氯和紫外消毒過程中胞外抗性基因的產生特征

付樹森,王 藝,王肖霖,王尚杰,程 遠,卞 博,張生博,袁青彬

氯和紫外消毒過程中胞外抗性基因的產生特征

付樹森,王 藝,王肖霖,王尚杰,程 遠,卞 博,張生博,袁青彬*

(南京工業大學環境科學與工程學院,江蘇 南京 211816)

考察了城市污水氯和紫外消毒過程中不同物理形態的胞外抗性基因的產生行為與及微生物群落的關聯特征.結果表明,氯消毒盡管使胞內抗性基因豐度下降,但使結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,而結合型胞外抗性基因豐度下降(0.3±0.1) log.氯消毒后, 結合型胞外DNA(a-eDNA)中變形菌門豐度下降而其他菌門的豐度上升,細菌多樣性指數由4.2上升到4.7;而游離型胞外DNA(f-eDNA)中變形菌門上升了6.6%,多樣性指數則從3.5降低到2.8.紫外消毒后,a-eDNA中變形菌門豐度下降了36.6%,多樣性則上升到4.8,而f-eDNA中細菌豐度變化較小.分子生態網絡分析揭示了抗性基因與細菌間廣泛的寄存關系,、、和分別與17、15、15和5種菌屬間存在共現性,表明抗性基因潛在宿主的變化是導致消毒后胞外抗性基因產生的關鍵原因.本研究表明氯和紫外消毒不能消除抗性基因風險,反而通過導致不同胞外抗性基因的大量產生,使風險的形式發生變化.

氯消毒；紫外消毒；胞外抗性基因；細菌群落；分子生態網絡

全球范圍抗生素的大量使用極大加劇了細菌抗藥性的發展,已成為威脅公共健康的嚴峻問題.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,已檢出數百種抗生素抗性基因(ARG)[5-7],成為環境中抗性基因的主要儲存庫.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程對ARG的去除效果也受到廣泛關注.很多研究發現氯和紫外消毒不能有效地去除ARG[8-12],去除效果遠低于其宿主耐藥細菌[14-16],消毒后出水抗性基因仍高達1010～1013copies/L[17].即便抗性基因豐度有所降低,胞內抗性基因(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受到關注.雖然位于細胞外,eARG并未失去生物活性,在適宜條件下仍可被吸收進入細胞表達抗性[20-21].相關研究將攜帶SHV基因的質粒成功轉化到受體DH 5a,轉移效率達(6.7±0.5)′102(CFU/ug DNA)[22].即使在自然環境中,eARG也可進入細胞轉化.如枯草芽孢桿菌可以吸收水中eARG至體內表達,效率達3.0′10-3(transformation CFU/total CFU)[23].此外,eARG可以不同的物理形態存在,如游離于水中(游離型胞外抗性基因(f-eARG))或附著于顆粒物(結合型胞外抗性基因(a-eARG ))[24-25],兩者的環境行為和風險可能存在顯著差異.如a-eARG更能抵抗環境的降解[26-28],而f-eARG可能更易遷移和水平轉移,研究表明f-eARG更容易與感受態細胞結合[29].同樣,Kunito等研究發現含NDM-1抗性基因的質粒與土壤吸附結合后轉化效率下降[30].因此,揭示不同形態eARG的歸趨對于準確揭示抗性基因的風險至關重要.

本研究考察了城市污水氯和紫外消毒過程中結合態和游離態eARG的產生特征,選取廣泛報道的兩類抗性基因,四環素類(A,X)和磺胺類抗性基因(I,II)[31-32]作為代表,同時考察了該過程結合態胞外DNA(f-eDNA)和游離態胞外DNA(a- eDNA)中細菌群落的變化,并借助分子生態網絡解析了兩類eARG與細菌群落間的關聯特征.研究結果將為揭示消毒過程中抗性基因的風險變化提供理論支撐.

1 材料與方法

1.1 消毒實驗

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內運送至實驗室4℃存儲,1周內分析完畢.對于氯消毒,采用1L滅菌燒杯作為反應容器,向500mL水樣中投加次氯酸鈉(NaClO)溶液(10mg/L),攪拌(300r/min)反應10min后加入硫代硫酸鈉(Na2SO3)(5mg/L)終止.對于紫外消毒,將40mL水樣置于一次性培養皿,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度為0.12mJ/cm2,通過控制紫外輻照時間[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DNA(iDNA)提取.胞外DNA(eDNA)的提取采用本實驗室開發的磁珠法[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提前預熱(55℃)的Elution Buffer,振蕩30s使其充分混合,隨后每隔1min震蕩30s,持續5min.將溶液與磁珠分離后即得到eDNA.對于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)操作說明書提取.

1.3 ARG豐度的測定

表1 各基因引物信息

應用熒光定量PCR(qPCR)測定樣品中ARG的豐度.首先,將高濃度的標準品進行10倍梯度連續稀釋5個梯度作為標準曲線,在qPCR儀(ABI7500,美國)上進行反應.qPCR的測定采用20μL反應體系,其中包括10μL 2×SuperReal PreMix Plus(Tiangen Biotech,中國),2μL 50×Rox染料(TiangenBiotech,中國),0.4μL上下引物、6.2μL ddH2O以及1μL標準品.反應條件為:95℃預熱15min→40個擴增循環(95℃ 10s,退火20s,64℃保持30s).樣品的測定步驟同標準曲線,并根據qPCR的擴增循環數Ct值計算ARG拷貝數.確保標準曲線的2>0.99.樣品3組平行間偏差<5%,所有樣品的擴增效率在90%～110%.各基因引物信息見表1.

1.4 細菌群落分析

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藥科技有限公司Illumina miseq平臺進行細菌16S rDNA高通量測序[35].即樣品首先進行16S rDNA PCR擴增,引物為515F(5'-GTGCCAGCMGCCGCGG-3')和806R(5'- GGACTACHVGGGTWTCTAAT-3'),隨后制備～ 400bp DNA片段的序列文庫并測序.細菌群落組成,Shannon指數分析均在MajorBio I-Sanger云平臺上進行(www.i-sanger.com).另外,采用分子生態網絡考察ARG和菌屬間的共現性,數據在公開網站Molecular Ecological Network Analysis Pipeline (http://ieg2.ou.edu/MENA)處理后,用Cytoscape軟件作圖[36-37].

2 結果與討論

2.1 氯和紫外消毒過程中eARG的產生特征

圖1展示了氯和UV消毒后3類ARG的豐度變化.可以發現,氯和UV消毒均使iARG的豐度下降.氯消毒之后,胞內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iARG的去除效率高于氯消毒,這可能和兩種消毒作用機理不同有關.氯消毒并不能直接作用于DNA,而是破壞細胞膜和蛋白質等,消毒后部分細菌可能處于VBNC(存活但不可培養)狀態[38-39]導致DNA仍位于胞內.相比之下,UV可直接作用于DNA[6]使ARG被破壞而無法被檢出.

圖1 氯和紫外消毒后iARG、a-eARG和f-eARG絕對豐度的變化

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

兩種eARG經氯和紫外消毒后變化趨勢存在顯著差異.對于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現,如Liu等[18]報道氯消毒后,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結合形成a-eARG.這可能是因為DNA在水中類似于聚合電解質,具有較高的表面電荷和分子柔性,可被顆粒物吸附[40]而變為a-eARG.另外,細菌作為表面含生物大分子的膠體[41],經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使ARG形成a-eARG.

與a-eARG不同,f-eARG經氯消毒之后,豐度下降(0.3±0.1)log;而經UV處理之后,豐度上升(0.4±0.2)log,其相對占比(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消毒機制有關.UV消毒是通過作用于胸腺嘧啶鍵形成二聚體,破壞DNA的結構[6],阻止遺傳物質的復制而引起細菌死亡.死亡的細菌懸浮在水體中,可能阻礙UV對DNA的破壞,從而使得f-eARG更容易存活下來.同時,與氯消毒相比,UV特定的DNA靶向作用機理使細菌較難破碎形成小分子膠體,導致DNA缺少吸附位點而以游離型為主,使f-eARG豐度升高.

圖2 氯和紫外消毒前后三種類型ARG (iARG、f-eARG和a-eARG)的相對百分比

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

2.2 氯和紫外消毒后細菌群落在3種DNA變化

變形菌門(Proteobacteria)、藍菌門(Cyanobacteria)、Patescibacter超門和擬桿菌門(Bacteroidetes)是消毒前iDNA的主要菌門,其相對豐度分別為45.0%、17.5%、16.9%和3.2%(圖3(a)).消毒之后,除變形菌門外,其他門相對豐度均降低,群落多樣性指數的降低也證實了這一點.氯和紫外消毒之后,iDNA香農指數從5.3分別下降到4.2和4.9.a-eDNA和f-eDNA中的變形菌門相對豐度在消毒之后分別升高93.1%和86.7%.此結果與本文之前報道的結果相符[25]:變形菌門在污水消毒過程中更容易釋放DNA成為eDNA.對a-eDNA和f-eDNA中細菌種屬(圖3(b))的進一步研究發現等菌屬的增加導致了變形菌門豐度的提高.

氯消毒使iDNA中的變形菌門和棲熱菌門(Deinococcus-Thermus)的豐度升高,這表明與其他菌門相比,這兩種菌門更難釋放DNA到環境當中.在a-eDNA中除變形菌門外絕大多數菌門豐度均上升,如Cyanobacteria的豐度由0.5%上升到5.0%,Bacteroidetes的豐度則上升了11.0%,而細菌濃度的上升可能會引起結合型ARG豐度的升高,而在f-eDNA中,變形菌門上升了6.6%,而其他菌門的豐度變化較小.與此對應,氯消毒之后f-eARG中細菌的多樣性指數從3.5降低到2.8,而a-eDNA中細菌多樣性指數則由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()和放線菌門()豐度上升,表明這兩種菌門比其它菌門更能耐受UV.

圖3 氯和UV消毒前后三種類型DNA中細菌群落在門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中變形菌門的豐度下降了36.6 %,其他菌門的豐度反而上升.而在f-eDNA中大多數的菌門豐度變化較小.與氯消毒相似的是,UV消毒之后f-eDNA的香農系數下降至2.9,而a-eDNA的則上升到4.8.同時,UV消毒降低了iDNA中人體致病菌的豐度,包括等.

2.3 ARG與菌屬間的共現性分析

(A) 為ARG和菌屬之間的共現性.(B) 為每種ARG和其相關菌屬的共現性,其中只統計了每個樣品中OTUs豐度大于1 %的菌屬,節點的大小表示豐度的大小

利用分子生態網絡圖考察了ARG和細菌菌屬之間的共線性,從而揭示可能存在的宿主關系[42].結果表明,A、X、I和II分別與17、15、15和5種菌屬存在共現性,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與細菌間的寄存關系也被前人報道,如A存在于[41]、[42]和[45],X存在于[46]、I存在于[47]和等.這種寄存關系意味著潛在宿主的類型及數量變化可能是消毒過程中eARG豐度變化的關鍵原因.如A與lI由于潛在宿主較多,消毒時可能有更多的iARG轉變會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在消毒過程中較難被釋放.此外,研究還發現ARG存在于部分人類致病菌,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,給公共健康帶來嚴重威脅.

3 結論

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中除變形菌門外大多數菌門的豐度均提高,細菌多樣性指數由4.2上升到4.7;f-eDNA中變形菌門提高6.6 %,多樣性指數則從3.5降至2.8.紫外消毒后a-eDNA中變形菌門豐度下降36.6 %,多樣性則上升到4.8,而f-eDNA中菌門豐度變化較小.

3.3A、X、I和II分別與17, 15, 15和5種菌屬之間存在共現性關系,表明ARG-細菌間廣泛的寄存關系.消毒過程中潛在宿主的變化是導致eARG豐度變化的關鍵原因.此外,ARG 還可寄存于和等病原菌,給公共健康帶來嚴重威脅.

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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

付樹森(1996-),男,江蘇徐州人,南京工業大學碩士研究生,研究方向為水環境中抗性基因的檢出、分布和去除技術.

2021-02-19

江蘇省自然科學基金資助項目(BK20201367);國家自然科學基金資助項目(51608260)

* 責任作者, 副教授, yuanqb@njtech.edu.cn