污泥蚯蚓堆肥对染色体和质粒上耐药基因归趋的影响

徐俊杰,夏 慧*,魏枫沂,陈 进,谢佳辰,黄 魁,2

污泥蚯蚓堆肥对染色体和质粒上耐药基因归趋的影响

徐俊杰1,夏 慧1*,魏枫沂1,陈 进1,谢佳辰1,黄 魁1,2

(1.兰州交通大学环境与市政工程学院,甘肃 兰州 730070；2.甘肃省黄河水环境重点实验室,甘肃 兰州 730070)

染色体和质粒分别介导污泥中的抗生素抗性基因(Antibiotic resistance genes, ARGs)进行垂直和水平转移,使ARGs在亲代或不同菌种之间传播,导致污泥蚯蚓堆肥对ARGs的削减有限.为了解决这个问题,本实验通过研究蚯蚓堆肥过程中染色体与质粒上ARGs和移动遗传元件(Mobile genetic elements, MGEs)的丰度变化,以无添加蚯蚓为对照,进行20d的蚯蚓堆肥,探究蚯蚓堆肥对污泥中ARGs的垂直和水平转移的影响.结果显示:前10d是污泥蚯蚓堆肥中ARGs转移的高峰期.除了,蚯蚓组其余ARGs丰度在质粒和染色体上均发生了显著的增加(<0.05).与对照组相比,质粒上的、、、的丰度在蚯蚓组显著增加了1.02倍、1.97倍、2.43倍、0.75倍(<0.05),而染色体上仅在蚯蚓组显著增加(<0.05).对于MGEs,质粒上的1丰度在蚯蚓组中比对照组显著增加了1.63倍(<0.05),而染色体上的却截然相反,是对照组大于蚯蚓组.堆肥的后10d,两组染色体和质粒中的MGEs和总ARGs的丰度均降低,且蚯蚓堆肥组降低速度更快.蚯蚓堆肥中,在质粒上MGEs与、、有显著的正相关性(<0.05),而在染色体上MGEs与所有ARGs均无显著相关性.冗余分析发现,ARGs的变化与MGEs、蚯蚓堆肥引起的环境变化有关,而且环境因素如电导率、有机质、氨氮和硝酸盐氮,对质粒上ARGs和MGEs的影响比对染色体的更为强烈.综上所述,携带MGEs的质粒介导的水平转移是蚯蚓堆肥中ARGs难以削减的的主要原因.

抗生素；抗性基因；遗传元件；剩余污泥；堆肥；蚯蚓

随着我国城市污水处理规模的提升,剩余污泥产量也逐年增加[1].然而,由于长期的“重水轻泥”,污泥处理处置形势非常严峻[2].污泥成分极为复杂,既含有碳、氮、磷等可利用物质,也含有重金属、有机污染物、微塑料、ARGs等污染物[3]. ARGs为环境中新型生物污染物,污泥中已发现有360种ARGs[4].污水中的ARGs积蓄在污泥中,其去除效果甚微[5].因此,控制和减少污泥资源化过程中的ARGs的传播和污染,成为亟待解决的问题.

蚯蚓堆肥是利用蚯蚓和微生物的协同作用,分解转化污泥中难降解有机物的一种资源化技术,其成本低,操作简单,可持续性处理污泥,同时蚯蚓粪富含较多的植物可利用的营养物质以及丰富的农业有益菌群,具有较高的市场价值[6-7].但是由于污泥来源的复杂性,蚯蚓堆肥对其中ARGs的削减并不显著[8-9].Huang等[10]发现蚯蚓堆肥可以选择性地清除剩余活性污泥中一些四环素和磺胺类抗性基因的相对丰度.Cui等[11]在蚓堆肥过程中发现喹诺酮类耐药基因被显著去除.然而也有报道污泥蚯蚓粪中、和的丰度在堆肥后显著增加[12-13].对ARGs在蚯蚓污泥堆肥中的传播认知不足,可能是难以控制ARGs的主要原因,因此研究ARGs在蚯蚓堆肥中的传播机制,对有效减低污泥蚯蚓粪中ARGs的环境风险尤为重要.

由于抗生素的滥用,导致抗性致病菌甚至超级细菌的滋生,ARGs的产生和传播扩散也成为一个备受瞩目的公共安全问题.ARGs的扩散传播分为垂直转移与水平转移.一方面,通过亲代遗传的垂直转移对接合子的形成和ARGs的扩散有重要作用[14].另一方面,在不同菌种之间,ARGs通过质粒进行的接合转移是水平转移的主要方式[15-16].有研究表明质粒几乎可以携带所有临床相关的抗生素抗性基因[17-18].虽然染色体和质粒在环境中均可携带ARGs进行传播,但区分染色体与质粒来研究ARGs极为鲜见.

本实验通过研究污泥蚯蚓堆肥前后染色体与质粒上ARGs的丰度变化,探究蚯蚓堆肥对ARGs的转移有何影响,为控制控制蚯蚓堆肥中ARGs的传播提出新思路.

1 材料和方法

1.1 供试材料和实验设置

实验所用新鲜脱水污泥(含水率64.54%)取自兰州市安宁区七里河污水厂,堆肥蚓种为赤子爱胜蚓(),经脱水污泥驯化7d后用于本实验.实验选用长方体塑料箱(58cm×38cm×25cm)作为堆肥反应器,供试污泥物理化学性质如表1所示.

表1 供试污泥物理化学性质

注:同列指标后存在相同字母表明两两之间不具有显著差异性(>0.05),同行字母之间无比较意义.

使用5mm×5mm金属方格网对新鲜脱水污泥进行造粒,然后在各反应器中投加12kg脱水污泥,并接种1200条蚯蚓(均重0.31g)开始堆肥实验.以无添加蚯蚓为对照组,每组设3个平行,堆肥共进行20d.所有反应器都使用遮阳布覆盖,并保持室温(20～25℃).为了保持水分,每3d喷洒一次自来水.为保持有氧条件,每隔一周手动翻堆减少污泥颗粒积压团聚.实验第0d、10d、20d各取样1次,每个反应器取2份样品,一份自然风干后研磨,过60目筛,置于4℃冰箱中保存,用于理化性质分析;另一份新鲜样品提取DNA,并置于-20℃冰箱中冷冻保存,用于DNA相关分析.

1.2 测定方法

1.2.1 理化性质分析 有机质含量采用恒重法,使用约2g新鲜样品在105℃环境下12h以烘干水分得到干样品,干样品在650℃的马弗炉中2h以测量有机物.将风干研磨样品与去离子水1:50(质量浓度)混匀,磁力搅拌30min后测定pH值(雷磁PHS-3C,上海)和电导率(雷磁DDS-307,上海).硝酸盐氮采用紫外分光光度法(HJ/T 346-2007),氨氮采用多参数水质分析仪(CNPN-7SII)测定.具体理化测试参照黄魁等[19]方法.

1.2.2 DNA和质粒提取及荧光定量PCR 取约0.25g新鲜污泥样品用DNeasy®Power Soil®Kit(Qiagen,德国)试剂盒提取DNA,并用1%琼脂糖凝胶电泳检测其浓度,所得DNA样品于-20℃冰箱保存备用.取25μL各样品DNA,使用SanPrep柱式质粒DNA小量抽提试剂盒(生工,上海)提取质粒.选用6种常见的ARGs和2种MGEs进行定量,其中包括四环素类抗性基因(、)、大环内酯类抗性基因(、)、磺胺类抗性基因(、)以及整合子()和整座子(-)引物序列及条件见文献[16].其中,和的耐药机制是靶点替换,和是靶点改变,是抗生素灭活,是靶点保护.定量反应为25μL体系:SYBR Green (艾科瑞,湖南)12.5μL,10μmoL上下游引物各1μL,DNA模板1μL, DNA-free超纯水9.5μL.所用引物均购置于生工生物工程(上海)股份有限公司,标准品为携带目的基因的质粒,详细制备过程见文献[20].

1.3 统计方法

使用Statistica 10.0统计软件对样品的理化性质、抗性基因数量在各组之间的差异进行单因素方差分析和相关性分析,显著性水平为0.05.各处理组堆肥前后ARGs的丰度图以及ARGs和MGEs之间的相关性热图使用OriginPro 2021绘制,用Canoco 4.5软件对环境因子、MGEs和ARGs之间的关系进行冗余分析.

2 结果与讨论

2.1 理化性质变化

蚯蚓堆肥引起的理化性质的变化不仅可以表征污泥稳定化效果,也有可能间接影响ARGs的丰度变化.有机质的降解和矿化作用可以直接反应蚯蚓堆肥过程中污泥的稳定性.由表1可知,堆肥第10d,对照组与蚯蚓组有机质含量比原污泥分别减少了11.40%和16.87%(<0.05).但堆肥的后10d,两组有机质含量趋于稳定,甚至在蚯蚓组出现小幅上升.电导率是反映有机质矿化程度的重要指标[21].在堆肥的前10d,电导率并没有因为有机质的降解而增加,反而出现下降趋势,表明堆肥中有机质降解后并没有及时转化为矿物盐或无机离子等物质.但在堆肥第20d,对照组与蚯蚓组电导率增加了32.58%和109.52%(<0.05).这表明在堆肥前期,有机质可能主要依靠蚯蚓的摄食作用降解为中间代谢产物或者大分子有机物等,而堆肥后期污泥中的微生物进一步降解生成小分子和无机盐物质.蚯蚓堆肥能够显著加快污泥堆肥的矿化进程,与先前研究结果一致[22].

氨氮和硝酸盐氮的含量是评估蚯蚓堆肥成熟度的重要指标.从表1可知,对照组和蚯蚓组的氨氮含量在前10d堆肥过程中持续下降,可能是堆肥初期的功能细菌AOA和AOB数量少且活性低,导致大量NH4+以NH3的形式损失[23].而堆肥的第20d,蚯蚓组比对照组氨氮显著增加了75.97%(<0.05),可能是蚯蚓的钻洞行为增加了污泥内部孔隙率并降低了厌氧率,从而促进了AOB的快速繁殖[19].堆肥前10d两组的硝酸盐氮含量持续增加但增幅较小,但后10d增加较快,堆肥前10d两组的硝酸盐氮增加较慢可能是由于堆肥前期蚯蚓摄食作用是主导者,微生物的作用并不占优势.而随着蚯蚓摄食、钻洞等行为,为硝化细菌的生长提供了良好的环境并逐渐繁殖[24],从而导致后10d硝酸盐氮快速增加.整个堆肥结束,对照组和蚯蚓组硝酸盐氮含量分别从0增加到356.87mg/kg和1253.16mg/kg,蚯蚓组的硝酸盐氮含量比对照组增加显著(<0.05),表明由于蚯蚓活动促进硝化反应的进行.所以,蚯蚓堆肥能够协同微生物促进有益氮循环,提高堆肥产物的利用价值.

2.2 ARGs丰度变化

由图1(a)染色体中ARGs丰度所示,堆肥至第20d,对于大环内酯类ARGs,蚯蚓组的和分别增加了37.45%和21.94%,而在对照组中,减少了20.16%,但增加了18.90%.堆肥至第20d,四环素类的两种ARGs呈现出截然不同的变化趋势.在蚯蚓组和对照组中分别增加了9.36倍和1.58倍,两组差异极为显著(<0.001).在蚯蚓组和对照组中分别减少了66.16%和52.48%,两组并无显著差异.对于磺胺类的ARGs,堆肥第10d,在蚯蚓组和对照组中分别增加了2.32倍和2.18倍,则分别增加了2.64倍和2.36倍.堆肥的后,10d,缓慢增加,蚯蚓组和对照组分别增加了8.70%和0.76%,而在蚯蚓组和对照组分别显著(<0.05)减少了20.84%和40.00%.

由图1(b)质粒中ARGs丰度可知,大环内酯类的两个ARGs在含量与变化趋势方面都比较相似.堆肥至第10d,蚯蚓组的和分别显著增加了94.63%(<0.01)和153.08%(<0.001),而在对照组中却分别减少了6.85%和14.75%.堆肥至第20d,相比于原始污泥,在蚯蚓组和对照组分别减少了56.23%和81.13%,分别减少了57.91%和68.63%.对于四环素类ARGs,堆肥至第20d,和在对照组中分别减少了53.85%和78.57%,而在蚯蚓组,增加了4.15倍,减少了83.87%.磺胺类ARGs与大环内酯类相似,都呈现出先上升再下降的趋势,堆肥至第20d,和在蚯蚓组中分别增加了104.27%(<0.01)和61.05%,而在对照组中基本没变化.

图1 污泥稳定化过程中各组的染色体(a)和质粒(b)上ARGs的绝对丰度

*<0.05, **<0.01, ***<0.001

结合图1(a)和图1(b)可知,同是四环素类ARGs的和呈现出截然不同的变化趋势,可能是因为两个抗性基因的抗性机制不同,是抗生素灭活基因,是靶点保护基因.值得注意的是磺胺类的两种抗性基因和丰度最高,比其他ARGs大了3个数量级左右.Luo等[17]在海河中检测ARGs也有类似的结果,这可能是由于磺胺类抗生素是人工合成的抑菌药且成本低、抗菌谱广、稳定性较高和亲水性较强[25].结合耐药机制看,污泥蚯蚓堆肥可能对靶点改变和靶点保护类ARGs削减效果较好,对靶点替换和抗生素灭活类ARGs削减效果较差.而本实验ARGs数据量较少,对耐药机制更深入的分析需要进一步的研究.

*<0.05

堆肥至第10d,蚯蚓组除了,其他ARGs在质粒和染色体中都发生了增加,且大多数ARGs较对照组增加显著(<0.05).而堆肥的后10d,质粒上的ARGs除了,其余的均处于减少状态,染色体上的ARGs除了和,其余的也均处于减少状态.大多数ARGs出现先增加后减少的现象,可能是堆肥前10d蚯蚓作用比较活跃,ARGs在蚯蚓肠道中传播比较剧烈,Cui等[11]在对剩余污泥进行蚯蚓堆肥时也发现了ARGs在前7d先增加后减少的现象.有研究表明,在蚯蚓胃中聚集的细菌群落会成为ARGs的受体[26].而后期ARGs减少可能是蚯蚓进食活动减少,细菌经过蚯蚓肠道厌氧环境的筛选,蚯蚓粪中多数为厌氧菌[27-28],排出体外后为好氧环境,厌氧菌受到抑制从而减少并影响了ARGs的传播.由图3可知,蚯蚓组对比对照组,质粒上的总ARGs和MGEs丰度在第10d和第20d分别显著(<0.05)增加82.6%和77.9%,而染色体上的增加不显著,表明蚯蚓堆肥中的ARGs很有可能是通过质粒传播的.在染色体和质粒上,总ARGs和MGEs的丰度均表现为蚯蚓组大于对照组,并且除了,蚯蚓组其他的ARGs比对照组均有增加.表明蚯蚓堆肥难以对ARGs有效削减,这与Huang等[10]通过宏基因组分析脱水污泥蚯蚓堆肥中ARGs的研究结果并不相同,这可能与堆肥周期长短有关.

*<0.05

2.3 MGEs对ARGs的影响

整合子(Ⅰ1)是一种可移动的DNA分子,具有特殊的结构,可捕获和整合外源性基因,特别是抗生素抗性基因、重金属抗性基因等,使之转变为功能性基因的表达单位[30-31].转座子(-)也能携带其他功能性外源基因在染色体和质粒间转移,其中以携带ARGs最为常见[32].所以MGEs在 ARGs的传播中发挥着重要作用[33].由图2可知,在染色体上,蚯蚓组的在堆肥的20d内持续增加,最终增加了1.37倍;而在对照组则是先增加后减少,最后比原污泥增加了2.64倍.-在蚯蚓组和对照组中均是先增加后减少,最终和原污泥相比基本上没变化,两组之间也并无差异(>0.05).两个MGEs在堆肥第10d均发生了显著增加,表明堆肥前10d可能是ARGs转移传播的高峰期.

在质粒上,堆肥至第10d,在蚯蚓组和对照组分别增加了2.61倍和0.37倍,两组有显著差异(<0.05);质粒上的-分别增加了43.73%和6.38%,两组并无显著差异.而堆肥结束后,相比原污泥,-在蚯蚓组和对照组分别减少了60.62%和70.63%,而在两组的丰度基本和原污泥相等.

结合图1和图2,质粒中的和-先增加后减少的趋势与大部分ARGs相同,且蚯蚓组丰度高于对照组,而染色体中MGEs的丰度则是对照组高于蚯蚓组.结果表明蚯蚓堆肥中质粒上的MGEs可能对ARGs的转移贡献度更大.

由图4所见,在质粒上,对照组中与、、呈显著正相关(<0.05),与有较强正相关(<0.01).而蚯蚓组中与、、有极为显著的正相关(<0.001),与有较强正相关性(<0.01).该结果与Duan等[34]研究的ARGs的增多可能与堆肥产物中的丰度有关相符.对照组中-与、有显著正相关(<0.05),与、、有极为显著的正相关(<0.001).蚯蚓组中-与有显著正相关(<0.05),与有较强正相关(<0.01),与、有极为显著的正相关(<0.001).以上结果显示,蚯蚓组的MGEs比对照组,与ARGs的相关性更加密切.蚯蚓组ARGs增加的比对照组更快,进一步表明蚯蚓堆肥体中ARGs的归趋受MGEs的影响[35-36].在染色体上,蚯蚓组的和-与所有ARGs都无显著正相关,说明MGEs对ARGs垂直转移的几乎无帮助.综上所述,携带着MGEs的质粒所介导的水平转移是蚯蚓堆肥中ARGs传播的重要方式.

2.4 环境因子、MGEs和ARGs之间的关系

由图5可知,堆肥至第10d,蚯蚓堆肥进程和、-在染色体和质粒中均呈现显著正相关(<0.05),而且蚯蚓堆肥进程与质粒中的、、、,染色体中的、、都呈现显著正相关(<0.05).而堆肥20d时,蚯蚓堆肥进程与上述指标几乎无相关性.这个现象与先前的数据结合,ARGs和MGEs在蚯蚓堆肥第10d显著增加,表明蚯蚓堆肥会增加ARGs传播的风险.由图5(b)可知,在ARGs激增的前10d,质粒中的ARGs与MGEs在蚯蚓堆肥组中比对照组中增加显著,进一步证明蚯蚓堆肥中ARGs的传播主要依靠的是质粒.质粒中的、、、和-与电导率、氨氮、硝酸盐氮呈负相关,而染色体中的这些ARGs与环境因子之间相关性很低.这表明环境因素主要影响的是质粒介导的水平转移[37],且电导率、氨氮和硝酸盐氮可能会对ARGs和MGEs的丰度有一定的抑制作用.因此,在污泥蚯蚓堆肥中,进一步加快有益氮循环,既可以增加蚯蚓粪的可利用价值,又可能对质粒上ARGs和MGEs的削减有所帮助.

有研究表明,环境因子对ARGs丰度的影响主要取决于它们对其潜在宿主细菌的影响[38-39].同时,质粒和染色体中的和-与、、都呈显著正相关(<0.05),表明这些ARGs可能在蚯蚓堆肥前10d借助MGEs快速传播.进一步说明MGEs在蚯蚓堆肥过程中ARGs的传播中发挥了重要作用[40].综上所述,环境因子会强烈地影响蚯蚓堆肥时质粒中ARGs和MGEs的变化.因此如何调控蚯蚓堆肥体中环境因子来阻控质粒介导的水平转移,有待进一步研究.

3 结论

3.1 蚯蚓堆肥的前10d是ARGs转移的高峰期,而后10d,ARGs的丰度会下降.

3.2 蚯蚓堆肥引起的环境因子变化会影响ARGs和MGEs的丰度,从而影响ARGs的水平转移.

3.3 质粒上的MGEs与ARGs丰度变化呈显著正相关,表明携带MGEs的质粒在ARGs的水平转移中起着至关重要的作用.

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Fate of antibiotic resistance genes on chromosomes and plasmids affected by earthworms during vermicomposting of dewatered sludge.

XU Jun-jie1, XIA Hui1*, WEI Feng-yi1, CHEN Jin1, XIE Jia-chen1, HUANG Kui1,2

(1.School of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China；2.Key laboratory of Yellow River Water Environment in Gansu Province, Lanzhou 730070, China)., 2023,43(2)：694～701

Chromosomes and plasmids mediate the vertical and horizontal transfer of antibiotic resistance genes (ARGs) in sludge, respectively, which allows ARGs to spread between parents or different bacteria strains, resulting in limited reduction of ARGs during vermicomposting. To address this issue, the effects of vermicomposting on vertical and horizontal transfer of ARGs in sludge were investigated by detecting the abundance changes of ARGs and mobile genetic elements (MGEs) on chromosomes and plasmids during vermicomposting for 20 days, with no addition of earthworms as the control. The results showed that the first 10d was the peak of ARGs transfer in sludge vermicomposting. Except forgene, a significant increase in the abundance of the remaining ARGs in the vermicomposting occurred on both plasmids and chromosomes (<0.05). Compared with the control, the gene abundances of,,, and1on plasmids significantly increased by 1.02-fold, 1.97-fold, 2.43-fold, and 0.75-fold in the vermicomposting (<0.05), while onlyon chromosomes significantly increased (<0.05). Compared with the control, the MGEs abundance of1 on plasmids significantly enriched by 1.63-fold in the vermicomposting (<0.05), while its abundance on chromosomes was diametrically opposite, its abundance in the control was larger than vermicomposting. In the 10～20 d of composting, the abundance of MGEs and total ARGs on chromosomes and plasmids decreased in both treatments, with a faster decrease in the vermicomposting. In addition, the MGEs had a significant positive correlation (<0.05) with,, and2 on plasmids, while no significant correlation among MGEs and all ARGs on chromosomes was recorded during vermicomposting. The redundancy analysis revealed that the changes of ARGs were related to the MGEs and environmental changes during vermicomposting, and the environmental factors such as conductivity, organic matter, ammonia and nitrate had a stronger effect on ARGs and MGEs on plasmids than those on chromosomes. This study suggests that the plasmids carrying MGEs mediated horizontal transfer is a major reason for hardly reducing ARGs in sludge vermicompost.

antibiotic；resistance gene；mobile genetic element；excesssludge；composting；earthworms

X171.5

A

1000-6923(2023)02-0694-08

徐俊杰(1998-),男,安徽宿州人,兰州交通大学硕士研究生,主要研究污泥资源化技术.发表论文4篇.

2022-07-01

国家自然科学基金资助项目(51868036;52000095);甘肃省科技计划资助项目(20JR2RA002);甘肃省优秀研究生“创新之星”项目(2022CXZX-559)

* 责任作者, 副教授, xiahui@mail.lzjtu.cn