水產養殖環境中抗生素抗性基因的研究進展

李十盛,高 會,趙富強,陳以芹,張克玉,黃加勁,那廣水,3*

水產養殖環境中抗生素抗性基因的研究進展

李十盛1,2,高 會2**,趙富強2,4,陳以芹2,張克玉1,2,黃加勁1,2,那廣水1,2,3*

(1.上海海洋大學海洋生態與環境學院,上海市 201306；2.國家海洋環境監測中心,遼寧 大連 116023；3.海南熱帶海洋學院生態環境學院,海南 三亞 572022；4.大連海洋大學海洋科技與環境學院,遼寧 大連 116023)

為摸清我國水產養殖環境抗生素抗性基因(Antibiotic resistance genes, ARGs)污染狀況,統計了當前水產養殖環境水、沉積物和養殖對象中七大類抗性基因的賦存狀況,進一步分析了影響其賦存的因素,對未來我國水產養殖ARGs污染研究進行了展望.水產養殖環境中ARGs主要以iDNA形式存在;磺胺類和四環素類抗性基因的豐度和檢出率較高,被認為是水產養殖環境主要的ARGs,已受到當前研究的廣泛關注;而喹諾酮類、氯霉素類、β-內酰胺類、大環內酯類和氨基糖苷類抗性基因報道的豐度和檢出率相對較低.養殖過程中,養殖用藥、飼料及養殖模式是影響水產養殖環境ARGs賦存的主要因素,但環境理化因子調節養殖環境ARGs的作用也不容忽視.

抗生素抗性基因(ARGs)；水產養殖環境；賦存狀況；影響因素

我國水產養殖養殖總面積高達719萬hm2[1],水產養殖產量占全球的61.2%[2].我國水產養殖用藥的種類繁多,常用的藥品有抗菌類藥物、水體消毒劑、抗寄生蟲藥物、抗真菌藥物等九類[3].其中我國批準應用于水產養殖的抗菌類藥物包括多甲砜霉素、四環素、恩諾沙星、氟苯尼考、磺胺嘧啶和甲氧芐啶[4]等數十種.但隨著水產養殖產業的飛速發展,為了提高水產養殖對象產量,降低養殖成本,抗生素作為養殖藥物或混合飼料大量投入使用,這和健康養殖相互矛盾[5].水產養殖環境抗生素被認為是自然水體抗生素主要來源之一[6-7],如長江流域和珠江下游抗生素濃度在N.D.～537ng/L之間[8-9];海河流域抗生素平均濃度為821ng/L[10];南黃海和東海沿岸抗生素濃度在N.D.～497.6ng/L之間[6,11],北部灣河口和沿海抗生素總濃度為1.81～118ng/L[7];黃海、渤海抗生素濃度僅N.D.～16.6ng/L[12].而黃海周邊典型水產養殖場和中國東南部海水養殖場中抗生素濃度在N.D.～ 995.02ng/L[13-14];北部灣海水養殖區抗生素總濃度達43.2～885ng/L,遠高于其周圍環境水體[7].抗生素的頻繁使用不僅降低了抗生素的藥效,還促進水產養殖環境和養殖生物體內抗性細菌及抗生素抗性基因(Antibiotic resistance genes, ARGs)的發生,加速了ARGs的傳播.導致水產養殖環境成為抗生素和ARGs的潛在儲藏庫[14-15].

ARGs是可使細菌能耐受更高濃度抗生素,或者其缺失會導致細菌對抗生素的易感性增加的基因,其可原本便存在于環境細菌的基因組中,也可細菌經過基因突變而獲得[16],通過垂直基因轉移[17]和水平基因轉移[18-19]傳播.但養殖環境中持續的選擇壓力(抗生素等),加速了ARGs出現、入侵和侵占這一過程[20].如果抗生素選擇壓力足夠大,很低概率的抗性也可被細菌選擇獲得和轉移[21].Davies等[22]表示ARGs污染及其在整個生物圈微生物種群中的分布是人類使用抗生素的持續選擇的結果,這不是自然過程,而是疊加在自然上的人為情況.

水產養殖環境中,ARGs隨著菌群遷移傳播,可直接進入養殖生物體內,最終通過食物鏈和人類腸道微生物群進行水平基因轉移[15,23-24].Yang等[25]發現海水養殖沉積物中質粒和轉座子攜帶的ARGs與人類致病菌中的ARGs序列高度相似.即使攜帶ARGs的細菌被殺滅,它仍可以釋放到環境中,并隨著養殖水體擴散或定期交換到自然水體當中[26],嚴重影響生態健康.Hedberg等[27]研究也表明水產養殖尾水是沿海水域ARGs的重要來源.由于ARGs在環境和生物體內的傳播長久而持續,造成生態和食品安全的不確定性風險,引發社會越來越多的關注.世界衛生組織(WHO)將抗生素耐藥性的傳播列為21世紀公共衛生面臨的三大最嚴重威脅之一[28].ARGs作為一類新型環境污染物,其在水產養殖環境介質中的傳播擴散可能比抗生素本身的危害更大[29].

與醫療[30-31]、污水處理[32-33]、禽畜糞便[34-35]等環境相比,水產養殖環境由于養殖模式、養殖品種、養殖階段等不同,甚至城市污水處理廠、畜禽養殖場未被完全清除的抗生素及ARGs排放到河流[36-38],隨著水體交換最終進入水產養殖環境,導致水產養殖中ARGs的研究及控制更難以開展.水產養殖環境中的ARGs作為影響人類健康、生態系統、養殖生產力重要節點[39](圖1),而其賦存狀況及影響因素研究相對較少.因此,本文以水產養殖環境為研究區域,結合近年相關文獻(以PCR檢測方法為基礎),分析了水產養殖環境中喹諾酮類、β-內酰胺類、大環內酯類、氯霉素類、四環素類、磺胺類(含甲氧芐氨嘧啶類)以及氨基糖苷類ARGs賦存狀況,探究了影響水產養殖環境ARGs賦存和傳播的影響因素,為未來水產養殖環境ARGs的深入研究和控制提供科學依據.

圖1 抗生素、抗性菌和ARGs在水產養殖環境中作用的因果關系

1 水產養殖環境中抗生素抗性基因賦存狀況

1.1 水產養殖環境中抗生素抗性基因存在形態

目前環境中的ARGs主要以兩種形式存在: iARGs和eARGs.iARGs是胞內DNA(iDNA)所攜帶的ARGs,主要存在抗生素抗性細菌體內,可以通過復制傳給下一代,也可以通過接合和轉導傳遞給其它物種[8,40].eARGs是游離態的胞外DNA(eDNA)所攜帶ARGs,可由死細菌裂解或活細菌分泌釋放,裸露于環境介質中,并具有很長的存在周期,可通過轉化方式進入到水環境的細菌中,使細菌獲得抗生素抗性[8,40].與iARGs相比,eARGs因為其特有的性質更容易在環境中傳播,對人體健康和生態系統產生嚴重的威脅,研究表明[41]豐富的eDNA導致了水生環境中ARGs高轉移頻率.可見,胞內外ARGs的傳播擴散形式大不相同.

研究發現在渤海海水,來自醫院、制藥廠、污水處理廠的污泥,以及渤海、海河、城市湖泊的沉積物中,eARGs的豐度皆高于iARGs,沉積物中的eDNA被認為可被粘土礦物、沙子和腐殖質吸收,保護它不受部分核酸酶失活和物理分離的攻擊[19,42-43].與上述區域不同,Yuan等[41]在汕頭的牛蛙和魚,蝦和蟹混養的養殖場沉積物中發現,ARGs主要以iARGs形式存在,iDNA中可檢測ARGs的平均檢測頻率(60.3%)高于eDNA(40.6%).同時,在牲畜糞便中,eDNA占污泥中總DNA的不到1.5%[35],與汕頭養殖場的ARGs的胞內胞外分布相似.這可能是eDNA主要來源是死細胞的裂解釋放[42],而牲畜和水產養殖場的營養豐富,能夠很好地支持活細胞的生長和存活.因此, iDNA可能是水產養殖環境中ARGs的主要存在形式.

1.2 水產養殖環境水體中抗生素抗性基因賦存狀況

ARGs在水產養殖環境水體中普遍存在,ARGs相對豐度水平在1.28×10-8～1.87×10-1copies/16s rRNA之間(見表1).目前水產養殖環境水體中ARGs研究集中在磺胺類(包括甲氧芐氨嘧啶)、四環素類、喹諾酮類、氯霉素類、β-內酰胺類、大環內酯類六大類.其中磺胺類ARGs(含甲氧芐氨嘧類)有;四環素類ARGs有;喹諾酮類ARGs有;氯霉素類ARGs有;β-內酰胺類ARGs有;大環內酯類ARGs有(見圖2).

在多數報道中,磺胺類豐度占養殖水總ARGs相對最高,被認為是中國海水養殖場中最常檢測到的抗性基因[14].其次是四環素類ARGs,其亞型最為豐富,和在韓國海水養殖場可占總ARG的74.8%～98.0%[44].但是不同養殖區域,養殖對象不同造成的ARGs賦存狀況存在很大差異.如喹諾酮類在珠江三角洲河口白腿蝦養殖區為主要檢出ARG[45];在杭州灣河口蝦養殖區相對豐度能達到9.97×10-3copies/16s rRNA[46],這可能是喹諾酮類抗生素在蝦養殖中大量使用,且喹諾酮類ARGs在沿海水域研究[47]中是主要的抗性基因.而氯霉素類ARGs,在數地[44-46,48]均有報道;水東灣蝦場蝦塘水中有極高豐度[48].β-內酰胺類ARGs的研究報道相對較少卻不容忽視,韓國沿海養殖場相對豐度能達到1.04×10-3copies/16s rRNA[44,49].

表1 水產養殖環境水體中ARGs賦存

續表1

注:a為相對豐度(copies/16S rRNA);b為絕對豐度(copies/mL;copies/g);-為數據未明確報道;( )內為檢出率;加粗為報道主要ARG.表2、3同上.

1.3 水產養殖環境沉積物中抗生素抗性基因賦存狀況

據統計水產養殖環境沉積物中ARGs相對豐度在1.25×10-6～1.08×10-1copies/16s rRNA之間(見表2).其中磺胺類ARGs(含甲氧芐氨嘧類)有、、、/、;四環素類ARGs有、、、、、、、、、、、、、、、-;喹諾酮類ARGs有、、、;氯霉素類ARGs有、、、、_、_、;β-內酰胺類ARGs有、-、-、-、、;大環內酯類ARGs有、、;氨基糖苷類ARGs有、、(見圖2).、豐度依然較高,被認為是珠江口水產養殖[45]沉積物中ARGs的潛在指標;被認為是珠江三角洲養殖場沉積物中四環素ARGs豐度的潛在指標[49].

表2 水產養殖環境沉積物中ARGs賦存

與水不同,沉積物是ARGs的一個重要的“匯”,且沉積物中ARGs存在持久性.越南集約化蝦養殖區[53]、海南東寨港[50]等地沉積物中的ARGs的賦存量均大于水.Yuan等[46]發現隨著時間的推移,ARGs的擴散導致其可在沉積物中積累,沉積物很可能增強抗生素抗性的傳播.但有研究[2]認為ARGs在沉積物群落中的“持久性”,是水生生物糞便中ARGs的不斷涌入所導致的.此外,不同的ARGs在水和沉積物中存在不同的賦存規律.統計發現如等ARGs僅在沉積物有報道,()()--等卻僅在水中有報道(圖2).Su等[48]研究也證實了在水產養殖環境中,ARGs的賦存在不同介質中有明顯差異.不過,PCR檢測的目標ARGs的局限性也可造成水和沉積物中ARGs統計結果產生差異,在煙臺海水養殖場[54]、瀛湖養殖區[55]運用宏基因組方法及高通量PCR方法檢出的ARGs則更加全面.

圖2 近年水產養殖環境中ARGs檢出報道頻次熱

1.4 水產養殖對象中抗生素抗性基因賦存狀況

水產養殖中抗生素主要作用于養殖生物,產生耐藥性不可避免.當前對水產養殖生物的ARGs的研究主要針對于其本身以及其腸道菌群.而鑒于運用PCR檢測方法研究水產養殖生物ARGs的報道相對較少,本文結合了運用宏基因組技術的相關研究.在水產養殖生物中,喹諾酮類、β-內酰胺類、大環內酯類、氯霉素類、四環素類、磺胺類、氨基糖苷類七大類ARGs皆有報道,其中被報道頻率較高(見圖2、表3).特別的,桿菌肽類、多重耐藥性和其他類ARGs(如等)豐度非常高[24,60].桿菌肽鋅作為禁用的飼料添加劑,其ARGs似仍以很高的量存在,但未發現有運用PCR技術針對性對其檢測的報道.而多重耐藥性和其他類ARGs的功能和特性復雜,可促使“超級細菌”形成,對人類健康及生態環境威脅更大.但目前對多重耐藥性ARGs的認識還相對較淺,其種類繁多,相關數據庫有待豐富,需更多研究作為基礎.

表3 水產養殖生物體中ARGs賦存

續表3

值得注意的是,抗生素在水生生物體內經食物鏈富集已經得到重視,但水產養殖環境和養殖生物體內賦存的ARGs之間的聯系的研究還處在起步階段.早在2003年,Furushita等[61]就發現在魚類細菌中檢測到的四環素耐藥基因與人類臨床分離株中的基因高度相似.當前認為水平基因轉移是ARGs水體、沉積物和水產品之間轉移的關鍵途徑[8,41,52],且可移動遺傳元件(Mobile Genetic Element,MGEs)對ARGs轉移起著重要作用[29,62],其本身可攜帶ARGs,也與ARGs有共發生現象,往往代表著ARGs的高遷移率.Su等[48]通過Source Tracer分析發現早期和中期的蝦腸道約70%的菌群起源于沉積物,且蝦腸道和沉積物賦存了相同的ARGs().由此,賦存在水產養殖水體和沉積物中的ARGs很可能最終富集到水產養殖對象的體內.

2 水產養殖環境中抗生素抗性基因的影響因素

2.1 養殖用藥及飼料對抗生素抗性基因的影響

養殖用藥及飼料的使用對ARGs發生及傳播的影響一直被密切關注.對于養殖用藥,ARGs在養殖水域大量存在和藥物在治療養殖生物疾病中大量使用是分不開的.ARGs與相應的同源抗生素顯著相關[66].從機制上,長期處于養殖用藥的環境中,細菌的SOS反應(由細菌DNA損傷引發的修補反應)會被激活,其可以通過產生氧離子而增加突變幾率,促使水平基因位移,活化整合酶,促使整合子重組,從而顯著增強ARGs的水平轉移[56-67].控制水產養殖中抗生素類用藥是減緩ARGs污染的重要手段.而對于飼料,市場上用于養殖水產品的飼料種類繁多,常混合抗生素使用.甲殼類水產品(日本沼蝦)長期暴露在飼料中抗生素(合法使用量)下,也會嚴重影響蝦腸道健康和ARGs豐度[68].其中,魚粉作為水產養殖飼料的主要基礎原料,含有豐富的抗生素及ARGs,其對沉積物中ARGs豐度的影響有明顯劑量效應,其中的營養物質可以驅動微生物群落結構來改變沉積物中ARGs[69].重要的,全球魚粉產量近70%被用于水產養殖,而中國是全球魚粉的主要消費國[70],“飼料禁抗”是大勢所趨.

但是水產養殖環境中養殖用藥和飼料中抗生素只是ARGs發生和傳播的主要應激源,抗生素與其相應的ARGs并非總是同時出現,單一類型的抗生素使用下,也可能存在復雜的ARGs污染[71-73]. ARGs也不是僅由于同源抗生素而引起.實際養殖過程中,盡管并未添加的抗生素,其ARGs照樣可以存在.如氯霉素、紅霉素類藥品早就被禁止用于水產養殖業,但水東灣蝦場中卻是蝦塘水中主要ARGs,在數地被檢出,也均以一定量存在[44-46,48].

2.2 養殖模式對抗生素抗性基因的影響

養殖模式是水產養殖獨有的ARGs影響因素.不同單一養殖模式下,ARGs的多樣性及豐度皆有差異.Gu等[55]對比了瀛湖圍欄養殖和箱型養魚ARGs的污染情況,發現箱型養殖的ARGs多樣性要比圍欄養殖更高,其認為由于不同魚類養殖方式,理化參數變化不同,從而引起的微生物群落變化導致,通過調節湖泊的某些因素是可以控制ARGs的.而相比于單一養殖模式,在當前循環資源的多營養級養殖發展趨勢下,混合養殖(鴨-魚池)水體相比于單一養殖更不易受到ARGs污染[49].Yuan等[41]也發現單一牛蛙養殖場的沉積物中ARGs的檢測頻率和豐度均高于混養養殖場沉積物.但是,混合養殖對ARGs的影響還取決于混養品種.杭州灣河口養殖區報道[46]在混養池塘中,與雜食性魚類相結合的蝦養殖可能會增加ARGs豐度,而與濾食性魚類養殖可能會減少ARGs豐度.合理安排混合養殖模式以及養殖品種是控制ARGs的重要手段.

2.3 環境理化因子對抗生素抗性基因影響

水產養殖環境的理化因子包括水溫、鹽度、酸堿性、化學耗氧量、溶解氧、營養物質和重金屬等.這些因子復雜多變,同氣候條件、放養密度、餌料種類和投餌量、生物排泄物等有密切關系.而針對水產養殖中環境理化因子對ARGs賦存狀況和傳播規律的影響的研究極少.養殖過程中某些環境理化因子是可控的,弄清其在水產養殖環境中影響,加以有效人為調控,可能達到控制ARGs污染的目的.

2.3.1 溫度、鹽度等對抗生素抗性基因影響 對于溫度,適宜的溫度更有助于水產品的生長.溫度也會影響質粒的轉移過程,37℃下抗性質粒更容易發生水平轉移[74].長江口及鄰近海域的研究[75]發現溫度與1和等基因的豐度之間呈顯著相關性(P<0.05).對于鹽度,其可調控微生物群落的組成和結構,從而調控環境中ARGs.河口開放養殖區最易受其影響,隨著鹽度的增加,垂直傳播的動態平衡會被打破,磺胺類ARGs會減少;鹽度影響的還具有季節性,不同季節,鹽度對于ARGs有不同的影響,所影響的ARGs類型也有所不同[76].

總有機碳(TOC)、化學需氧量(COD)、總磷(TP)、總氮(TN)等水質指標,代表著養殖環境的污染狀況,其皆影響ARGs.水東灣蝦塘水中和TOC呈正相關(<0.05);和TOC呈極強正相關(<0.01);與COD極強相關(<0.01)[48].東江流域[18]、長江口[77]的研究也表明TS、TOC、TN和等ARGs呈極顯著相關(<0.01).懸浮顆粒物(SS)還被認為可能是ARGs的載體,其豐度與、的豐度均存在顯著相關性(<0.05)[48,75].但在水產養殖過程中,TOC、COD、TP、TN等從機制上如何影響ARGs,還有待研究.適宜的含量既可保證水產品生長,水質良好,又能降低ARGs的威脅.

至于酸堿性,養殖環境中pH值一般變化幅度較小,但不同ARGs受pH值變化影響有不同的機制.如Huang等[78]發現厭氧處理過程中酸性條件下,噬菌體DNA含量會增加,且更有利于eDNA的排泄,因此四環素ARGs水平轉移的數量和其持基因能力大大提高;而堿性條件下,接合轉移可能受到限制;Lin等[34]研究卻發現糞肥中,酸化降低了磺胺類ARGs的水平轉移,pH可誘導厚壁菌門的富集,增強磺胺類抗生素降解,從而抑制的水平基因轉移.

2.3.2 重金屬對抗生素抗性基因影響 重金屬對水產養殖環境中ARGs賦存及傳播有極大影響,部分微量金屬元素也是水產品生長所必須的因子之一[79].螯合銅、硫酸銅等重金屬鹽作為消毒劑也在水產養殖中廣泛應用[80].研究發現重金屬可與抗生素共同對微生物造成選擇壓力,且重金屬可以抵抗降解,選擇壓力可以長期施加,在同一質粒中重金屬甚至可和ARGs共同存在[31,71,81].江蘇省白馬湖水產養殖場[52]評估Cu2+與ARGs呈明顯的正相關關系;東江流域[18]大部分ARGs與Cu、Zn等重金屬之間存在顯著的相關性.其中,弱酸提取態的重金屬被認為更容易從環境中釋放而被微生物利用.弱酸提取態Zn的含量與ARGs之間的顯著相關性更強(<0.01), Zn離子被認為是調控ARGs賦存的重要金屬元素之一[8,75].在機制上,已有銀、鋅影響機制的報道,但并不針對水產養殖環境.據報道,AgNPs和Ag+是通過誘導ROS過量產生,觸發SOS反應,增加細胞膜通透性來促進接合基因轉移,從而加強ARGs的傳播[82];天然閃鋅礦和ZnS納米顆粒則是促進細菌之間質粒介導的ARGs水平轉移,增強抗性編碼質粒的接合轉移[83].這從機制上解釋了重金屬對ARGs傳播的影響,為在水產養殖環境中進行類似研究提供了參考.而實際養殖過程中,重金屬在水產養殖環境中易富集、有毒性、去除難度高,除了對ARGs產生影響,對水產品和整個水產養殖環境也產生嚴重危害.探究重金屬對水產養殖環境中ARGs賦存和傳播的影響具有重要意義.

3 結語

為了從根本上防止抗生素耐藥性的發生和傳播,未來水產養殖環境中ARGs的研究可抓住以下幾點:

1) 深入解析ARGs在水產養殖環境、水產品以及人體致病菌之間的關系.盡管目前已發現水產養殖中的ARGs能通過食物鏈對人類造成威脅,和人類腸道菌群、致病菌所攜帶ARGs具有相似性,但缺乏更多證據及相關機制的研究.

2) 合理選擇水產養殖環境中代表性污染ARGs,開發適合水產養殖環境準確、快捷的ARGs監測方法,系統的獲取水產養殖環境中ARGs的相關數據,制定針對水產養殖環境ARGs污染的標準.傳統PCR檢測ARGs具有局限性,而宏基因組和高通量PCR在水產養殖環境的研究中相對較少,且檢測成本高.選定代表性的ARGs優先控制,將ARGs污染監測常規化,是控制ARGs污染的必要手段

3) 探究水產養殖過程中主流養殖模式、溫度、重金屬鹽消毒劑等可調因素對ARGs發生和傳播的影響,了解其影響機制,有效的把控養殖過程中ARGs的發生、傳播乃至去除.

4) 建立ARGs生態風險評估方法及預警體系,從而對水產養殖環境中ARGs對人類健康和生態環境的威脅科學的評估、預防ARGs污染的發生.

[1] 農業農村部漁業漁政管理局.2019中國漁業統計年鑒 [M]. 北京:中國農業出版社, 2019.

Bureau of Fisheries, Ministry of Agriculture and Rural Affairs, China. 2019 China Fisheries Statistical Yearbook [M]. Beijing: China Agricultural Press, 2019.

[2] FAO yearbook Fishery and Aquaculture Statistics 2017 FAO annuaire [M]. Rome, Italy: Food and agriculture organization of the united nations, 2017.

[3] 胡升翔.常用漁藥分類 [J]. 漁業致富指南, 2012,(3):54-55.

Hu S X. Classification of common fishing drugs [J]. Fishery Guide to be Rich, 2012,(3):54-55.

[4] Liu X, Steele J C, Meng X Z. Usage, residue, and human health risk of antibiotics in Chinese aquaculture: A review [J]. Environmental Pollution, 2017,223:161-169.

[5] Suzuki S, Nakanishi S, Tamminen M, et al. Occurrence of sul and tet(M) genes in bacterial community in Japanese marine aquaculture environment throughout the year: Profile comparison with Taiwanese and Finnish aquaculture waters [J]. Science of the Total Environment, 2019,669:649-656.

[6] Du J, Zhao H, Liu S, et al. Antibiotics in the coastal water of the South Yellow Sea in China: Occurrence, distribution and ecological risks [J]. Science of The Total Environment, 2017,595:521-527.

[7] Zhang R, Pei J, Zhang R, et al. Occurrence and distribution of antibiotics in mariculture farms, estuaries and the coast of the Beibu Gulf, China: Bioconcentration and diet safety of seafood [J]. Ecotoxicology and Environmental Safety, 2018,154:27-35.

[8] 郭行磐.長江口濱岸水環境中抗生素抗性基因的賦存特征 [D]. 上海:華東師范大學, 2019.

Guo X P, Characterization of the occurrence of antibiotic resistance genes in the coastal water environment of the Yangtze estuary[D]. Shanghai: East China Normal University, 2019.

[9] Li S, Shi W, Li H, et al. Antibiotics in water and sediments of rivers and coastal area of Zhuhai City, Pearl River estuary, south China [J]. Science of The Total Environment, 2018,636:1009-1019.

[10] Lei K, Zhu Y, Chen W, et al. Spatial and seasonal variations of antibiotics in river waters in the Haihe River Catchment in China and ecotoxicological risk assessment [J]. Environment International, 2019,130.

[11] Li F, Chen L, Chen W, et al. Antibiotics in coastal water and sediments of the East China Sea: Distribution, ecological risk assessment and indicators screening [J]. Marine Pollution Bulletin, 2020,151:110810.

[12] Zhang R, Tang J, Li J, et al. Antibiotics in the offshore waters of the Bohai Sea and the Yellow Sea in China: Occurrence, distribution and ecological risks [J]. Environmental Pollution, 2013,174:71-77.

[13] Han Q F, Zhao S, Zhang X R, et al. Distribution, combined pollution and risk assessment of antibiotics in typical marine aquaculture farms surrounding the Yellow Sea, North China [J]. Environment International, 2020,138:105551.

[14] Chen C Q, Zheng L, Zhou J L, et al. Persistence and risk of antibiotic residues and antibiotic resistance genes in major mariculture sites in Southeast China [J]. Science of the Total Environment, 2017,580:1175-1184.

[15] 趙 帝,徐在言,吳山功,等.草魚腸道微生物抗生素抗性基因研究 [J]. 水生態學雜志, 2019,40(6):111-116.

Zhao D, Xu Z Y, Wu S G, et al. Study of antibiotic resistance genes in grass carp gut microorganisms [J]. Journal of Hydroecology, 2019, 40(6):111-116.

[16] Martínez J L, Baquero F, Andersson D I. Predicting antibiotic resistance [J]. Nature Reviews Microbiology, 2007,5(12):958-965.

[17] Li B, Qiu Y, Song Y, et al. Dissecting horizontal and vertical gene transfer of antibiotic resistance plasmid in bacterial community using microfluidics [J]. Environment International, 2019,131:105007.

[18] Su H C, Pan C G, Ying G G, et al. Contamination profiles of antibiotic resistance genes in the sediments at a catchment scale [J]. Science of the Total Environment, 2014,490:708-714.

[19] Zhang Y, Niu Z, Zhang Y, et al. Occurrence of intracellular and extracellular antibiotic resistance genes in coastal areas of Bohai Bay (China) and the factors affecting them [J]. Environmental Pollution, 2018,236:126-136.

[20] Baquero F, Lanza V F, Cantón R, et al. Public health evolutionary biology of antimicrobial resistance: priorities for intervention [J]. Evol Appl, 2015,8(3):223-39.

[21] Martínez J L. Antibiotics and antibiotic resistance genes in natural environments [J]. Science, 2008,321(5887):365.

[22] Davies J, Davies D. Origins and evolution of antibiotic resistance [J]. Microbiol. Mol. Biol. Rev., 2010Sep;74(3):417-33.

[23] 梁惜梅,聶湘平,施 震.珠江口典型水產養殖區抗生素抗性基因污染的初步研究 [J]. 環境科學, 2013,34(10):4073-4080.

Liang Xi M, Nie X P, Shi Z. Preliminary study of antibiotic resistance gene contamination in typical aquaculture areas of the Pearl River Estuary [J]. Environmental Science, 2013,34(10):4073-4080.

[24] 劉可心.南美白對蝦腸道細菌抗生素抗性基因的研究 [D]. 廣州:華南理工大學, 2018.

Liu K X. Study of antibiotic resistance genes in intestinal bacteria of South American white shrimp [D]. Guangzhou: South China University of Technology, 2018.

[25] Yang J, Wang C, Wu J, et al. Characterization of a multiresistant mosaic plasmid from a fish farmsp. isolate reveals aggregation of functional clinic-associated antibiotic resistance genes [J]. Appl. Environ. Microbiol., 2014,80(4):1482-8.

[26] 付佳倫.細菌耐藥基因在斑馬魚體內定植、轉移規律及機制研究 [D]. 北京:中國人民解放軍軍事醫學科學院, 2017.

Fu J L. Study on the pattern and mechanism of bacterial drug resistance gene colonization and transfer in zebrafish [D]. Beijing: Chinese People's Liberation Army Academy of Military Medical Sciences, 2017.

[27] Hedberg N, Stenson I, Pettersson M N, et al. Antibiotic use in Vietnamese fish and lobster sea cage farms; implications for coral reefs and human health (vol 495, pg 366, 2018) [J]. Aquaculture, 2019, 500:679-679.

[28] World Health O. Antimicrobial resistance: global report on surveillance [M]. Geneva: World Health Organization, 2014.

[29] 楊鳳霞,毛大慶,羅 義,等.環境中抗生素抗性基因的水平傳播擴散 [J]. 應用生態學報, 2013,24(10):2993-3002.

Yang F X, Mao D Q, Luo Y, et al. Horizontal dispersal of antibiotic resistance genes in the environment [J]. Chinese Journal of Applied Ecology, 2013,24(10):2993-3002.

[30] Zhang G, Zheng G, Zhang Y, et al. Evaluation of a micro/nanofluidic chip platform for the high-throughput detection of bacteria and their antibiotic resistance genes in post-neurosurgical meningitis [J]. International Journal of Infectious Diseases, 2018,70:115-120.

[31] Zhai Y, He Z, Kang Y, et al. Complete nucleotide sequence of pH11, an IncHI2plasmid conferring multi-antibiotic resistance and multi- heavy metal resistance genes in a clinical Klebsiella pneumoniae isolate [J]. Plasmid, 2016,86:26-31.

[32] Nnadozie C F, Kumari S, Bux F. Status of pathogens, antibiotic resistance genes and antibiotic residues in wastewater treatment systems [J]. Reviews in Environmental Science And Bio-Technology, 2017,16(3):491-515.

[33] Jiao Y N, Chen H, Gao R X, et al. Organic compounds stimulate horizontal transfer of antibiotic resistance genes in mixed wastewater treatment systems [J]. Chemosphere, 2017,184:53-61.

[34] Lin H, Sun W, Yu Q, et al. Acidic conditions enhance the removal of sulfonamide antibiotics and antibiotic resistance determinants in swine manure [J]. Environmental Pollution, 2020,263:114439.

[35] Zhang Y, Snow D D, Parker D, et al. Intracellular and extracellular antimicrobial resistance genes in the sludge of livestock waste management structures [J]. Environmental Science & Technology, 2013,47(18):10206-10213.

[36] 羅 曉,張文麗,袁立霞,等.納污河流抗性基因和微生物群落相關性 [J]. 中國環境科學, 2019,39(6):2606-2613.

Luo X, Zhang W L, Yuan L X, et al. Resistance genes and microbial community correlation in polluted rivers [J]. China Environmental Science, 2019,39(6):2606-2613.

[37] 黃福義,安新麗,李 力,等.生活垃圾填埋場對河流抗生素抗性基因的影響 [J]. 中國環境科學, 2017,37(1):203-209.

Huang F Y, An X L, Li L, et al. Effects of domestic waste landfills on antibiotic resistance genes in rivers [J]. China Environmental Science, 2017,37(1):203-209.

[38] Sabri N A, Holst S V, Schmitt H, et al. Fate of antibiotics and antibiotic resistance genes during conventional and additional treatment technologies in wastewater treatment plants [J]. Science of the Total Environment, 2020.

[39] Williams-Nguyen J, Sallach J B, Bartelt-Hunt S, et al. Antibiotics and antibiotic resistance in agroecosystems: State of the science [J]. Journal of Environmental Quality, 2016,45(2):394-406.

[40] 張永鵬.渤海灣近岸海域抗生素及抗生素抗性基因的污染特征研究 [D]. 天津:天津大學, 2018.

Zhang Y P. Pollution characteristics of antibiotics and antibiotic resistance genes in the near-shore waters of Bohai Bay [D]. Tianjin: Tianjin University, 2018.

[41] Yuan K, Wang X, Chen X, et al. Occurrence of antibiotic resistance genes in extracellular and intracellular DNA from sediments collected from two types of aquaculture farms [J]. Chemosphere, 2019,234: 520-527.

[42] Dong P, Wang H, Fang T, et al. Assessment of extracellular antibiotic resistance genes (eARGs) in typical environmental samples and the transforming ability of eARG [J]. Environment International, 2019, 125:90-96.

[43] Mao D, Luo Y, Mathieu J, et al. Persistence of extracellular DNA in river sediment facilitates antibiotic resistance gene propagation [J]. Environmental Science & Technology, 2014,48(1):71-78.

[44] Jang H M, Kim Y B, Choi S, et al. Prevalence of antibiotic resistance genes from effluent of coastal aquaculture, South Korea [J]. Environmental Pollution, 2018,233:1049-1057.

[45] Su H, Liu S, Hu X, et al. Occurrence and temporal variation of antibiotic resistance genes (ARGs) in shrimp aquaculture: ARGs dissemination from farming source to reared organisms [J]. Science of the Total Environment, 2017,607:357-366.

[46] Yuan J, Ni M, Liu M, et al. Occurrence of antibiotics and antibiotic resistance genes in a typical estuary aquaculture region of Hangzhou Bay, China [J]. Marine Pollution Bulletin, 2019,138:376-384.

[47] Lu J, Zhang Y, Wu J. Continental-scale spatio-temporal distribution of antibiotic resistance genes in coastal waters along coastline of China [J]. Chemosphere, 2020,247:125908-125908.

[48] Su H, Hu X, Wang L, et al. Contamination of antibiotic resistance genes (ARGs) in a typical marine aquaculture farm: source tracking of ARGs in reared aquatic organisms [J]. Journal of Environmental Science And Health Part B-Pesticides Food Contaminants And Agricultural Wastes, 2020,55(3):220-229.

[49] Huang L, Xu Y B, Xu J X, et al. Antibiotic resistance genes (ARGs) in duck and fish production ponds with integrated or non-integrated mode [J]. Chemosphere, 2017,168:1107-1114.

[50] 姜春霞,黎 平,李森楠,等.海南東寨港海水和沉積物中抗生素抗性基因污染特征研究 [J]. 生態環境學報, 2019,28(1):128-135.

Jiang C X, Li P, Li S N, et al. Characterization of antibiotic resistance gene contamination in seawater and sediment of Dongzhai Port, Hainan [J].Ecology and Environmental Sciences, 2019,28(1):128-135.

[51] Wu J, Mao C, Deng Y, et al. Diversity and abundance of antibiotic resistance of bacteria during the seedling period in marine fish cage-culture areas of Hainan, China [J]. Marine Pollution Bulletin, 2019,141:343-349.

[52] Shen X, Jin G, Zhao Y, et al. Prevalence and distribution analysis of antibiotic resistance genes in a large-scale aquaculture environment [J]. Science of the Total Environment, 2020,711.

[53] Thi Thu Hang P, Rossi P, Hoang Dang Khoa D, et al. Analysis of antibiotic multi-resistant bacteria and resistance genes in the effluent of an intensive shrimp farm (Long An, Vietnam) [J]. Journal of Environmental Management, 2018,214:149-156.

[54] Wang J H, Lu J, Zhang Y X, et al. Metagenomic analysis of antibiotic resistance genes in coastal industrial mariculture systems [J]. Bioresource Technology, 2018,253:235-243.

[55] Gu J, Zhang L, Wang X, et al. High-throughput analysis of the effects of different fish culture methods on antibiotic resistance gene abundances in a lake [J]. Environmental Science And Pollution Research, 2019,26(6):5445-5453.

[56] 李云莉.中國沿海典型養殖水域抗生素及其抗性基因污染的初步研究 [D]. 上海:上海海洋大學, 2017.

Li Y L Preliminary study on antibiotics and their resistance gene contamination in typical coastal aquaculture waters in China [D]. Shanghai: Shanghai Ocean University, 2017.

[57] Gao Q, Li Y, Qi Z, et al. Diverse and abundant antibiotic resistance genes from mariculture sites of China's coastline [J]. Science of the Total Environment, 2018,630:117-125.

[58] Bueno I, Travis D, Gonzalez-Rocha G, et al. Antibiotic resistance genes in freshwater trout farms in a watershed in Chile [J]. Journal of Environmental Quality, 2019,48(5):1462-1471.

[59] Gao P, Mao D, Luo Y, et al. Occurrence of sulfonamide and tetracycline-resistant bacteria and resistance genes in aquaculture environment [J]. Water Research, 2012,46(7):2355-2364.

[60] Zhao Y, Zhang X X, Zhao Z, et al. Metagenomic analysis revealed the prevalence of antibiotic resistance genes in the gut and living environment of freshwater shrimp [J]. Journal of Hazardous Materials, 2018,350:10-18.

[61] Furushita M, Shiba T, Maeda T, et al. Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates [J]. Applied and Environmental Microbiology, 2003, 69(9):5336-5342.

[62] 趙小慧,蘇 潔,樊景鳳,等.海洋環境中細菌耐藥性研究進展 [J]. 中國抗生素雜志, 2019,(4):406-412.

Zhao S H, Su J, Fan J F, et al. Advances in the study of bacterial drug resistance in the marine environment [J]. Chinese Journal of Antibiotics, 2019,(4):406-412.

[63] 閆倩倩,李 彬,廖梅杰,等.山東主要刺參養殖區幼參腸道抗生素耐藥菌及耐藥基因分布特征 [J/OL]. 漁業科學進展, 1-12[2021- 08-14].https://doi.org/10.19663/j.issn2095-9869.20190424001.

Yan Q Q, Li B, Liao M J et al. Characteristics of antibiotic-resistant bacteria and resistance genes in the intestinal tract of juvenile cucumbers from major sashimi culture areas in Shandong [J/OL]. Progress in Fishery Sciences,1-12 [2021-08-14]. https://doi.org/ 10.19663/j.issn2095-9869.20190424001.

[64] Zhou R, Zeng S, Hou D, et al. Temporal variation of antibiotic resistance genes carried by culturable bacteria in the shrimp hepatopancreas and shrimp culture pond water [J]. Ecotoxicology and Environmental Safety, 2020,199:110738.

[65] Niu G, Wongsathein D, Boonyayatra S, et al. Occurrence of multiple antibiotic resistance and genotypic characterization inisolated from cage-cultured hybrid red tilapia (sp.) in the Ping River, Northern Thailand [J]. Aquaculture Research, 2019,50(12):3643-3652.

[66] Xu K, Wang J, Gong H, et al. Occurrence of antibiotics and their associations with antibiotic resistance genes and bacterial communities in Guangdong coastal areas [J]. Ecotoxicology And Environmental Safety, 2019,186.

[67] Beaber J W, Hochhut B, Waldor M K. SOS response promotes horizontal dissemination of antibiotic resistance genes [J]. Nature, 427(6969):72-74.

[68] Sun S, Korheina D K A, Fu H, et al. Chronic exposure to dietary antibiotics affects intestinal health and antibiotic resistance gene abundance in oriental river prawn (), and provokes human health risk [J]. Science of The Total Environment, 2020,720:137478.

[69] 韓 瑩.魚粉對海水養殖沉積物中抗生素抗性基因的影響和機理研究 [D]. 大連理工大學, 2019.

Han Y. Study on the effect and mechanism of fishmeal on antibiotic resistance genes in mariculture sediments [D]. Dalian University of Technology, 2019.

[70] Tacon A G J, Metian M. Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: Trends and future prospects [J]. Aquaculture, 2008,285(1):146-158.

[71] Zhang Q Q, Tian G M, Jin R C. The occurrence, maintenance, and proliferation of antibiotic resistance genes (ARGs) in the environment: influencing factors, mechanisms, and elimination strategies [J]. Applied Microbiology And Biotechnology, 2018,102(19):8261-8274.

[72] Chen B, Lin L, Fang L, et al. Complex pollution of antibiotic resistance genes due to beta-lactam and aminoglycoside use in aquaculture farming [J]. Water Research, 2018,134:200-208.

[73] Wang J H, Lu J, Wu J, et al. Proliferation of antibiotic resistance genes in coastal recirculating mariculture system [J]. Environmental Pollution, 2019,248:462-470.

[74] 李千偉.近岸海洋環境中典型抗生素抗性污染特征與傳播規律 [D]. 上海:上海海洋大學, 2018.

Li Q W. Characteristics and transmission patterns of typical antibiotic resistance pollution in the nearshore marine environment [D]. Shanghai: Shanghai Ocean University, 2018.

[75] 陸大培.長江口及鄰近海域沉積物中抗生素抗性基因時空分布特征及其與環境因子相關性研究 [D]. 上海:華東師范大學, 2019.

Lu D P. Spatial and temporal distribution characteristics of antibiotic resistance genes in sediments of the Yangtze River estuary and adjacent waters and their correlation with environmental factors [D]. Shanghai: East China Normal University, 2019.

[76] Lu Z, Na G, Gao H, et al. Fate of sulfonamide resistance genes in estuary environment and effect of anthropogenic activities [J]. Science of The Total Environment, 2015,527-528:429-438.

[77] Guo X P, Yang Y, Lu D P, et al. Biofilms as a sink for antibiotic resistance genes (ARGs) in the Yangtze Estuary [J]. Water Research, 2018,129:277-286.

[78] Huang H, Chen Y, Zheng X, et al. Distribution of tetracycline resistance genes in anaerobic treatment of waste sludge: The role of pH in regulating tetracycline resistant bacteria and horizontal gene transfer [J]. Bioresource Technology, 2016,218:1284-1289.

[79] 焦樂飛,代恬夢,金 敏,等.對蝦微量元素研究進展 [J]. 水產學報, 2019,43(10):2093-2101.

Jiao L F, Dai T M, Jin M, et al. Progress in the study of trace elements in shrimp [J].Journal of Fisheries of China, 2019,43(10):2093-2101.

[80] 董曉慧,楊原志,鄭石軒,等.飼料中不同銅源和水平對凡納濱對蝦生長、免疫和組織銅含量的影響 [J]. 大連水產學院學報, 2007, (5):377-383.

Dong X H, Yang Y Z, Zheng S X, et al. Effects of different sources and levels of copper in feed on growth, immunity and tissue copper content of Penaeus vannamei [J]. Journal of Dalian Ocean University, 2007,(5):377-383.

[81] Li A D, Li L G, Zhang T. Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants [J]. Frontiers in Microbiology, 2015,6.

[82] Lu J, Wang Y, Jin M, et al. Both silver ions and silver nanoparticles facilitate the horizontal transfer of plasmid-mediated antibiotic resistance genes [J]. Water Research, 2020,169.

[83] Li G, Chen X, Yin H, et al. Natural sphalerite nanoparticles can accelerate horizontal transfer of plasmid-mediated antibiotic- resistance genes [J]. Environment International, 2020,136.

Research progress on the occurrence and influencing factors of antibiotic resistance genes in aquaculture environment.

LI Shi-sheng1,2, GAO Hui2**, ZHAO Fu-qiang2,4, CHEN Yi-qin2, ZHANG Ke-yu1,2, HUANG Jia-jin1,2, NA Guang-shui1,2,3*

(1.College of Marine Ecology and Environment, Shanghai Ocean University, Shanghai 201306, China；2.National Marine Environmental Monitoring Center, Dalian 116023, China；3.College of Ecology and Environment, Hainan Tropical Ocean University, Sanya 572022, China；4.College of Marine Technology and Environment, Dalian Ocean University, Dalian 116023, China)., 2021,41(11)：5314～5325

It is important to clarify the contamination status of antibiotic resistance genes (ARGs) in the aquaculture environment in China.In this paper, the current status of the occurrence of seven major classes of resistance genes in aquaculture environmental water, sediment and aquatic products was counted. The factors influencing their occurrence were further analyzed. The future research on the pollution of ARGs in aquaculture in China was prospected. ARGs in the aquaculture environment are mainly in the form of iDNA.Sulfonamide and tetracycline resistance genes are considered to be the major ARGs in aquaculture environments due to their high abundance and detection rates, while the abundance and detection rate of quinolones, chloramphenicol, β-lactams, macrolides and aminoglycosides resistance genes are reported to be relatively low. In the process of aquaculture, aquaculture drugs, feed and culture mode are the main factors affecting the occurrence of ARGs in aquaculture environment, but the role of environmental physical and chemical factors in regulating ARGs in aquaculture environment cannot be ignored.

antibiotic resistance genes；aquaculture environment；occurrence state；influencing factors

X52

A

1000-6923(2021)11-5314-12

李十盛(1997-),男,吉林白山人,上海海洋大學碩士研究生,主要從事抗生素及抗生素抗性基因研究.

2021-04-07

國家重點研發計劃項目(2019YFD0901104);國家自然科學基金資助項目(41976222,42006195);自然資源部海洋大氣化學與全球變化重點實驗室開放基金(GCMAC2010)

* 責任作者, 研究員, gsna@nmemc.org.cn; **工程師, hgao@nmemc. org.cn