我國養豬業廢棄物中四環素類、磺胺類抗生素及相關抗性基因污染研究進展

王健，賁偉偉,*，強志民，潘尋

1. 中國科學院生態環境研究中心 中國科學院飲用水科學與技術重點實驗室，北京 100085 2. 環境保護部環境保護對外合作中心，北京 100035

我國養豬業廢棄物中四環素類、磺胺類抗生素及相關抗性基因污染研究進展

王健1，賁偉偉1,*，強志民1，潘尋2

1. 中國科學院生態環境研究中心 中國科學院飲用水科學與技術重點實驗室，北京 100085 2. 環境保護部環境保護對外合作中心，北京 100035

我國是世界養豬第一大國，生豬飼養量和豬肉產量均位居世界第一。養豬業每年所產生的糞便、廢水中含有大量畜用抗生素及其代謝產物，使養豬業廢棄物成為環境中重要的抗生素污染源之一，隨之產生的抗性基因污染及傳播問題也不容忽視。本文結合近年來國內外的研究數據，對我國養豬業廢棄物中四環素類、磺胺類抗生素及其相關抗性基因的檢測方法、污染狀況及影響抗性基因傳播的因素進行了分析，并基于控制我國養豬行業抗生素及抗性基因污染的目的，提出了今后的研究重點。

抗生素；抗性基因；四環素；磺胺；豬糞；養豬廢水

基于預防疾病和促進生長的目的，抗生素被大量應用于動物養殖業中。在我國，抗生素的過量使用在養殖業中普遍存在[1]。據報道，2013年我國抗生素使用量在16.2萬t以上，其中有52%用于畜禽養殖業[2]。我國是全世界最大的豬肉生產國，2014年我國豬肉產量5 671萬t，占我國所有肉類總產量的66.4%，養豬業也成為抗生素的“使用大戶”。然而，經口服或注射所施用的抗生素僅有小部分能被動物吸收代謝，而約有30%～90%以原形態或絡合物形態隨動物排泄物排出，使得養豬業廢棄物成為環境中抗生素污染的重要來源[3]。排放到環境中的抗生素除對動、植物體存在直接的生物毒性外[4]，還將給環境中的細菌帶來選擇壓力，造成抗性細菌的積累及抗性基因(antibiotic resistance genes, ARGs)的傳播。攜帶有ARGs的細菌最終可能通過食物鏈進入人體，引發難以治愈的細菌性疾病，給人類健康帶來嚴重威脅。近年來，我國養豬業中抗生素及ARGs的污染及其對周邊土壤、水體環境的影響日益受到關注。本文根據國內外最新的研究進展及我國養豬業現狀，以最常用的四環素類和磺胺類抗生素及其ARGs為分析和總結對象，對我國養豬業廢棄物中抗生素及ARGs的檢測、污染狀況及傳播進行了綜述，并指出今后的研究重點和方向。

1 養豬業廢棄物中抗生素及ARGs的檢測方法(Methods for detection of antibiotics and ARGs in swine waste)

養豬業廢棄物(糞便、廢水)成分復雜、干擾物質多，加大了抗生素的檢測難度。因此通常采用一系列的前處理步驟，最大程度地去除基質中的干擾物質，同時保證抗生素的萃取效率及有效富集。這使得前處理成為養殖廢棄物中抗生素檢測的關鍵步驟，對于最終結果的準確性及靈敏度至關重要。對于廢水樣本，通常采用固相萃取技術(solid phase extraction, SPE)，將廢水通過吸附小柱，采用一系列清洗、選擇性洗脫的方式達到富集、分離和純化樣本中抗生素的目的[5]。對于糞便及土壤樣本，首先需要采用機械振蕩、超聲等方式使基質中有機質充分解離釋放，再采用SPE進行抗生素的富集[6]。樣本通過前處理之后，所得到的含有抗生素的富集溶液即可通過儀器進行檢測。為達到多種抗生素同步檢測的目的，最常采用高效液相色譜(high performance liquid chromatography, HPLC)對抗生素進行分離，檢測器多采用靈敏度和選擇性較高的質譜(mass spectrometry, MS)及串聯質譜(MS/MS)[7-9]。作者通過改進的SPE-HPLC/MS方法，成功檢測出養豬廢水中9種常見的抗生素[10]，并通過溶劑萃取、超聲等前處理方法，對養豬廢水固相、豬糞中抗生素的提取過程進行了優化，使回收率達到了理想范圍，構建了一套適用于養豬業廢棄物中抗生素的定性、定量檢測方法體系[6,11]。

同抗生素的檢測方法類似，ARGs的檢測也需要首先對其進行富集。所采用的富集方法通常分為傳統的依賴細菌培養和不依賴細菌培養的方法，二者各有優缺點。依賴細菌培養的方法采用含有抗生素的培養基選擇性純化培養帶有抗生素抗性的細菌，再通過分子生物學方法分析菌株中的ARGs的基因型及其定位，該方法優勢在于可以直接得到特定菌株的抗性基因型信息[12-14]，但自然界中只有少數細菌生物是可培養的，因此導致依賴細菌培養的檢測方法具有很大的局限性[15]。不依賴于細菌培養的方法，即通過直接提取環境樣本中的DNA[16-19]，或者通過細菌篩選富集獲得細菌群體[20]，再以分子生物學方法對樣本進行分析，這一方法雖然無法得知ARGs的具體宿主來源，但由于可以獲得樣本的總體數據，因此目前被廣泛應用于環境樣本中ARGs的定性和定量檢測。由于養豬糞便、廢水中含有大量未消化的有機質，故需采用有效的方法予以去除，否則會嚴重影響后續的生物學分析。因此，在提取樣本總DNA時，多采用針對性強的DNA提取試劑盒來獲得高純度的樣本總DNA[16,21-23]；對于細胞篩選，則應首先采用恰當方法對環境樣本中的細菌進行分離純化后用于下一步分析[20]。而檢測ARGs所常用的分子生物學方法包括有聚合酶鏈反應(polymerase chain reaction, PCR)、探針雜交、實時熒光定量PCR及新一代DNA測序技術等。

1.1 PCR、Southern blot和DNA芯片技術

PCR技術以其簡便快速、特異性強等特點，在ARGs的定性檢測方面得到了廣泛應用[24-25]。Wu等[16]使用PCR方法在我國北京、天津、浙江等地的9家養豬場周邊土壤中檢出了15種常見的四環素類抗性基因(tetracycline resistance genes, TRGs)；Barkovskii和Bridges[26]使用PCR方法在美國3家養豬場的糞便、土壤、廢水樣本中檢出了14種TRGs。此外，改進的PCR方法，如multiplex PCR、nested-PCR等，能夠大幅度提高PCR的使用范圍和精確度，達到快速定性檢測ARGs的目的[27-28]，因此也被用于畜禽養殖相關樣本中ARGs的檢測。Garofalo等[29]利用nested-PCR技術，直接檢測了雞肉、豬肉及動物糞便中的11種ARGs；Khan等[30]利用multiplex PCR技術，在從牛奶、雞舍廢物中分離得到的腸球菌中檢出了萬古霉素類ARG(vanC1)。

具有一定同源性的核苷酸序列在一定條件下可以按堿基互補配對原則特異性地雜交形成雙鏈，Southern blot即是基于這一原理，通過設計并合成特異性DNA探針，可以定性地檢測與探針DNA序列互補的片斷。將Southern blot技術配合PCR方法使用，可使得到的結果可信度更高。Heuer和Smalla[19]采用該方法在豬糞施肥土壤中檢出了3種磺胺類抗性基因(sulfonamide resistance genes, SRGs)；Moura等[31]通過此方法檢測了屠宰場污水處理系統中的整合子(integrons)相關基因。

DNA芯片技術(DNA microarray)是高密度、高通量的分子雜交檢測技術，其克服了傳統Southern blot操作繁瑣、耗時長的缺點，可以在短時間內同時檢測多種基因。Perreten等[32]通過微陣列技術成功檢測了革蘭氏陽性菌中90種ARGs；Frye等[33]則更進一步，將該技術擴展到了革蘭氏陰性菌，并認為通過該技術可以檢測所有抗性細菌的ARGs。綜上所述，PCR結合相關DNA同源性雜交檢測技術可以達到對于基因的準確定性，但不能對序列同源性較近的基因進行準確的區分，只能進行粗略的半定量，因此在大多數情況下只能作為快速定性的方法。

1.2 實時熒光定量PCR技術

實時熒光定量PCR技術(Real-time PCR)是一種在普通PCR反應體系中加入熒光基團，利用熒光信號積累實時監控整個PCR進程，最后通過內參基因或標準曲線對未知模板進行定量分析的方法，它不僅實現了對DNA模板的定量，而且靈敏度更高、特異性和可靠性更強，使之在ARGs的定量檢測方面得到廣泛使用。國內外學者采用該方法，對豬、牛、雞等多類畜禽養殖相關樣本(包括養殖廢水、厭氧塘沉積物、糞便、堆肥以及施肥土壤等)中的TRGs和SRGs的分布進行了定量檢測[16-18,23,34]。

1.3 新一代DNA測序技術

近年來，基于焦磷酸測序技術[35-36]及循環芯片測序策略(cyclic-array sequencing)的新一代DNA測序技術趨于成熟[37]。該技術通過在芯片上同時運行的數百萬個測序反應，得到大量的長度在幾十到幾百個堿基對范圍的短序列，再將短序列拼接組裝從而得到完整的樣本基因組序列信息。與傳統Sanger測序技術相比，新一代測序技術具有成本更低、數據量更大、信息更全面等優點，在ARGs的定性、定量檢測方面已有一些應用[38-41]。同時，定性、定量PCR及探針雜交方法僅能檢測已知ARGs，而新一代測序技術則能通過功能宏基因組學分析尋找到新的潛在ARGs[42]。因此，新一代測序技術將會在未來ARGs的分析研究中發揮重要作用。

2 我國養豬業廢棄物中四環素類及磺胺類抗生素的污染現狀(Contamination of tetracyclines and sulfonamides in the waste from Chinese pig industry)

有關檢測數據顯示，我國豬糞樣本中四環素類抗生素的檢出濃度多在1～100 mg·kg-1濃度范圍[43-47]，與奧地利、丹麥等歐洲國家豬糞樣本中的檢出值(<46 mg·kg-1)相比稍高[48-49]；磺胺類抗生素的檢出濃度范圍多在0.1～10 mg·kg-1濃度范圍[50-51]，同國外檢出數據基本持平[49,52]。山東是我國養殖業規模較大的省份，Pan等[11]選取了山東21家典型集約化養豬場，采集并分析了126個豬糞樣本的抗生素濃度，發現四環素類檢出值和檢出率均高于其他種類抗生素，檢出率在84.9%～96.8%之間，其中金霉素的檢出濃度最高，達到了764.4 mg·kg-1，是目前為止四環素類抗生素在豬糞中檢測到的最高濃度；磺胺類抗生素的檢出率在0.9%～51.6%之間，其中磺胺二甲嘧啶檢出濃度最高，達到28.7 mg·kg-1。

由于我國養豬場多采用水沖糞工藝，致使養豬場廢水中抗生素的檢出率和檢出濃度也較高，并可隨廢水排放遷移至鄰近地表水中。Wei等[53]對江蘇省21家養豬場的廢水及鄰近河流水進行了分析，發現在廢水中四環素類和磺胺類是檢出率最高的抗生素，最高濃度分別達到了在72.9 μg·L-1(土霉素)和211 μg·L-1(磺胺二甲嘧啶)，在河流水中2類抗生素的最高檢出濃度也分別達到了2.42 μg·L-1(金霉素)和4.66 μg·L-1(磺胺二甲嘧啶)。Tong等[54]檢測了武漢市2家養豬場廢水中的四環素類、磺胺類抗生素，發現磺胺甲嘧啶和土霉素的濃度較高，最高檢出濃度均超過10 μg·L-1。Ben等[10,55]對北京、山東共20余家養豬場的廢水進行了常用抗生素的濃度測定，結果顯示北京地區樣本中磺胺類和四環素類的最高檢出濃度分別達到14.05 μg·L-1(磺胺間二甲氧嘧啶)和32.67 μg·L-1(金霉素)，在山東地區的樣本中，磺胺二甲嘧啶、土霉素和金霉素的濃度較高，三者的檢出中值濃度分別為14.56、8.05和6.01 μg·L-1，其中土霉素的最高檢出濃度達到了2.02 mg·L-1。以上數據同國外檢出數據相比，大致處于同一數量級[56-57]。

養豬業所產生的糞便和廢水中殘留的抗生素可通過施肥、灌溉、非人為擴散等多種方式向土壤中遷移。四環素類及磺胺類抗生素在我國土壤中的殘留現象較為普遍。在我國北方采集的施肥土壤樣本中，2類抗生素的最高檢出濃度分別為2 683和32.7 μg·kg-1，同時研究顯示，由于施肥多在冬季進行，導致冬季土壤中的抗生素檢出濃度遠高于夏季[58]。在珠江三角洲地區采用豬糞施肥的菜田土壤中，2類抗生素的最高檢出濃度分別為242.6和321.4 μg·kg-1[59]；在從福建沿海多個城市農田土壤中的濃度分析結果顯示，四環素類抗生素的最高檢出濃度也達到了2 669 μg·kg-1[60]。以上報道所調查的濃度數據同國外數據相比處于同一水平，但最高檢出濃度略高于國外[61-62]。

3 我國養豬業廢棄物中TRGs和SRGs的污染特征、傳播及影響因素(Contamination and dissemination of TRGs and SRGs and related impact factors in the waste from Chinese pig industry)

細菌對四環素類抗生素的抗性機制主要分為3種[63-65]：核糖體保護機制(ribosomal protection proteins, RPP)，如tetM、O、Q、S、T、W等；外排泵機制(efflux pumps proteins, EFP)，如tetA、B、C、G、K、L等；酶學修飾機制(enzymatic inactivation, EI)，如tetX等。細菌對磺胺類抗生素的抗性機制主要是靠獲得表達產物可以避免磺胺類抗生素侵害的二氫葉酸合成酶(dihydropteroate synthase, DHPS)突變基因，主要指sul1、sul2和sul3，其中sul1和sul2是環境中存在最為普遍、豐度較高的SRGs[64]。

近年來關于我國養豬廢棄物中ARGs的污染情況也有所報道。從現有調查數據可知，豬糞、廢水中含有較高濃度的TRGs和SRGs，且不同地域間的差別較小。各類TRGs的相對豐度(ARG與16s rDNA豐度的比值)略有差異，糞便中RPP-TRGs和EI-TRGs通常較高，約在10-4～10-1之間，EFP-TRGs則相對較低，相對豐度約在10-5～10-3左右[34,66]；廢水中3種TRGs的水平基本持平，均在10-4～10-1范圍[34]。SRGs(sul1和sul2)在糞便中的相對豐度在10-4～10-3左右，而廢水中的豐度可達10-2～10-1[34]。在土壤中，TRGs和SRGs豐度約在10-5～10-2范圍波動，與養豬廢棄物的施用方式密切相關[16,67]。

ARGs在環境中的傳播擴散除靠抗性細菌的自身繁殖外，還借助于各種基因水平轉移方式，包括細菌之間的質粒接合轉移、噬菌體介導的轉導作用、及細菌直接攝取裸露DNA從而獲得ARGs的自然轉化作用[68-70]。這3種方式中，噬菌體轉導具有高度的宿主特異性，使ARGs的轉移限制于同種細菌間；自然轉化作用在環境中的發生具有一定的隨機性，需要依靠具有天然轉化能力的受體細菌，而此類細菌種類稀少，同時游離DNA穩定性差，增加了ARGs通過自然轉化作用傳播的局限性；相比而言，質粒的宿主范圍廣，通常含有多種ARGs，并且質粒的接合轉移是細菌之間基因交流的主動方式，加之質粒中通常含有其他能夠介導基因獲取及轉移的相關基因元件從而促進ARGs的傳播，使之成為環境中介導ARGs水平轉移的主要方式[71-72]。豬糞、養豬廢水是抗性質粒的重要載體，已有研究報道表明其中含有多種類型的、可在細菌間傳播的抗性質粒[73-74]。當這些廢棄物通過排放、灌溉、施肥等不同途徑與環境水體或土壤接觸時，會將攜帶有ARGs的抗性細菌帶入這些環境介質中，并通過質粒及其他基因元件如整合子[19,34]、轉座子[17,75]等，促進其中ARGs的擴散[76-78]。

養豬業廢棄物(糞便、廢水)通常存在較高濃度的抗生素殘留，并且由于持續排放，使其在被污染的土壤、水體中逐漸累積，直接造成抗性選擇壓力導致環境中相關抗性細菌及ARGs的增殖。Heuer和Smalla[19]將含有磺胺嘧啶的豬糞施肥于土壤，發現其中sul1基因的豐度在至少2個月內有所上升；在畜禽廢水中，SRGs的相對豐度與磺胺類抗生素的殘留濃度呈現較強的相關性[18]；我國學者對浙江、北京、天津、福建沿海等地養豬場周邊的土壤樣本的分析結果顯示，其中所含有TRGs的相對豐度與四環素類抗生素的殘留濃度也存在著顯著正相關性[16,60]。同時，一種抗生素的存在也可以影響其他非針對于此抗生素的ARGs[18]，這可能與細菌多重抗藥性相關[41]。除抗生素之外，糞便、廢水中含有的常規污染物，如COD、氮、磷等，也對于ARGs的積累有促進作用[18,79]；一些重金屬元素與抗生素存在抗性共選擇效應，因此這些金屬元素的存在也對于ARGs的積累起到了促進作用[17-18]。水分是維持細菌生命活動正常進行的最根本條件，干燥條件會使攜帶有ARGs的細菌失水死亡[80]。溫度也對ARGs有一定影響，低溫(0～5 °C)可顯著延長攜帶有ARGs的細菌在環境中的存活時間從而有利于ARGs的駐留[80]；高溫則有助于消減ARGs，例如高溫堆肥不僅可以消減糞便中的抗生素[81-87]，對ARGs也有較為明顯的消減效果[21,88-89]。另外，日照也是影響ARGs豐度水平的因素之一，據報道在養殖廢水處理塘中，日照時間與其中的TRGs豐度呈現負相關[23,90]。

4 結論與展望(Conclusions and prospects)

由于技術、經濟和管理等的局限性，我國大多數養豬場廢棄物并未得到妥善處理，廢水目前仍以直排為主，而糞便則多采用簡單室內堆放風干處理，僅少數進行堆肥或發酵處理。鑒于養豬廢棄物所造成的抗生素及ARGs污染十分值得關注，針對此問題，本文提出如下建議：

(1)應針對污染物從養殖源(動物腸道、糞便、養殖廢水等)到環境介質(土壤、水體等)的排放途徑，深入研究抗生素的環境行為、降解途徑、機理及產物，以及ARGs的演化、擴散規律及關鍵影響因素。

(2)通過對養豬糞便、廢水及施肥土壤中ARGs的種類分布與豐度的調查，得到ARGs的生態毒理基礎數據，結合目前一些已知有效的控制抗生素及ARGs的方法，建立起一套適應我國國情的養豬廢棄物控制策略，從而更好的控制ARGs在環境中的擴散，促進我國養豬業合理、健康的發展。

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Contamination of Tetracyclines, Sulfonamides and Corresponding Resistance Genes in the Waste from Chinese Pig Industry

Wang Jian1, Ben Weiwei1,*, Qiang Zhimin1, Pan Xun2

1. Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China2. Foreign Economic Cooperation Office, Ministry of Environmental Protection of Peoples' Republic of China, Beijing 100035, ChinaReceived 29 May 2015 accepted 6 July 2015

China has the world’s largest pig industry in the number of hogs and the amount of pork production. The swine manure and wastewater produced from the pig industry contain a number of veterinary antibiotics and their metabolites, which make the swine waste an important pollution source of antibiotics to the environment. The subsequent contamination and dissemination of antibiotic resistance genes (ARGs) cannot be overlooked. Based on the research data in recent years, the detection methods and pollution status of tetracyclines, sulfonamides and the corresponding ARGs, as well as the impact factors on the dissemination of ARGs in the Chinese pig industry, were summarized in this paper. Moreover, the focus of further research is also proposed for the purpose of controlling the contamination of antibiotics and ARGs caused by the Chinese pig industry.

antibiotics; antibiotic resistance genes (ARGs); tetracyclines; sulfonamides; swine manure; swine wastewater

國家自然科學基金項目(21107127)

王健(1985-)，男，博士研究生，研究方向為新型污染物控制，E-mail: tingwj110@163.com；

*通訊作者(Corresponding author)， E-mail: wwben@rcees.ac.cn

10.7524/AJE.1673-5897.20150529001

2015-05-29錄用日期：2015-07-06

1673-5897(2015)5-002-09

X171.5

A

賁偉偉(1982-)，女，環境工程博士，助理研究員，主要研究方向環境微量污染物控制。

王健, 賁偉偉，強志民，等. 我國養豬業廢棄物中四環素類、磺胺類抗生素及相關抗性基因污染研究進展[J]. 生態毒理學報，2015, 10(5): 2-10

Wang J, Ben W W, Qiang Z M, et al. Contamination of tetracyclines, sulfonamides and corresponding resistance genes in the waste from Chinese pig industry [J]. Asian Journal of Ecotoxicology, 2015, 10(5): 2-10 (in Chinese)