭示了抗性基因與細(xì)菌間廣泛的寄存關(guān)系,、、和分別與17、15、15和5種菌屬間存在共現(xiàn)性,表明抗性基因潛在宿主的變化是導(dǎo)致消毒后胞外抗性基因產(chǎn)生的關(guān)鍵原因.本研究表明氯和紫外消毒不能消除抗性基因風(fēng)險(xiǎn),反而通過(guò)導(dǎo)致不同胞外抗性基因的大量產(chǎn)生,使風(fēng)險(xiǎn)的形式發(fā)生變化.

氯消毒；紫外消毒；胞外抗性基因；細(xì)菌群落；分子生態(tài)網(wǎng)絡(luò)

全球范圍抗生素的大量使用極大加劇了細(xì)菌抗藥性的發(fā)展,已成為威脅公共健康的嚴(yán)峻問(wèn)題.抗性基因被列為一種新興污染物[1],在河流[2]、空氣[3]和土壤[4]等各種環(huán)境中大量檢出.其中污水處理廠作為各種廢水的匯集地,已檢出數(shù)百種抗生素抗性基因(ARG)[5-7],成為環(huán)境中抗性基因的主要儲(chǔ)存庫(kù).作為生物污染的主要控制單元,以氯和紫外消毒為代表的污水消毒過(guò)程對(duì)ARG的去除效果也受到廣泛關(guān)注.很多研究發(fā)現(xiàn)氯和紫外消毒不能有效地去除ARG[8-12],去除效果遠(yuǎn)低于其宿主耐藥細(xì)菌[14-16],消毒后出水抗性基因仍高達(dá)1010～1013copies/L[17].即便抗性基因豐度有所降低,胞內(nèi)抗性基因(iARG)也未被徹底破壞,而僅被釋放到胞外,變成胞外抗性基因(eARG)[18-19].

eARG作為一種新形式抗性基因近年來(lái)受到關(guān)注.雖然位于細(xì)胞外,eARG并未失去生物活性,在適宜條件下仍可被吸收進(jìn)入細(xì)胞表達(dá)抗性[20-21].相關(guān)研究將攜帶SHV基因的質(zhì)粒成功轉(zhuǎn)化到受體DH 5a,轉(zhuǎn)移效率達(dá)(6.7±0.5)′102(CFU/ug DNA)[22].即使在自然環(huán)境中,eARG也可進(jìn)入細(xì)胞轉(zhuǎn)化.如枯草芽孢桿菌可以吸收水中eARG至體內(nèi)表達(dá),效率達(dá)3.0′10-3(transformation CFU/total CFU)[23].此外,eARG可以不同的物理形態(tài)存在,如游離于水中(游離型胞外抗性基因(f-eARG))或附著于顆粒物(結(jié)合型胞外抗性基因(a-eARG ))[24-25],兩者的環(huán)境行為和風(fēng)險(xiǎn)可能存在顯著差異.如a-eARG更能抵抗環(huán)境的降解[26-28],而f-eARG可能更易遷移和水平轉(zhuǎn)移,研究表明f-eARG更容易與感受態(tài)細(xì)胞結(jié)合[29].同樣,Kunito等研究發(fā)現(xiàn)含NDM-1抗性基因的質(zhì)粒與土壤吸附結(jié)合后轉(zhuǎn)化效率下降[30].因此,揭示不同形態(tài)eARG的歸趨對(duì)于準(zhǔn)確揭示抗性基因的風(fēng)險(xiǎn)至關(guān)重要.

本研究考察了城市污水氯和紫外消毒過(guò)程中結(jié)合態(tài)和游離態(tài)eARG的產(chǎn)生特征,選取廣泛報(bào)道的兩類(lèi)抗性基因,四環(huán)素類(lèi)(A,X)和磺胺類(lèi)抗性基因(I,II)[31-32]作為代表,同時(shí)考察了該過(guò)程結(jié)合態(tài)胞外DNA(f-eDNA)和游離態(tài)胞外DNA(a- eDNA)中細(xì)菌群落的變化,并借助分子生態(tài)網(wǎng)絡(luò)解析了兩類(lèi)eARG與細(xì)菌群落間的關(guān)聯(lián)特征.研究結(jié)果將為揭示消毒過(guò)程中抗性基因的風(fēng)險(xiǎn)變化提供理論支撐.

1 材料與方法

1.1 消毒實(shí)驗(yàn)

水樣取自南京市某城市污水處理廠二沉池出水,裝于20L聚乙烯塑料桶中,1h內(nèi)運(yùn)送至實(shí)驗(yàn)室4℃存儲(chǔ),1周內(nèi)分析完畢.對(duì)于氯消毒,采用1L滅菌燒杯作為反應(yīng)容器,向500mL水樣中投加次氯酸鈉(NaClO)溶液(10mg/L),攪拌(300r/min)反應(yīng)10min后加入硫代硫酸鈉(Na2SO3)(5mg/L)終止.對(duì)于紫外消毒,將40mL水樣置于一次性培養(yǎng)皿,以低壓紫外平行光儀(30% UV-C, TL 120W/01,Philips)作為紫外消毒光源消毒,光源功率為120W,光束在樣品表面中心處的照射強(qiáng)度為0.12mJ/cm2,通過(guò)控制紫外輻照時(shí)間[33]使最終消毒劑量為100mJ/cm2.

1.2 DNA的分離和提取

將10mL樣品經(jīng)濾膜(0.22μm)抽濾后,濾液用于f-eDNA提取,另外將抽濾后的濾膜浸入10mL磷酸鹽緩沖溶液中,振蕩(250r/min)10min后再次經(jīng)濾膜(0.22μm)抽濾,得到的濾液用于a-eDNA提取,而兩次抽濾的兩張濾膜用于胞內(nèi)DNA(iDNA)提取.胞外DNA(eDNA)的提取采用本實(shí)驗(yàn)室開(kāi)發(fā)的磁珠法[25].即取2mL濾液加入15mL離心管,

隨后加入4mLBuffer CL(含蛋白酶K(20mg/L)和鹽酸胍(2mol/L))以及3mL異丙醇手搖混合,漩渦振蕩2min.加入30μL懸浮磁珠,漩渦振蕩4min使磁珠與樣品中 DNA充分混合.將離心管放置于磁力架上靜置70s后棄掉上清液.加入600μL Buffer CW (含7mol/L鹽酸胍的異丙醇溶液),用移液槍打勻后振蕩1min,繼續(xù)放置到磁力架上靜置70s,隨后棄去液體.用75%乙醇清洗磁珠2次.隨后將離心管于室溫下靜置10min,加入30μL提前預(yù)熱(55℃)的Elution Buffer,振蕩30s使其充分混合,隨后每隔1min震蕩30s,持續(xù)5min.將溶液與磁珠分離后即得到eDNA.對(duì)于iDNA,將濾膜剪碎到提取管中,按照土壤DNA提取試劑盒(FastDNATMSPIN kit for soil, MPbio,美國(guó))操作說(shuō)明書(shū)提取.

1.3 ARG豐度的測(cè)定

表1 各基因引物信息

應(yīng)用熒光定量PCR(qPCR)測(cè)定樣品中ARG的豐度.首先,將高濃度的標(biāo)準(zhǔn)品進(jìn)行10倍梯度連續(xù)稀釋5個(gè)梯度作為標(biāo)準(zhǔn)曲線,在qPCR儀(ABI7500,美國(guó))上進(jìn)行反應(yīng).qPCR的測(cè)定采用20μL反應(yīng)體系,其中包括10μL 2×SuperReal PreMix Plus(Tiangen Biotech,中國(guó)),2μL 50×Rox染料(TiangenBiotech,中國(guó)),0.4μL上下引物、6.2μL ddH2O以及1μL標(biāo)準(zhǔn)品.反應(yīng)條件為:95℃預(yù)熱15min→40個(gè)擴(kuò)增循環(huán)(95℃ 10s,退火20s,64℃保持30s).樣品的測(cè)定步驟同標(biāo)準(zhǔn)曲線,并根據(jù)qPCR的擴(kuò)增循環(huán)數(shù)Ct值計(jì)算ARG拷貝數(shù).確保標(biāo)準(zhǔn)曲線的2>0.99.樣品3組平行間偏差<5%,所有樣品的擴(kuò)增效率在90%～110%.各基因引物信息見(jiàn)表1.

1.4 細(xì)菌群落分析

將氯和紫外消毒前后的iDNA、f-eDNA和a-eDNA送至上海美吉生物醫(yī)藥科技有限公司Illumina miseq平臺(tái)進(jìn)行細(xì)菌16S rDNA高通量測(cè)序[35].即樣品首先進(jìn)行16S rDNA PCR擴(kuò)增,引物為515F(5'-GTGCCAGCMGCCGCGG-3')和806R(5'- GGACTACHVGGGTWTCTAAT-3'),隨后制備～ 400bp DNA片段的序列文庫(kù)并測(cè)序.細(xì)菌群落組成,Shannon指數(shù)分析均在MajorBio I-Sanger云平臺(tái)上進(jìn)行(www.i-sanger.com).另外,采用分子生態(tài)網(wǎng)絡(luò)考察ARG和菌屬間的共現(xiàn)性,數(shù)據(jù)在公開(kāi)網(wǎng)站Molecular Ecological Network Analysis Pipeline (http://ieg2.ou.edu/MENA)處理后,用Cytoscape軟件作圖[36-37].

2 結(jié)果與討論

2.1 氯和紫外消毒過(guò)程中eARG的產(chǎn)生特征

圖1展示了氯和UV消毒后3類(lèi)ARG的豐度變化.可以發(fā)現(xiàn),氯和UV消毒均使iARG的豐度下降.氯消毒之后,胞內(nèi)A,X,I和II的豐度平均降低(0.2±0.1) log;而紫外消毒后四種抗性基因的豐度則平均降低(0.6±0.3)log.顯然在常用劑量下,紫外對(duì)iARG的去除效率高于氯消毒,這可能和兩種消毒作用機(jī)理不同有關(guān).氯消毒并不能直接作用于DNA,而是破壞細(xì)胞膜和蛋白質(zhì)等,消毒后部分細(xì)菌可能處于VBNC(存活但不可培養(yǎng))狀態(tài)[38-39]導(dǎo)致DNA仍位于胞內(nèi).相比之下,UV可直接作用于DNA[6]使ARG被破壞而無(wú)法被檢出.

圖1 氯和紫外消毒后iARG、a-eARG和f-eARG絕對(duì)豐度的變化

(Blank:空白,Cl:次氯酸鈉(100mg Cl×min/L),UV:紫外(100mJ/cm2),Absolute Abundance:絕對(duì)豐度(copies/L))

兩種eARG經(jīng)氯和紫外消毒后變化趨勢(shì)存在顯著差異.對(duì)于a-eARG,氯消毒后4種ARG豐度顯著提高了(0.7±0.1)log,同樣a-eARG的相對(duì)占比(圖2)分別由1.1%、1.7%、3.0%和7.0%提高至8.0%、 8.3%、22.0%和52.0%.而經(jīng)UV處理之后,a-eARG豐度下降(0.1±0.2)log. a-eARG豐度的上升在前人研究中也被發(fā)現(xiàn),如Liu等[18]報(bào)道氯消毒后,a-eARG的濃度上升了7.8倍.這表明,氯消毒后釋放至胞外的抗性基因傾向于與水中物質(zhì)結(jié)合形成a-eARG.這可能是因?yàn)镈NA在水中類(lèi)似于聚合電解質(zhì),具有較高的表面電荷和分子柔性,可被顆粒物吸附[40]而變?yōu)閍-eARG.另外,細(xì)菌作為表面含生物大分子的膠體[41],經(jīng)氯消毒氧化后可能破碎形成眾多小顆粒膠體,從而提供更多吸附位點(diǎn)使ARG形成a-eARG.

與a-eARG不同,f-eARG經(jīng)氯消毒之后,豐度下降(0.3±0.1)log;而經(jīng)UV處理之后,豐度上升(0.4±0.2)log,其相對(duì)占比(圖2)同樣上升3.0%～25.0%.表明UV消毒更有利于f-eARG的形成.這可能仍與UV消毒機(jī)制有關(guān).UV消毒是通過(guò)作用于胸腺嘧啶鍵形成二聚體,破壞DNA的結(jié)構(gòu)[6],阻止遺傳物質(zhì)的復(fù)制而引起細(xì)菌死亡.死亡的細(xì)菌懸浮在水體中,可能阻礙UV對(duì)DNA的破壞,從而使得f-eARG更容易存活下來(lái).同時(shí),與氯消毒相比,UV特定的DNA靶向作用機(jī)理使細(xì)菌較難破碎形成小分子膠體,導(dǎo)致DNA缺少吸附位點(diǎn)而以游離型為主,使f-eARG豐度升高.

圖2 氯和紫外消毒前后三種類(lèi)型ARG (iARG、f-eARG和a-eARG)的相對(duì)百分比

Blank:空白, Cl:次氯酸鈉(100mg Cl min/L)UV:紫外(100mJ/cm2)

2.2 氯和紫外消毒后細(xì)菌群落在3種DNA變化

變形菌門(mén)(Proteobacteria)、藍(lán)菌門(mén)(Cyanobacteria)、Patescibacter超門(mén)和擬桿菌門(mén)(Bacteroidetes)是消毒前iDNA的主要菌門(mén),其相對(duì)豐度分別為45.0%、17.5%、16.9%和3.2%(圖3(a)).消毒之后,除變形菌門(mén)外,其他門(mén)相對(duì)豐度均降低,群落多樣性指數(shù)的降低也證實(shí)了這一點(diǎn).氯和紫外消毒之后,iDNA香農(nóng)指數(shù)從5.3分別下降到4.2和4.9.a-eDNA和f-eDNA中的變形菌門(mén)相對(duì)豐度在消毒之后分別升高93.1%和86.7%.此結(jié)果與本文之前報(bào)道的結(jié)果相符[25]:變形菌門(mén)在污水消毒過(guò)程中更容易釋放DNA成為eDNA.對(duì)a-eDNA和f-eDNA中細(xì)菌種屬(圖3(b))的進(jìn)一步研究發(fā)現(xiàn)等菌屬的增加導(dǎo)致了變形菌門(mén)豐度的提高.

氯消毒使iDNA中的變形菌門(mén)和棲熱菌門(mén)(Deinococcus-Thermus)的豐度升高,這表明與其他菌門(mén)相比,這兩種菌門(mén)更難釋放DNA到環(huán)境當(dāng)中.在a-eDNA中除變形菌門(mén)外絕大多數(shù)菌門(mén)豐度均上升,如Cyanobacteria的豐度由0.5%上升到5.0%,Bacteroidetes的豐度則上升了11.0%,而細(xì)菌濃度的上升可能會(huì)引起結(jié)合型ARG豐度的升高,而在f-eDNA中,變形菌門(mén)上升了6.6%,而其他菌門(mén)的豐度變化較小.與此對(duì)應(yīng),氯消毒之后f-eARG中細(xì)菌的多樣性指數(shù)從3.5降低到2.8,而a-eDNA中細(xì)菌多樣性指數(shù)則由4.2上升到4.7.值得注意的是,氯消毒降低了iDNA中一些致病菌的豐度,例如和.紫外消毒使iDNA中擬桿菌門(mén)()和放線菌門(mén)()豐度上升,表明這兩種菌門(mén)比其它菌門(mén)更能耐受UV.

圖3 氯和UV消毒前后三種類(lèi)型DNA中細(xì)菌群落在門(mén)和屬水平的豐度變化

(Blank:空白,Cl:次氯酸鈉(100mg Cl min/L),UV:紫外(100mJ/cm2)

與之相比,UV消毒后a-eDNA中變形菌門(mén)的豐度下降了36.6 %,其他菌門(mén)的豐度反而上升.而在f-eDNA中大多數(shù)的菌門(mén)豐度變化較小.與氯消毒相似的是,UV消毒之后f-eDNA的香農(nóng)系數(shù)下降至2.9,而a-eDNA的則上升到4.8.同時(shí),UV消毒降低了iDNA中人體致病菌的豐度,包括等.

2.3 ARG與菌屬間的共現(xiàn)性分析

(A) 為ARG和菌屬之間的共現(xiàn)性.(B) 為每種ARG和其相關(guān)菌屬的共現(xiàn)性,其中只統(tǒng)計(jì)了每個(gè)樣品中OTUs豐度大于1 %的菌屬,節(jié)點(diǎn)的大小表示豐度的大小

利用分子生態(tài)網(wǎng)絡(luò)圖考察了ARG和細(xì)菌菌屬之間的共線性,從而揭示可能存在的宿主關(guān)系[42].結(jié)果表明,A、X、I和II分別與17、15、15和5種菌屬存在共現(xiàn)性,這表明A,X均可能寄存于和等菌屬,而I和II可能分別在和等菌屬中.上述多種ARG與細(xì)菌間的寄存關(guān)系也被前人報(bào)道,如A存在于[41]、[42]和[45],X存在于[46]、I存在于[47]和等.這種寄存關(guān)系意味著潛在宿主的類(lèi)型及數(shù)量變化可能是消毒過(guò)程中eARG豐度變化的關(guān)鍵原因.如A與lI由于潛在宿主較多,消毒時(shí)可能有更多的iARG轉(zhuǎn)變會(huì)eARG.而A和X的宿主有[46]和[49]等耐氯菌,I的宿主有耐UV菌屬[50],可能使iARG在消毒過(guò)程中較難被釋放.此外,研究還發(fā)現(xiàn)ARG存在于部分人類(lèi)致病菌,如A、X與[51],I、II與[50]及人畜共患病原菌[52]間存在寄存關(guān)系.病原菌中ARG的存在可能使抗生素治療失效,給公共健康帶來(lái)嚴(yán)重威脅.

3 結(jié)論

3.1 氯和紫外消毒后兩種eARG的變化存在顯著差異:氯消毒(100mg Cl×min/L)后a-eARG豐度上升(0.7±0.1) log,而f-eARG豐度下降(0.2±0.1) log;紫外消毒(100mJ/cm2)后a-eARG豐度下降0.3±0.1log,而f-eARG豐度上升(0.4±0.2 )log.

3.2 氯消毒后a-eDNA中除變形菌門(mén)外大多數(shù)菌門(mén)的豐度均提高,細(xì)菌多樣性指數(shù)由4.2上升到4.7;f-eDNA中變形菌門(mén)提高6.6 %,多樣性指數(shù)則從3.5降至2.8.紫外消毒后a-eDNA中變形菌門(mén)豐度下降36.6 %,多樣性則上升到4.8,而f-eDNA中菌門(mén)豐度變化較小.

3.3A、X、I和II分別與17, 15, 15和5種菌屬之間存在共現(xiàn)性關(guān)系,表明ARG-細(xì)菌間廣泛的寄存關(guān)系.消毒過(guò)程中潛在宿主的變化是導(dǎo)致eARG豐度變化的關(guān)鍵原因.此外,ARG 還可寄存于和等病原菌,給公共健康帶來(lái)嚴(yán)重威脅.

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Generation of extracellular antibiotic resistance genes during municipal wastewater chlorination and UV disinfection.

FU Shu-sen, WANG Yi, WANG Xiao-lin, WANG Shang-jie, CHENG Yuan, BIAN Bo, ZHANG Sheng-bo, YUAN Qing-bin*

(College of Environmental Science and Engineering, Nanjing Tech University, Nanjing 211816, China)., 2021,41(10)：4756～4762

In this study, the generation of eARGs in different physical forms during wastewater chlorination and ultraviolet (UV) disinfection was investigated, and the correlation with microbial communities was explored. Results indicated that though chlorination decreased the abundance of intracellular ARGs, absorbed eARGs was significantly amplified by (0.7±0.1)log, while the abundance of free eARGs decreased by (0.2±0.1)log. UV disinfection also decreased the abundance of intracellular ARGs, but caused a significant increase of free eARGs ((0.4±0.2)log) and a decrease of absorbed eARGs ((0.3±0.1)log). Post chlorination, the abundance of most phyla increased whiledecreased in the absorbed extracellular DNA (a-eDNA), resulting in a increase of the bacterial diversity index from 4.2 to 4.7. Whereas,increased by 6.6% in the free extracellular DNA (f-eDNA) after chlorination, causing a decrease of the bacterial diversity index from 3.5 to 2.8. Post UV disinfection, the abundance ofin a-eDNA decreased by 36.6%, while the bacterial diversity index increased to 4.8; the abundance of bacteria in f-eDNA changed slightly. The molecular ecological network analysis indicated a wide hosting relationship between ARGs and bacteria genera.,,andwere correlated with 17, 15, 15 and 5genera respectively, suggesting changes in potential hosts post disinfection were essential mechanisms of the eARGs generation. This study shows that chlorination and UV disinfection can’t eliminate the risk of antibiotic resistance but only change patterns of the risk by inducing the generation of adsorbed and free eARGs.

chlorination；ultraviolet disinfection；extracellular antibiotic resistance genes；bacterial community；molecular ecological network

X172

A

1000-6923(2021)10-4756-07

付樹(shù)森(1996-),男,江蘇徐州人,南京工業(yè)大學(xué)碩士研究生,研究方向?yàn)樗h(huán)境中抗性基因的檢出、分布和去除技術(shù).

2021-02-19

江蘇省自然科學(xué)基金資助項(xiàng)目(BK20201367);國(guó)家自然科學(xué)基金資助項(xiàng)目(51608260)

* 責(zé)任作者, 副教授, yuanqb@njtech.edu.cn