2024, Vol. 45
2. 长江水环境教育部重点实验室, 上海 200092
2. Key Laboratory of Yangtze River Water Environment, Ministry of Education, Shanghai 200092, China
抗生素广泛应用于治疗细菌感染等疾病, 但抗生素的滥用促进了抗生素耐药性细菌（antibiotic resistant bacteria, ARB）和抗生素抗性基因（antibiotic resistance genes, ARGs）的增殖与传播[1~3], 严重威胁环境与人类健康.抗生素耐药性已成为人类的主要死亡原因[4], 而中国是抗生素耐药性发生率最高的国家之一[5], 目前使用的大多抗生素都可以在环境中找到对应的ARB[6].ARGs是ARB拥有抗生素耐药性的原因, 是环境中广泛存在的一种新污染物, 具有迁移转化途径复杂和种类多样等特点, 对人类健康存在威胁, 成为世界各地面临的日益严峻的挑战[7~9].ARGs主要依靠水平基因转移（horizontal gene transfer, HGT）在不同微生物之间传播抗生素耐药性, 导致ARB的增殖[10, 11].ARGs可分为胞内抗生素抗性基因（intracellular antibiotic resistance genes, iARGs）和胞外抗生素抗性基因（extracellular antibiotic resistance genes, eARGs）[12].iARGs主要存在于染色体等胞内DNA（iDNA）中, eARGs主要存在于水中的整合子、转座子或质粒等胞外DNA（eDNA）上[13].虽然eARGs逐渐受到越来越多的关注[14], 但相较于iARGs, 水中eARGs的研究较少[15], 原因之一是目前eARGs回收方法存在不足[16], 未能广泛适用于水中较低丰度的eARGs环境[17].
在水中分布广泛的eARGs是ARB活体分泌或死后释放的一种物质[18], 具有与传统污染物截然不同的独特环境行为[19], 可存在数月且不易被降解[20].eARGs是水体、生物膜和沉积物等环境中ARGs的主要存在形式[21].eARGs可导致细菌向ARB转化[22], 因此在ARB的传播扩散方面发挥重要作用, 对人类健康和生态系统存在潜在威胁[23].然而因eARGs检测分析方法存在不足等因素, 缺乏如eARGs对ARB的传播与转移作用等方向的研究[20, 24], 因此需对eARGs开展深入研究[25].基于抗生素在全球广泛使用及ARB与ARGs安全风险健康隐患现状, 开展水中eARGs的相关研究具有理论与实践意义.本文探讨了水中的eARGs污染来源、特征与研究价值, 讨论并分析了当下从水中富集回收eARGs的方法及其优缺点, 举例探讨其在不同水中的应用案例, 以期为科学评估水中eARGs的污染现状与环境风险提理论借鉴与供技术依据.
1 水环境eARGs污染来源与污染特征 1.1 水中eARGs的污染来源医院和牲畜养殖等产生的富含未被利用抗生素的污水汇集于污水处理厂, 对污水处理厂水中的细菌产生较强的进化压力, 推动并加速了ARB与ARGs的传播与扩散, 甚至产生多重耐药的超级细菌[26, 27].污水厂对ARGs的去除率约13%~97%, 未能有效去除[28].此外, 污水厂消毒工艺会导致ARB裂解, 致使iARGs转化为eARGs[29]. Dong等[30]发现eDNA丰度与eDNA中16S rRNA基因的丰度存在显著相关性（R2=0.887 4, P < 0.05）, 证明eDNA主要由细胞裂解产生, 同样表明ARB的裂解可导致iARGs转化为eARGs.由此, 污水处理厂出水的eARGs丰度升高[31, 32], 致使接纳水体的eARGs丰度提高[33].因此污水处理厂是水环境ARB与ARGs的重要来源.
未经污水厂处理而直接排入自然水体的抗生素同样可导致环境中的非抗性细菌向ARB转化并分泌eARGs, 水环境（如管壁等）的生物膜对抗生素的富集作用加剧了这一过程[34]. Liu等[21]证明较低的抗生素质量浓度（约1 μg·L-1）有利于eARGs在水中和生物膜中的传播.
畜牧养殖大量使用的抗生素亦能导致eARGs的传播与扩散.家畜肠道内的ARB与ARGs随粪便排至土壤等环境, 可在降雨等环境因素的作用下以约8%的平均迁移率转化为水中的eARGs[19, 35].此外, 由牲畜粪便等制成的有机肥中仍存在约25%的eARGs残留[36], 在施肥后同样可通过雨水或灌溉等途径转化为地表水或地下水中的eARGs.有研究表明, 畜牧业——特别是水产养殖业, 抗生素的滥用造成水中ARB与ARGs的增殖与流行, 这在亚洲国家尤为突出[37].
1.2 eARGs的污染特征目前全球各类水体均检测出eARGs, 且eARGs种类繁多, 包括大环内酯类抗性基因（ermA和ermB等）、磺胺类抗性基因（sul1和sul2等）、β-内酰胺类抗性基因（blaTEM和apmC等）和四环素类抗性基因（tetA、tetB和tetM等）等（表 1）.此外, 我国海水[38]和管网末梢水[39]等水体已发现诸如mcr-1这类可能催生超级耐药细菌的超级抗性基因（SARGs）.这表明我国水环境存在不同程度的eARGs污染, 且包括消毒在内的水厂常规处理手段无法有效去除水中的eARGs污染[40~42].
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表 1 水中eARGs污染现状 Table 1 Current pollution status of eARGs in water |
eDNA可以与其他矿物或有机物颗粒结合形成复合物, 为eDNA提供保护以防止被环境中的核酸酶降解[17], 因此吸附在颗粒物上的eDNA可以在水中存在数月乃至两年[18, 20], 而游离的eDNA几天之内就会迅速降解[49].但Zhang等[50]的研究表明, 吸附在某些特殊粘土颗粒（如高岭土）上的eDNA则更易被光降解.
由于水环境营养匮乏且细菌寿命普遍较短, 因此其丰度与iARGs丰度受到限制；相反, 可在水中存在较长时间的eARGs就会因细菌裂解等因素而逐渐富集, 导致eARGs丰度超过iARGs[21]：清河水中eARGs丰度约为iARGs丰度的1.5~4倍[46], 污水厂出水中eARGs相对丰度可达90%[49], 长江河口生物膜和沉积物中eARGs相对丰度普遍约为50%~95%[51].
大量使用的塑料制品使水中广泛存在微塑料污染, 这可能对包括eARGs在内的基因表达产生影响, 且导致eARGs在微塑料上选择性富集[52, 53], 使游离态eARGs转变为吸附态eARGs, 延长eARGs的存在时间.微塑料对eARGs的富集能力可达iARGs的13.1倍[54], 从而推动生长在微塑料上生物膜中的细菌向ARB转化[55]. eDNA是生物膜的重要组成成分[56], 可用作生物膜的营养物质和能量来源[57], 对生物膜的生长和脱落等因素存在重要影响. Rice等[58]的研究证实, 缺乏控制细胞裂解的cidA基因可导致eDNA的释放量降低, 从而减少生物膜量, 阻碍生物膜中ARB的增殖与ARGs的扩散.
水厂处理中常见的氯消毒和紫外消毒等消毒工艺未能有效去除eARGs, 甚至可促进eARGs的释放与传播：Yuan等[49]发现紫外消毒对吸附态eARGs去除量不足30%, 反而可提高游离态eARGs的丰度；Li等[59]表明氯胺消毒中较高浓度的NH4+可促进活体细菌吸收水中的eARGs；Jin等[60]证明氯消毒可促进eARGs释放, 推动不同细菌之间的ARGs交换；Yuan等[22]发现氯消毒使细菌对eARGs的亲和力增加了1.6~5.8倍, 氯化产生的细菌碎片可吸附eARGs, 其转化率为游离态eARGs转化率的2.9~7.2倍.
eARGs可在适宜条件下通过HGT重新进入活体细胞表达其抗药性, 如HB101型大肠杆菌对eARGs的转化率约10-5~10-4, 转化率与消毒方式等因素有关：紫外消毒比氯消毒的转化率低了18%~56%[22].环境中eARGs的自然转化可发生在各种环境区域[57], 包括河流及其沉积物、生物膜、饮用水处理厂和废水处理厂等[30, 61], 对人类健康构成潜在威胁[62].
2 水中eARGs富集回收方法与应用目前学者已建立了多种环境样品中富集回收并定量eDNA的方法[63], 不同方法均存在不足或缺陷[49], 目前尚无针对水中eARGs富集回收的标准化方法.水中常用的eARGs富集回收方法大致可分为化学试剂法、磁珠提取法和吸附-洗脱法这3类.
2.1 化学试剂法化学试剂法是利用乙酸钠（CH3COONa）和十六烷基三甲基溴化铵（CTAB）等盐类回收水样中的eARGs[64].图 1展示了使用乙酸钠进行eDNA回收的流程, 即在过滤后的水样中以0.05∶0.5∶1.1的比例分别加入3 mol·L-1乙酸钠（pH 5.2）、水样和无水乙醇, 在-20℃下过夜储存后离心, 弃掉上清液后即可使用试剂盒提取沉淀当中的DNA[65, 66].
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图 1 乙酸钠回收eDNA流程 Fig. 1 Illustration of eDNA extraction by sodium acetate |
Corinaldesi等[67]建立的CTAB法可有效去除样品中的多糖和蛋白质等杂质, 不仅可应用于水环境, 也可用于生物膜和污泥等固体样品的eARGs回收[64].Mao等[68]改良过的CTAB法相当于在CTAB法之上增加了预处理步骤.相比使用乙酸钠进行核酸沉淀, CTAB法相对较为繁琐.由于存放DNA的容器多为聚丙烯管, 且DNA在储存过程中会与聚丙烯结合导致DNA的变性与损失, 而较高的离子强度可以加剧这种效应[69], 因此化学试剂法中乙酸钠或CTAB等盐类化学剂的使用会促进eDNA的损失, 降低eDNA的回收率[65].
Zhang等[48]实验表明CTAB法对水中eARGs可能具有较好的回收效果（66%~91%）, 但Yuan等[49]实验结果却显示乙酸钠法与CTAB法对水中eARGs的回收率均不足10%.这可能是因为他们在所检测的水样和实验操作等因素上存在差异.因此, 虽然化学试剂法对水中游离态或吸附态的eARGs均可进行回收[30], 但回收效果不稳定, 无法富集水样中的eARGs[64], 故更适于沉积物、污泥和生物膜等固体样品的eARGs回收[70].
2.2 磁珠提取法磁珠提取法是一种针对小体积样品中eARGs的回收方法[49].磁珠提取法的流程如图 2所示, 即：向5 mL过滤后的水样中加入10 mL异丙醇和7.5 mL Cl缓冲液后, 再加入20 μL磁珠液并涡旋.静置沉淀以弃去上清液, 再用1 mL CW1缓冲液（7 mol·L-1盐酸胍, 溶于50%异丙醇中）洗涤, 再次沉淀去除上清液.用1 mL CW2缓冲液（75%乙醇）洗涤两次后静置5 min.加入30 μL洗脱缓冲液, 涡旋5 min后放置在磁性架上沉淀并收集上清液进行后续DNA分析.
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图 2 磁珠回收eDNA流程 Fig. 2 Illustration of eDNA extraction by magnetic beads |
磁珠提取法基于磁珠对DNA有相较于蛋白质和腐植酸更强的亲和力的原理, 能对DNA进行可逆吸附, 因此可不受样品条件限制, 有效富集回收污水和污泥等样本中的eARGs, 回收率可达85%[64].磁珠法操作步骤相对简易且回收能力强, 可回收5 mL样品中的高质量浓度（78 ng·mL-1）的DNA, 回收能力为化学试剂法或试剂盒的20倍[49].然而磁珠法对大体积或更小体积样品的回收效果欠佳, 且存在部分干扰[16, 49].虽然磁珠法无需额外的缓冲液洗脱样品, 但仍可用缓冲液洗脱Na+离子以提高磁珠的回收率[49].由于磁珠比表面积较大, 能结合更多的DNA[71], 因此具有较高的材料利用率.然而磁珠提取法价格较为昂贵[72].
由于磁珠法同样无法富集eARGs且处理水样体积较小, 因此更适合从医院和污水厂等eARGs丰度较高的水体富集回收eARGs, 而不适用于给水厂和供水管网等eARGs丰度较低的环境.
2.3 吸附-洗脱法利用核酸吸附颗粒（NAAPs）进行吸附-洗脱富集eARGs需将过滤后的水样匀速通过NAAP吸附柱, 随后用洗脱液洗脱并收集.洗脱液借助异丙醇沉淀DNA后用70%乙醇洗涤并用TE缓冲液重悬, 再使用试剂盒纯化即可进行后续分析[18], 其流程如图 3所示.
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图 3 NAAPs柱吸附-洗脱回收eDNA流程 Fig. 3 Illustration of eDNA extraction by adsorption-elution |
利用NAAPs吸附-洗脱可对水中游离的eDNA进行富集[43].该方法可从水样中获得95%的eDNA回收率, 对质粒和短链DNA均有较好的富集回收效果[18], 能够从细菌和eDNA丰度较低的自来水等水样富集eDNA（检出限为10-1 copies·mL-1）[73], 适用于生活饮用水、河流、湖泊和雨水等多种环境水体[18, 39, 40].影响该方法对eARGs回收率的主要因素是pH、高锰酸盐指数和eARGs丰度等.NAAPs富集水样的最适pH为6~9；高锰酸盐指数（以O2计）低于11.32 mg·L-1时, 该方法对eARGs的富集回收效果最优；然而本方法仅可对较低丰度的eARGs实现有效回收, 当水样中eARGs的丰度高于103 copies·mL-1时, 该方法的回收率降低[18].
也有研究者对吸附-洗脱法进行改良[57], 先使用0.22 μm的亲水性聚醚砜过滤器（PES）对水样连续过滤, 再利用NAAPs对eARGs吸附-洗脱进行富集回收, 最高可获得99%的eARGs回收率.
虽然本方法对高eARGs丰度的水样回收效果欠佳, 但可对水样进行富集, 因此更适用于自来水、地下水和雨水等eARGs丰度较低的水体, 而不适用于污废水等eARGs丰度较高的水样.
2.4 3种方法在不同水样的应用比对综上所述, 3种富集回收水中eARGs的方法均存在一定的优势与不足.有研究表明, 每个回收方法的不同处理步骤均会对实验结果造成一定偏差[74], 采用不同的方法也可能会导致微生物种属检测的偏差[75], 目前尚无可以涵盖整个微生物多样性的方法[63].因此在针对不同的水体样本时, 应综合分析水样所适用的预处理方法和eARGs回收方法, 选择恰当的方式开展实验, 否则可能会因为错误的预处理（例如使用超声波处理可能会导致细菌裂解, 从而使iARGs变为eARGs[49]）或回收方法导致结果产生偏差或误差.
表 2简要概括了上文所述的3种方法目前在不同水样中的应用案例, 并列举出实验者所测得的eARGs丰度及回收率, 以期为读者提供参考.
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表 2 3种方法在不同水样的应用案例1） Table 2 Application examples of three methods to different water samples |
2.5 其他方法与新兴方法
除上述3种常用的回收富集方法, Liang等[78]也通过特殊的过滤装置回收水中的eARGs.然而本方法回收率低（不足20%）, 回收体积较小（100 mL）.该方法的eARGs回收率受总悬浮固体浓度（TSS）影响, TSS越大, eARGs回收率越高.Yuan等[49]的实验表明过滤所截留的固体颗粒存在吸附态的eARGs, 而游离态的eARGs则存在于滤后水中, 这可能解释了Liang等[78]过滤法回收率低且回收效果与TSS呈正相关的原因.然而目前缺乏有关过滤法回收水中eARGs更广泛的应用案例.
目前回收水中eARGs的方法逐步完善改进, 有研究者制作了基于分子印记技术的腺嘌呤印记珠[16]和孔径在3.0 nm左右的磁性纳米颗粒[79]用于回收水中的eARGs.腺嘌呤印记珠可快速、方便并高选择性地回收eARGs, 可避免常见竞争化合物的干扰, 回收率较传统方法有所提高, eARGs回收率在83%~96%之间, 使得该方法成为从复杂的环境样品中回收eARGs的优秀方法[16]；由于磁性纳米颗粒对DNA具有较高的亲和力, 因此能有效抵抗竞争化合物的干扰, 应用范围广[79].然而目前尚缺乏两项技术在水中的相关应用案例, 因此两者的实用价值有待进一步评估.
3 展望我国内陆水域、近海海水、井水和生活饮用水等水环境中均存在不同程度的eARGs污染, 然而受限于eARGs回收方法（如吸附-洗脱法仅可回收游离态的eARGs）等因素, 有关eARGs环境行为的深入研究或吸附态与游离态eARGs行为差异研究等鲜见报道, 部分研究甚至不会对游离态与吸附态的eARGs进行区分.然而水中iARGs、游离态eARGs与吸附态eARGs的丰度相当[49], 故需对水中游离态eARGs与吸附态eARGs开展深入研究分析, 以充分探讨两者在不同水环境中行为与对ARB的传播扩散能力等方面的差异.
随着习近平总书记提出“双碳”理论, 资源的可回收与再利用成为主流.由于污水处理厂出水水质达到地表水环境质量标准GB3838的相关要求, 故作为回用水应用于景观娱乐、绿化浇灌和农业灌溉等, 因此回用水中存在的ARGs污染与ARB等微生物安全问题不容忽视[3, 80]. eARGs和iARGs作为ARGs的两种不同存在形式, 均可导致ARB的传播扩散, 然而目前未建立有关eARGs的污染控制标准[81], 且常规途径对eARGs的去除效果不佳[40], 因此需探究eARGs对微生物生态的影响, 更好地了解eARGs对ARB增殖传播的能力, 为抑制生物膜的生长与ARB的扩散提供理论依据, 并探究eARGs有效易行的去除方法, 为城市供水、污水外排与再生水回用等水中eARGs风险监测与评估提供理论借鉴, 从而有助于制定合理有效的监控措施, 保护人类生命健康与用水安全.
4 结论（1）各类自然水体中存在广泛的eARGs污染, 这些eARGs可在水体中长期存在, 导致ARB的传播与扩散, 威胁人类健康, 然而目前缺乏有关eARGs的深入研究与污染控制标准.
（2）目前回收富集水中eARGs的常见方法为：化学试剂法、磁珠提取法和吸附-洗脱法这3类, 3种方法各有优势与不足, 在回收富集eARGs时应结合水质合理选用.
（3）基于腺嘌呤印记珠和磁性纳米颗粒的eARGs回收方法具有较高的回收率和抗干扰性, 然而缺乏广泛的应用案例, 实用价值有待进一步评估.
致谢: 感谢同济大学环境科学与工程学院马丽清与牛江月等同学对本论文的大力支持.
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