氣溶膠中抗生素抗性基因研究進(jìn)展：以養(yǎng)殖場和醫(yī)院為例

薛銀剛，劉菲，王利平,#，江曉棟，王倩，施昕瀾，薛柯，金珊，姜逸

1. 常州大學(xué)環(huán)境與安全工程學(xué)院，常州 213164 2. 常州市環(huán)境監(jiān)測中心，江蘇省環(huán)境保護(hù)水環(huán)境生物監(jiān)測重點(diǎn)實(shí)驗(yàn)室，常州 213001

抗生素作為人類醫(yī)學(xué)的重要發(fā)明，自問世以來創(chuàng)造了很多醫(yī)學(xué)奇跡。除了醫(yī)療領(lǐng)域，由于抗生素具有預(yù)防疾病和刺激生長的作用，以亞治療劑量長期添加于動物飼料中，被廣泛應(yīng)用于養(yǎng)殖業(yè)[1-3]。我國是抗生素生產(chǎn)和使用大國。據(jù)報道，我國年產(chǎn)抗生素原料約21萬t，3萬t出口，國內(nèi)使用18萬t，其中約一半用于臨床，一半用于畜禽養(yǎng)殖業(yè)[4]。抗生素的濫用和過度使用在微生物抗性發(fā)展中起著至關(guān)重要的作用[5]，由此產(chǎn)生的抗生素耐藥菌(antibiotic resistant bacteria，ARB)和抗生素抗性基因(antibiotic resistance genes，ARGs)污染問題引起人們的廣泛關(guān)注[6-7]。

當(dāng)ARGs作為一種新型環(huán)境污染物提出之后[8]，有關(guān)其在環(huán)境中來源、傳播和污染的報道日益增多。迄今已有很多種ARGs在世界范圍內(nèi)的水和廢水[9-13]、土壤[14-15]和沉積物[16-17]等介質(zhì)中被相繼檢出，主要檢出的是四環(huán)素類、磺胺類、喹諾酮類、大環(huán)內(nèi)酯類和氨基糖苷類等。目前關(guān)于ARGs的研究主要集中于ARGs的來源探究、ARGs的環(huán)境地球化學(xué)行為與傳播機(jī)制、細(xì)菌耐藥對人體健康的影響以及ARGs的環(huán)境污染控制等方面[9, 11-13]，且大多數(shù)研究主要圍繞水和土壤中ARGs展開。相對于水、土壤和沉積物等環(huán)境介質(zhì)，關(guān)于ARGs在空氣介質(zhì)中污染現(xiàn)狀的研究信息還較為缺乏，而空氣中攜帶ARGs的致病菌一旦被人吸入，可能對人體造成直接的健康危害。Pal等[18]通過研究發(fā)現(xiàn)相比泥土和水等環(huán)境介質(zhì)，北京霧霾空氣樣品中檢出的ARGs種類最多，平均有64.4種，表明氣溶膠是ARGs的潛在儲存庫和傳播的載體。然而，國內(nèi)外對大氣氣溶膠中ARGs的研究相對較少。

本文就養(yǎng)殖場和醫(yī)院2個抗生素大量使用的典型場所展開討論，根據(jù)近年來國內(nèi)外的文獻(xiàn)資料，對ARGs的污染現(xiàn)狀、檢測技術(shù)和對人體的健康風(fēng)險進(jìn)行綜述，并對今后開展氣溶膠中ARGs研究提出幾點(diǎn)建議。

1 養(yǎng)殖場氣溶膠中ARGs污染現(xiàn)狀(Status of ARGs pollution in livestock farms aerosols)

獸用抗生素進(jìn)入動物體內(nèi)，便會誘導(dǎo)產(chǎn)生抗性菌株[19]，這些腸道抗性菌株隨糞便排入環(huán)境中，在糞便處理和動物移動期間很容易被氣溶膠化[20]，研究表明動物糞便及其接觸過的土壤中都含有大量ARGs，經(jīng)過氣流作用會懸浮于空氣中[21]。同時，養(yǎng)殖過程中產(chǎn)生的廢水以及養(yǎng)殖場廢物處理過程中都含有大量ARGs，通過蒸發(fā)、厭氧消化和發(fā)酵過程都會產(chǎn)生氣溶膠[22]。目前，養(yǎng)殖場氣溶膠中已發(fā)現(xiàn)的ARGs主要為四環(huán)素類和大環(huán)內(nèi)酯類，高的檢出率與這2類抗生素在養(yǎng)殖場的大量使用密不可分[23-24]。養(yǎng)殖場氣溶膠中已檢出的ARGs種類見表1。

四環(huán)素類抗生素是一類重要的廣譜抗菌藥物，由于具有預(yù)防疾病和促進(jìn)動物生長等作用，被廣泛用于集約化畜禽養(yǎng)殖場[25]，許多致病菌由于產(chǎn)生了tet-R基因而對四環(huán)素類抗生素產(chǎn)生抗性[26]。目前發(fā)現(xiàn)于畜禽養(yǎng)殖場氣溶膠中的四環(huán)素類抗性基因已達(dá)十余多種。在養(yǎng)雞場氣溶膠中發(fā)現(xiàn)tetW、tetL、tetA、tetA/P、tetX、tetG、tetA/C、tetY、tetB、tetH、tetZ、tetQ和tetO 13種四環(huán)素類抗性基因[22,27-28]。并且四環(huán)素類抗性基因廣泛存在于各類養(yǎng)殖環(huán)境的氣溶膠中，Mceachran等[29]研究肉牛飼養(yǎng)場附近空氣顆粒物樣品，利用實(shí)時熒光定量PCR技術(shù)(real-time quantitative PCR，qPCR)檢測出6種四環(huán)素類ARGs，并發(fā)現(xiàn)相比上風(fēng)向，下風(fēng)向氣溶膠中四環(huán)素類抗性基因的豐度是顯著增加的。高達(dá)13種四環(huán)素類ARGs也被發(fā)現(xiàn)存在于養(yǎng)豬場環(huán)境產(chǎn)生的氣溶膠中[30-34]。

大環(huán)內(nèi)酯類抗生素由于可以有效地抑制某些革蘭氏陽性菌和革蘭氏陰性菌，從而被廣泛地應(yīng)用于動物細(xì)菌感染的治療和預(yù)防[35]，Just等[28]在來源于籠養(yǎng)和散養(yǎng)的舍內(nèi)微生物氣溶膠中發(fā)現(xiàn)了ermA、ermB 2種大環(huán)類酯類抗性基因，其中ermA只存在于散養(yǎng)雞舍的氣溶膠中，可以看出用于散養(yǎng)雞的抗生素主要用于動物生長，可能更有助于紅霉素耐藥性形成。金黃色葡萄球菌是畜禽養(yǎng)殖環(huán)境中常見的病原體[36]，Liu等[32]通過從6個養(yǎng)豬場的149株金黃色葡萄球菌氣溶膠中檢出了ermC，同時大多數(shù)菌株表現(xiàn)出多重抗藥性，有3株菌株抗9種抗生素。ermF、mefA也被發(fā)現(xiàn)存在于養(yǎng)豬場的腸球菌和鏈球菌等含致病菌的氣溶膠中[34]。由表1可知ermA、ermB是畜禽養(yǎng)殖場最常見的大環(huán)內(nèi)酯類抗性基因。

表1 養(yǎng)殖場和醫(yī)院氣溶膠中檢出的抗生素抗性基因(ARGs)Table 1 Antibiotic resistance genes (ARGs) detected in aerosols of livestock farms and hospitals

除了2類檢出率較高的ARGs，在畜禽養(yǎng)殖場的氣溶膠還發(fā)現(xiàn)了1種桿菌肽鋅抗性基因bcrR[28]，2種喹諾酮類抗性基因qnrS、parC[27]，1種氨基糖苷類抗性基因aac6′-aph2″[32]，8種β-內(nèi)酰胺類抗性基因blaCARB-4、blaCTX-M、blaampC、blaOXAⅠ、blaOXA-18、blaOXAⅡ、blaPSE、blaTEM*和3種sul2、sul3、dfrA1磺胺類抗性基因[27]。

2 醫(yī)院氣溶膠中ARGs污染現(xiàn)狀(Status of ARGs pollution in hospitals aerosols)

近年來，醫(yī)院環(huán)境的細(xì)菌耐藥性明顯升高[37]。氟喹諾酮類是用于治療幾種革蘭氏陰性和革蘭氏陽性細(xì)菌感染的廣譜抗生素，其抗藥性首次在耐甲氧西林金黃色葡萄球菌的治療中被發(fā)現(xiàn)[38]。有研究表明，在過去10年中，大腸桿菌對氟喹諾酮類(如環(huán)丙沙星)的耐藥率有所提高[39]；一些致病革蘭氏陰性細(xì)菌菌株(大腸桿菌、產(chǎn)氣腸桿菌和肺炎克雷伯桿菌)幾乎抵抗所有當(dāng)代抗生素[40]；Santoro等[41]從醫(yī)院廢水處理設(shè)施收集廢水樣品，其中22.2%的菌株被列為多重耐藥。Tang等[42]對上海第一人民醫(yī)院的細(xì)菌抗生素敏感性進(jìn)行調(diào)查，發(fā)現(xiàn)該醫(yī)院的抗生素抗藥性持續(xù)增加，大腸桿菌、鮑曼不動桿菌和金黃色葡萄球菌是最普遍的抗性菌株。

醫(yī)院內(nèi)的感染對病患和雇員構(gòu)成健康威脅，特別是抗生素抗性在細(xì)菌之間的傳播問題，接觸傳播和空氣微生物傳播是醫(yī)院細(xì)菌感染的較為常見的傳播方式[43]。已有研究表明，從醫(yī)院呼吸科住院部的微生物氣溶膠中分離的許多菌株抗一種或多種抗生素，所有與少動鞘氨醇單胞菌有關(guān)的分離株均檢出萬古霉素抗性基因vanB，另有一些菌株含四環(huán)素類抗性基因tetA、tetC和紅霉素抗性基因ermX[44]。耐甲氧西林金黃色葡萄球菌可導(dǎo)致醫(yī)院感染的傳播[43]，Drudge等[45]從醫(yī)院各個部門(急診、普通門診、重癥監(jiān)護(hù)病房、兒科和胸肺科門診)的獨(dú)立空氣凈化裝置中收集灰塵樣本，利用PCR技術(shù)在多個樣品中檢測到aac6′-aph2″、ermA和mecA抗性基因。與醫(yī)院環(huán)境接觸的人員高度暴露于多重耐藥細(xì)菌，并且在醫(yī)生和護(hù)士家中，空氣中耐甲氧西林菌株的檢出率更高，同時所有分離的凝血酶陰性葡萄球菌菌株均對萬古霉素、利福平和利奈唑胺敏感[46]。上述研究表明，醫(yī)院氣體環(huán)境是ARGs和致病菌的儲存庫，但是醫(yī)院環(huán)境生物氣溶膠中ARGs和致病菌的污染特征研究僅僅處于起步階段，而國內(nèi)還鮮少涉及該方面的研究工作。

3 氣溶膠中ARGs的樣品采集與檢測技術(shù)(Sampling and detecting technology of ARGs in aerosol)

3.1 氣溶膠中ARGs的樣品采集技術(shù)

攜帶ARGs的微生物氣溶膠可借助空氣介質(zhì)進(jìn)行傳播和擴(kuò)散，其中粒徑為0.1～20.0 μm的氣溶膠與人類健康密切相關(guān)[47]。氣溶膠中ARGs的采集一般是借鑒微生物氣溶膠的采集方法，目前已用于氣溶膠中ARGs的采集方法主要有固體撞擊式采樣法、液體捕捉式采樣法、膜過濾采樣法和離心式采樣法。

固體撞擊式采樣法是指空氣中微生物氣溶膠在獲得足夠的慣性后，脫離氣流而撞擊于固體平板上，通常以培養(yǎng)基為收集介質(zhì)，使該類采樣方法更適于微生物的培養(yǎng)和鑒定[48]。固體撞擊式采樣器最常用的是安德森(Andersen)采樣器，Andersen采樣器通過模擬人的呼吸道解剖結(jié)構(gòu)和動力學(xué)特征，采用分粒徑撞擊原理設(shè)計(jì)[49]，采集效率高且采集粒徑范圍寬。Andersen二級采樣器(第一級>8 μm；第二級0.8～8 μm)已被用于養(yǎng)豬場ARB的采集，其細(xì)菌的抗生素耐藥性利用K-B紙片擴(kuò)散法確定[50]。Andersen六級采樣器是一種6級篩板式空氣微生物采樣器，第1～6級捕獲的微粒直徑分別為>7.0 μm；4.7～7.0 μm；3.3～4.7 μm；2.1～3.3 μm；1.1～2.1 μm和0.65～1.1 μm。由于其具有微生物存活率高、采集粒譜范圍寬、采樣效率高以及適用范圍廣等特點(diǎn)，被廣泛應(yīng)用于醫(yī)院和養(yǎng)殖場環(huán)境氣溶膠中ARGs的采集[31-32, 44]。另外，Andersen八級采樣器(空氣動力學(xué)直徑：>9.0 μm；5.8～9.0 μm；4.7～5.8 μm；3.3～4.7 μm；2.1～3.3 μm；1.1～2.1 μm；0.7～1.1 μm和0.4～0.7 μm)被用于研究集約化養(yǎng)殖過程中與粒徑相關(guān)的細(xì)菌多樣性和四環(huán)素抗性基因(tetW、tetL)豐度[22]。

液體捕捉式采樣法可以完成氣溶膠到水溶膠的采樣過程，主要采用的是液體沖擊式采樣器，收集介質(zhì)通常是無菌水、緩沖生理鹽水或營養(yǎng)液等液體，該類收集介質(zhì)可以起到緩沖作用，減少微生物的損傷[48]。常用的液體沖擊式采樣器有AGI系列的全玻璃液體沖擊式采樣器AGI-30和SKC公司生產(chǎn)的BioSampler液體生物氣溶膠取樣器，由于長時間的采樣會引起收集介質(zhì)的蒸發(fā)而增加試驗(yàn)誤差，所以該類采樣器適用于短時間收集樣品[51-52]，其收集的采樣液可以用于進(jìn)一步的微生物培養(yǎng)或分子生物學(xué)檢測。AGI-30采樣器已被用于采集養(yǎng)豬場氣溶膠中多重耐藥的腸球菌、凝固酶陰性葡萄球菌和鏈球菌[34,53]，適用于收集室內(nèi)環(huán)境氣載多重耐藥細(xì)菌。Brooks等[54]為研究家禽舍內(nèi)生物氣溶膠的抗生素抗性成分，利用BioSampler采集肉雞廠室內(nèi)和室外的氣溶膠樣品，結(jié)果表明ARB高度集中于室內(nèi)環(huán)境中，且多為耐藥葡萄球菌。Ling等[30]利用BioSampler和Omni 3000濕式濃縮采樣器采集養(yǎng)豬場室內(nèi)PM10(收集于無菌的磷酸鹽緩沖液PBS)，檢測出tetX、tetW和intI1。

膜過濾式采樣法是指空氣中的微生物顆粒在抽氣裝置的作用下被阻留在濾材上，所使用的采樣器結(jié)構(gòu)主要包括抽氣裝置和裝有多孔濾膜的收集裝置，是較為簡單的一類采樣方法[49]。膜過濾式采樣器的特點(diǎn)是效率高，在氣溶膠中ARGs的樣品采集過程中有利于收集不可培養(yǎng)的細(xì)菌，這對全面掌握ARGs的種類和豐度等信息具有重要意義，但是該類方法在溫度過低或過高時均不利于生物粒子的捕捉，也不適于長時間采樣[55]。膜過濾式采樣法常用的濾膜材料包括玻璃纖維、聚氯乙稀和聚碳酸酯等，生物氣溶膠的采集效率與所選濾膜類型、孔徑和氣體流速有關(guān)。Hi-Q CF-902便攜式大流量空氣取樣器結(jié)合玻璃纖維濾膜[29]和Maple多級沖擊式采樣器配備聚氯乙烯濾膜[28]分別用于牛飼養(yǎng)場和家禽舍內(nèi)生物氣溶膠中ARGs的樣品采集，經(jīng)qPCR技術(shù)檢出多種ARGs (tetB、tetL、tetM、tetO、tetQ、tetW、tetA/C、tetG、ermA、ermB和bcrR)，表明該類方法的適用性。

離心式采樣法是指生物氣溶膠顆粒利用旋轉(zhuǎn)運(yùn)動產(chǎn)生的離心力獲得一定動量，并因其慣性偏離氣體流線，撞擊于采集面上[47]，所用采樣器有我國制造的LWC-I空氣浮游菌采樣器和德國制造的RCS離心式空氣微生物采樣器。Li等[56]利用RCS離心式空氣微生物采樣器采集污水處理廠空氣樣本，利用PCR結(jié)合凝膠電泳技術(shù)檢出空氣樣本中存在sul2和intI1。目前關(guān)于利用離心式采樣法進(jìn)行氣溶膠中ARGs的樣品采集的研究還較少，同時有研究表明，在微生物氣溶膠的回收率方面，離心式空氣微生物采樣器比Andersen采樣器略低[57]。

AGI-30采樣器和Andersen 6級樣器是國際空氣生物學(xué)學(xué)會推薦使用的采樣器，也是氣溶膠中ARGs的樣品采集常用的2類采樣設(shè)備，但是未有研究針對不同類型采樣器的性能、適用性、收集效率和后續(xù)ARGs定性定量檢測等方面進(jìn)行比較評價，使氣溶膠中ARGs的檢出結(jié)果存在較大的不確定性。不同的樣品采集條件適用于不同的采樣器，Chang等[58]采用了過濾法(聚碳酸酯濾膜)、液體捕捉式(Andersen單級采樣器)和固體撞擊式(AGI-30采樣器)3類方法收集養(yǎng)豬場生物氣溶膠，其中AGI-30采樣器收集的可培養(yǎng)細(xì)菌濃度最高，過濾法收集到的可培養(yǎng)真菌濃度最高。研究者們只有充分了解各種采樣方法以及采樣器的特點(diǎn)，才能使試驗(yàn)分析結(jié)果更具準(zhǔn)確性，而目前關(guān)于氣溶膠中ARGs的研究還較少，不同采樣方法對ARGs種類和濃度的分析結(jié)果可能產(chǎn)生的影響還不明確，有待開展進(jìn)一步的研究。

3.2 氣溶膠中ARGs的樣品檢測技術(shù)

微生物是ARGs的宿主，抗生素抗性的傳統(tǒng)檢測技術(shù)主要是基于微生物培養(yǎng)，以抗性表型來評價其抗性類型[59]。傳統(tǒng)的抗菌藥敏試驗(yàn)包括紙片擴(kuò)散試驗(yàn)，E檢驗(yàn)梯度擴(kuò)散試驗(yàn)和肉湯稀釋敏感性試驗(yàn)[60]。藥敏試驗(yàn)被用于評價多種環(huán)境中微生物對抗生素的抗性強(qiáng)弱，如檢測出污水和污泥中大腸桿菌對四環(huán)素類抗生素抗藥率高達(dá)57%[61]以及海水和沙粒中腸球菌屬的抗紅霉素和四環(huán)素的頻率最高[62]。藥敏試驗(yàn)也被用于氣溶膠中細(xì)菌的抗性表征，Gandolfi等[63]根據(jù)臨床實(shí)驗(yàn)室標(biāo)準(zhǔn)研究所協(xié)議，使用紙片擴(kuò)散法研究PM10中細(xì)菌的抗生素抗藥性。Liu等[32]利用Mueller-Hinton瓊脂試驗(yàn)對養(yǎng)豬場室內(nèi)氣溶膠進(jìn)行抗生素敏感性檢測，發(fā)現(xiàn)了多重耐藥金黃色葡萄球菌。Sapkota等[34]使用最小抑菌濃度瓊脂稀釋法在養(yǎng)豬場的室內(nèi)空氣中篩選多重耐藥腸球菌屬和鏈球菌屬，發(fā)現(xiàn)其多重耐藥性是由多個大環(huán)內(nèi)酯類和四環(huán)素類抗性基因編碼。

藥敏實(shí)驗(yàn)法需要24～48 h純化細(xì)菌，另24～72 h完成敏感性試驗(yàn)[64]，為了提高敏感性測試時間，PCR技術(shù)這一快速平臺在臨床實(shí)驗(yàn)室獲得認(rèn)可[60]。PCR技術(shù)是一種用于放大擴(kuò)增特定的DNA片段的分子生物學(xué)技術(shù)，可使微量的DNA大幅增加，無需對微生物進(jìn)行分離培養(yǎng)，從而被廣泛地應(yīng)用于ARGs的研究[65-66]。PCR技術(shù)可與微生物培養(yǎng)相結(jié)合用于檢測ARGs，Lopes等[67]結(jié)合瓊脂盤擴(kuò)散法和PCR技術(shù)檢測豬身上分離的鼠傷寒沙門氏菌所攜帶的ARGs，其中多重抗藥性的菌株由blaTEM、catA1、floR、sul1、sul2、sul3、tetA和tetB共同編碼。Schoenfelder等[68]結(jié)合藥敏實(shí)驗(yàn)法和PCR技術(shù)研究凝血酶陰性葡萄球菌在家畜環(huán)境中的抗生素抗性譜。鑒于PCR技術(shù)無法對ARGs進(jìn)行準(zhǔn)確定量，qPCR結(jié)合熒光能量傳遞技術(shù)，利用熒光信號累積實(shí)時監(jiān)測整個PCR進(jìn)程以實(shí)現(xiàn)抗性基因的精確定量。qPCR技術(shù)在糞便、土壤、水和垃圾固體等多種樣品的ARGs的定量方面發(fā)揮了巨大優(yōu)勢[69-72]。普通PCR和qPCR技術(shù)已被廣泛應(yīng)用于氣溶膠中ARGs的定量檢測，如表1所示。高敏等[27]結(jié)合PCR和qPCR技術(shù)獲得了養(yǎng)雞場空氣樣品完整的抗性數(shù)據(jù)，實(shí)現(xiàn)ARGs的定性定量分析。

4 氣溶膠中ARGs對人體健康的危害(The harm of ARGs in aerosols to human health)

4.1 ARGs的傳播擴(kuò)散

由于大氣間細(xì)菌的行為、生存能力和運(yùn)輸受到缺乏信息的限制，空氣中的微生物對人體健康造成的實(shí)際風(fēng)險的評估仍然具有挑戰(zhàn)性[63]。空氣傳播可能是已知具有相對較高抗生素抗性水平的細(xì)菌引起重要疾病的主要傳播途徑[73-74]。Gibbs等[50]在距離養(yǎng)豬場的25 m、50 m、100 m和150 m處分別設(shè)置采樣點(diǎn)，在采集到的氣溶膠樣品中發(fā)現(xiàn)耐藥性金黃色葡萄球菌的濃度隨著距離的增加而減少。產(chǎn)生于畜禽糞便或畜禽本身的致病菌逸散到養(yǎng)殖場環(huán)境中形成氣溶膠，便會由傳統(tǒng)通風(fēng)方式從舍內(nèi)傳播到舍外，一旦進(jìn)入舍外環(huán)境，風(fēng)會將源于畜禽場的氣溶膠傳播開來。ARGs具有廣泛的傳播潛力，有證據(jù)表明畜禽場抗生素使用量與人類病原體中相關(guān)ARGs含量的上升有關(guān)[75]。ARGs存在于氣溶膠中可以進(jìn)行長距離的傳播[36, 76]，其在環(huán)境中的持久性殘留、傳播和擴(kuò)散比抗生素本身的危害還要大[77]。ARGs可以整合到質(zhì)粒[78]、轉(zhuǎn)座子[79]、整合子[80]等可移動遺傳元件實(shí)現(xiàn)在不同的菌株(包括致病菌)之間的橫向轉(zhuǎn)移，進(jìn)入環(huán)境的抗性基因可以在不同介質(zhì)中遷移和轉(zhuǎn)化，最終很可能進(jìn)入人體，使人體對各種抗生素的耐藥性有所增加[76]。目前關(guān)于氣溶膠中抗性基因與可移動遺傳元件的關(guān)系還不明確，有待開展進(jìn)一步的研究工作。Létourneau等[31]收集養(yǎng)殖場工人的鼻分泌物，在1名和10名工作者的鼻腔菌群分別檢出了tetA/C和tetG，由此可見，工人可以通過呼吸獲取空氣中的微生物[81]，暴露于氣溶膠化的人類病原體和抗生素抗性細(xì)菌，而其攜帶的抗性基因很可能會傳播給其他人員。

圖1 養(yǎng)殖場和醫(yī)院攜帶ARGs氣溶膠的產(chǎn)生與傳播Fig. 1 Formation and transmission of aerosols carrying ARGs in livestock farms and hospitals

4.2 致病菌耐藥性

WHO在2015年的喬治敦大學(xué)“全球未來行動”講座中提到“耐藥性的超級細(xì)菌令世界各地急診室和重癥監(jiān)護(hù)病房不堪重負(fù)”[82]，充分表明致病菌耐藥性對人體健康造成了極大威脅。目前在畜禽養(yǎng)殖環(huán)境的空氣中已檢出多種多重耐藥致病菌；Chapin等[53]分析了養(yǎng)豬場氣溶膠中致病菌耐藥性情況，結(jié)果表明，98%的分離株至少對常用的2種抗生素表現(xiàn)出高水平的抗性，而人體吸入這些空氣可能是多重耐藥性病原體從動物轉(zhuǎn)移到人類的暴露途徑。Friese等[83]在土耳其火雞和肉雞場的空氣中檢測到耐甲氧西林金黃色葡萄球菌，檢出率高達(dá)77.8%。氣溶膠中ARGs對人體的健康風(fēng)險在于致病菌一旦攜帶ARGs后容易被人體吸入，從而直接對人體造成健康危害。研究表明從豬和雞糞中釋放的大多數(shù)細(xì)菌氣溶膠中含大量病原體，其中大約80%的生物氣溶膠是可呼吸的[20]。PM2.5具有較強(qiáng)的吸附能力，是多種污染物(化學(xué)和生物源性污染物)的集合體[84-85]，由于其粒徑小，進(jìn)入機(jī)體部位深，對人體的暴露程度很高，從而產(chǎn)生較大的毒害作用[86-87]。分布在畜禽場氣溶膠(粒徑<2.5 μm)中的致病菌可能穿過并沉積在氣管、支氣管和肺泡區(qū)更深處[88]，存在于細(xì)顆粒物中的ARGs很可能是向人體呼吸系統(tǒng)傳播的重要途徑。張?zhí)m河等[89]發(fā)現(xiàn)動物舍內(nèi)的四環(huán)素和紅霉素耐藥菌氣溶膠主要沉降在人體的咽喉和支氣管，這無疑危害著動物飼養(yǎng)員的生命健康。Gao等[22]利用Andersen 8級采樣器采集畜禽場空氣樣品，在不同粒徑的顆粒物中均檢出了tetW和tetL，存在于較小顆粒中攜帶抗性基因的大腸桿菌能夠穿透并沉積在氣管和原發(fā)性支氣管中，這對養(yǎng)殖環(huán)境中易感人群的健康構(gòu)成威脅。

5 結(jié)語與展望(Conclusion and outlook)

我國是抗生素生產(chǎn)和使用大國，而畜禽養(yǎng)殖業(yè)和醫(yī)療行業(yè)作為抗生素大量使用的場所，長期以來存在大劑量和不科學(xué)用藥的情況，造成ARGs這一新型污染物在各種環(huán)境中累積、傳播。目前對于氣溶膠中ARGs的研究較少，我國尚處于起步階段。氣溶膠作為ARGs的潛在儲存庫缺乏有關(guān)種類、濃度、來源以及健康風(fēng)險等系統(tǒng)的研究數(shù)據(jù)，本文通過綜述養(yǎng)殖場和醫(yī)院2個典型場所氣溶膠中ARGs的研究現(xiàn)狀，對未來開展相關(guān)的研究提出了以下幾點(diǎn)建議：

(1)開展養(yǎng)殖場和醫(yī)院等典型場所ARGs的關(guān)聯(lián)性研究工作，致力于探討不同場所氣溶膠中ARGs污染的來源、種類、濃度、傳播擴(kuò)散機(jī)制(宿主菌和可移動遺傳元件)及關(guān)鍵影響因素(抗生素和氣溶膠中的化學(xué)成分)。

(2)由于空氣樣品所含生物量通常較低，而ARGs的采集技術(shù)和處理方法對檢測結(jié)果影響很大，統(tǒng)籌生物氣溶膠采樣、樣品預(yù)處理和ARGs鑒定(PCR和qPCR)等步驟，建立氣溶膠中ARGs的樣品采集和定性定量檢測方法具有重要意義。

(3)建立氣溶膠中ARGs風(fēng)險評估方法，主要分為劑量-效應(yīng)評估和呼吸系統(tǒng)暴露評估。通過動物實(shí)驗(yàn)和分子生態(tài)毒理學(xué)研究，確定ARGs通過呼吸系統(tǒng)進(jìn)入體內(nèi)的劑量反應(yīng)與人體耐藥水平和疾病暴露的關(guān)系；開展相關(guān)實(shí)驗(yàn)并結(jié)合歷史資料進(jìn)行典型場所氣溶膠中ARGs的呼吸系統(tǒng)暴露評估，評估重點(diǎn)為人體暴露于氣體環(huán)境中的ARGs濃度、頻率和持續(xù)時間，并考慮不同場所氣溶膠中ARGs污染程度和受試人群的差異。

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