基于宏基因组学探讨东平湖水库的菌群结构、耐药基因谱及其公共健康风险

水库中长时间滞留的水体会成为耐药基因的存储库,因此研究其菌群结构、耐药基因谱及其潜在的健康风险具有重要的公共卫生学意义.本研究以东平湖水库的水体和沉积物为研究对象,采用宏基因组测序技术,系统分析其在旱季和雨季中的菌群结构、耐药基因谱、耐药基因水平转移以及携带耐药基因的致病菌类型.结果表明,与水库沉积物相比,水体中的菌群结构和耐药基因谱受季节影响较大,且旱季的总耐药基因丰度明显高于雨季;水库沉积物中总耐药基因丰度高于水体,但水体中耐药基因的水平转移高于沉积物.本研究共检测到20个耐药基因型,377个耐药基因亚型;杆菌素和万古霉素耐药基因分别是水和沉积物中主要的耐药基因型,变形杆菌和放线菌分别是水体和沉积物中主要携带耐药基因的菌群.此外,本研究还鉴定出30株携带耐药基因的致病菌,包括大肠杆菌、肺炎克雷伯菌、铜绿假单胞菌、肠炎沙门氏菌以及鲍曼不动杆菌,其中有2株大肠杆菌携带毒力因子.综上所述,东平湖水库中的耐药基因不仅种类繁多,而且还存在潜在的公共健康风险;因此定期检测水体环境的菌群结构及其耐药基因谱具有重要的科学价值和现实意义.     

Metagenomic insights into the profile of antibiotic resistomes in sediments of aquaculture wastewater treatment system

To meet the rapidly growing global demand for aquaculture products, large amounts of antibiotics were used in aquaculture, which might accelerate the evolution of antibioticresistant bacteria(ARB) and the propagation of antibiotic genes(ARGs). In our research, we revealed the ARGs profiles, their co-occurrence with mobile genetic elements(MGEs), and potential hosts in sediments of a crab pond wastewater purification system based on metagenomic analysis. The residual antibiotic seems to increase ...     

Coastal mudflats as reservoirs of extracellular antibiotic resistance genes: Studies in Eastern China

Despite coastal mudflats serving as essential ecological zones interconnecting terrestrial/freshwater and marine systems, little is known about the profiles of antibiotic resistance genes (ARGs) in this area. In this study, characteristics of typical ARGs, involving both intracellular (iARGs) and extracellular ARGs (eARGs) at different physical states, were explored in over 1000 km of coastal mudflats in Eastern China. Results indicated the presence of iARGs and eARGs at states of both freely present or attached by particles. The abundance of eARGs was significantly higher than that of iARGs (87.3% vs 12.7%), and their dominance was more significant than those in other habitats (52.7%-76.3%). ARG abundance, especially for eARGs, showed an increasing trend (p < 0.05) from southern (Nantong) to northern (Lianyungang) coastal mudflats. Higher salinity facilitated the transformation from iARGs to eARGs, and smaller soil particle size was conducive to the persistence of eARGs in northern coastal mudflats. This study addresses the neglected function of coastal mudflats as eARGs reservoirs.     

Temporal variation and sharing of antibiotic resistance genes between water and wild fish gut in a peri-urban river

Antibiotic resistance genes (ARGs) as emergence contaminations have spread widely in the water environment. Wild fish may be recipients and communicators of ARGs in the water environment, however, the distribution and transmission of ARGs in the wild fish and relevant water environment were rarely reported. Here, we have profiled ARGs and bacterial communities in wild freshwater fish and relevant water in a peri-urban river using high-throughput qPCR and 16S rRNA gene sequence. A total of 80 and 220 unique ARG subtypes were identified in fish and water samples. Fish and water both showed significant ARG seasonal variations (P < 0.05). The highest absolute abundance of ARGs in fish and water occurred in summer (1.32 × 10⁹ copies per g, on average) and autumn (9.04 × 10⁶ copies per mL), respectively. In addition, the bipartite network analysis showed that 9 ARGs and 1 mobile genetic element continuously shared in fish and water. Furthermore, bacteria shared in fish and water were found to significantly correlate with shard ARGs. The findings demonstrate that bacteria and ARGs in fish and water could interconnect and ARGs might transfer between fish and water using bacteria as a spreading medium.     

尾矿库水体环境抗生素抗性基因的分布特征

抗生素抗性基因是近年来人们普遍关注的新型环境生物污染物,也是科学家们研究的热点.为了探究尾矿库水体环境抗生素抗性基因的污染特征,揭示尾矿库水体环境抗生素抗性基因的影响因素,本文采用超高通量定量PCR技术（HT-qPCR）,研究了某尾矿库的4个点位的水体抗生素抗性基因的多样性、丰度和驱动机制.结果表明,尾矿库水体环境具有种类多样的抗生素抗性基因,库区水体（WK0）、主坝排渗水（WK1）、副坝排渗水（WK2）和河水（HS）分别检测出抗生素抗性基因97、52、44和56种,可移动遗传元件分别检测到11、6、3和6种;在绝对丰度水平上,库区水体（WK0）、主坝排渗水（WK1）、副坝排渗水（WK2）和河水（HS）分别有1.75×10⁸、6.39×10⁷、9.90×10⁷和7.37×10⁷ copies·L^-1,库区水体的抗性基因丰度最高,表明库区是一个潜在的抗性基因存储库;冗余分析（RDA）表明,铜Cu、总有机碳TOC和可移动遗传元件（MGEs）与抗生素抗性基因呈显著正相关的关系,这3个因素对抗生素抗性基因变化的解释量达到了61.64%（前两轴）,因此Cu、TOC和MGEs对尾矿库水体环境抗生素抗性基因的赋存和演变可能具有重要作用.     

环境中抗生素抗性基因丰度与抗生素和重金属含量的相关性分析:基于Web of Science数据库检索

抗生素和重金属是两种重要的环境污染物和耐药性选择压力源,但低含量水平下其对环境微生物种群耐药性水平的影响尚不明确.本文全面检索了截至2020年1月1日发表在Web of Science数据库中同时监测了抗生素抗性基因（ARGs）丰度、抗生素含量或重金属含量的研究文献,提取相关数据,利用统计回归方法调查环境介质中3个参数的相关关系.结果表明,除废水外,地表水、沉积物和土壤等环境介质中,即使在非常低的抗生素含量水平下,抗生素选择压力也会对ARGs丰度的增加产生显著的统计效应（P<0.05）.不同类别抗生素对耐药基因表现出不同程度的选择和富集作用.重金属含量以及抗生素含量和重金属含量的交互作用对ARGs的传播也具有显著影响（P<0.05）.水相和固相环境样本的多变量统计回归模型的最终R²分别为0.482和0.707,比单变量回归的R²更高,能更好地解释ARGs丰度的差异,但环境中还存在其他可能影响ARGs丰度的因素.本文的研究结果可为环境中抗生素耐药性风险评估及传播控制提供支撑.     

微塑料对河口沉积物抗生素抗性基因的影响

微塑料和抗生素抗性基因都是环境中的新兴污染物,也是近年来的研究热点.为探究微塑料对河口沉积物抗生素抗性基因的影响,在沉积物中添加3种不同的微塑料进行培养实验,主要采用高通量定量PCR方法研究河口沉积物抗性基因种类、丰度、多样性及其变化情况.结果表明,微塑料显著改变了沉积物中抗生素抗性基因结构组成,两种难降解微塑料PVC和PE使得沉积物抗生素抗性基因组成结构显著改变,而可溶性微塑料PVA使得沉积物抗生素抗性基因种类数显著减少;添加PVC、PE和PVA微塑料的沉积物抗生素抗性基因的绝对丰度显著增加,分别为4. 1×10~9、8. 1×10~9和2. 0×10~9copies·g~(-1),添加PE微塑料的沉积物抗生素抗性基因丰度增加了近一个数量级,微塑料显著增加了沉积物抗生素抗性基因的绝对丰度; OLS回归分析显示,抗生素抗性基因丰度与转座子、整合子基因显著正相关,表明可移动遗传元件可能促进了抗生素抗性基因的迁移、传播和扩散.     

酸性矿山废水对沉积物真核微生物群落的影响

对受酸性矿山废水污染的橫石河上游到下游水体和沉积物中理化参数以及沉积物中真核微生物的多样性进行系统调查,使用斯皮尔曼相关性分析和典范对应分析（CCA）甄别真核微生物群落的主要环境胁迫因子.结果表明：橫石河中真核微生物优势种群为真菌门（Fungi）（4.51%~86.69%）、绿藻门（Chlorophyta）（0.61%~77.36%）和纤毛虫门（Ciliophora）（0.81%~34.91%）.沉积物中真核微生物群落Alpha多样性随酸性矿山废水污染梯度降低而逐渐升高.与原核微生物群落不同,酸性矿山废水环境沉积物中真核微生物群落结构的变化主要受硫酸根和电导率的影响.     

福建省敖江下游抗生素抗性基因分布特征

河流流域的下游往往是各种大小城镇聚集之处,快速城市化等人类活动导致了城市河流环境剧烈改变.抗生素抗性基因是一种新型污染物,城市河流中抗生素抗性基因的传播和富集可能对城市人口健康和城市生态安全造成潜在的危害.本研究采用高通量定量PCR技术,研究福建省连江县城区上游、城区下游和河流入海口这3个采样点的抗生素抗性基因污染情况和分布特征.结果表明:(1)敖江流域连江县城区下游河流抗生素抗性基因丰度达到了3.9×10~(10)copies·L~(-1),显著高于城区上游和河流入海口的丰度(P0.05);(2)城区下游检测出了129种抗生素抗性基因,显著高于城区上游检测出的79种,而河流入海口的抗性基因种类仍然高达118种,表明河流水体环境中抗生素抗性基因一旦出现,就很难自然消减;(3)城市河流环境是抗生素抗性基因的一个重要存储库.     

渔业复垦塌陷地抗生素抗性基因与微生物群落

水产养殖行业抗生素的广泛使用引起了抗生素抗性基因（antibiotic resistance genes,ARGs）污染问题.为探究渔业复垦塌陷地ARGs污染特征,利用宏基因组学技术检测分析了渔业复垦塌陷地ARGs的相对丰度和微生物群落结构.研究区域共检测出29种ARGs,bacA在所有样品中相对丰度最高,达1.96×10^-5~1.19×10^-4.沉积物中磺胺类和四环素类ARGs相对丰度较高,井水中多药类ARGs相对丰度较高.微生物群落结构表明变形菌门（Proteobacteria）在所有样品中为最优势细菌门,沉积物中绿弯菌门（Chloroflexi）和广古菌门（Euryarchaeota）较为优势.属水平上,硫杆菌属（Thiobacillus）为沉积物中最优势细菌属,不动杆菌属（Acinetobacter）和假单胞菌属（Pseudomonas）为井水中优势细菌属.ARGs与微生物相关性分析表明,菌属与ARGs间主要呈中度相关,多种菌属与ARGs显著正相关,ARGs的分布受微生物群落结构的重要影响.渔业复垦塌陷地沉积物与井水均受到ARGs污染,应加强相应控制措施保护区域环境.     

好氧-厌氧两相堆肥过程中抗生素耐药基因的变化特征及影响因素研究

针对堆肥处理过程中抗生素耐药基因（antibiotic resistance genes,ARGs）去除效率不高、诱导潜在致病菌传播扩散等环境安全问题,采用好氧-厌氧两相堆肥处理猪粪、秸秆,探讨ARGs丰度及微生物群落的变化特征,并明确影响ARGs相对丰度变化的关键因子.结果表明:与单一好氧相比,好氧-厌氧两相堆肥能更有效地降低部分ARGs及潜在致病菌的相对丰度,其中ermB、tetK的相对丰度分别降低了39.68%、72.19%,假单胞菌属（Pseudomonas）、梭菌属（Clostridium_sensu_stricto_1）的相对丰度分别降低了96.09%、90.07%.主成分分析（Principal Component Analysis,PCA）及冗余分析（Redundancy analysis,RDA）得出,微生物群落和环境因子均显著影响ARGs相对丰度的变化（P < 0.05）,贡献率分别为82.0%、71.2%.其中,梭菌属（Clostridium_sense_stricto_1）、黄杆菌属（Flavobacterium）、尿素芽孢杆菌属（Ureibacillus）和海洋杆菌属（Oceanobacillus）是影响ARGs相对丰度变化的主要微生物菌属;温度、电导率、含水率和pH是影响ARGs相对丰度变化的关键环境因子,贡献率分别为22.0%、16.7%、16.2%和15.0%.研究显示:与好氧堆肥相比,好氧-厌氧两相堆肥能更有效地去除ARGs,并抑制潜在致病菌的繁殖扩散,从而减少环境风险;然而部分ARGs和潜在致病菌仍存在增殖现象,因此亟需进一步优化好氧-厌氧两相堆肥工艺.      

Profiling the antibiotic resistome in soils between pristine and human-affected sites on the Tibetan Plateau

With increasing pressure from anthropogenic activity in pristine environments, the comprehensive profiling of antibiotic resistance genes(ARGs) is essential to evaluate the potential risks from human-induced antibiotic resistance in these under-studied places. Here, we characterized the microbial resistome in relatively pristine soil samples collected from four distinct habitats on the Tibetan Plateau, using a Smart chip based high-throughput q PCR approach. We compared these to soils from the s...     

Effective removal of antibiotic resistance genes and potential links with archaeal communities during vacuum-type composting and positive-pressure composting

As a major reservoir of antibiotics, animal manure contributes a lot to the augmented environmental pressure of antibiotic resistance genes (ARGs). This might be the first study to explore the effects of different ventilation types on the control of ARGs and to identify the relationships between archaeal communities and ARGs during the composting of dairy manure. Several ARGs were quantified via Real-time qPCR and microbial communities including bacteria and archaea were analyzed by High-throughput sequencing during vacuum-type composting (VTC) and positive-pressure composting (PPC). The total detected ARGs and class I integrase gene (intI1) under VTC were significantly lower than that under PPC during each stage of the composting (p < 0.001). The relative abundance of potential human pathogenic bacteria (HPB) which were identified based on sequencing information and correlation analysis decreased by 74.6% and 91.4% at the end of PPC and VTC, respectively. The composition of archaeal communities indicated that methane-producing archaea including Methanobrevibacter, Methanocorpusculum and Methanosphaera were dominant throughout the composting. Redundancy analysis suggested that Methanobrevibacter and Methanocorpusculum were positively correlated with all of the detected ARGs. Network analysis determined that the possible hosts of ARGs were different under VTC and PPC, and provided new sights about potential links between archaea and ARGs. Our results showed better performance of VTC in reducing ARGs and potential HPB and demonstrated that some archaea could also be influential hosts of ARGs, and caution the risks of archaea carrying ARGs.     

Film mulching reduces antibiotic resistance genes in the phyllosphere of lettuce

Phyllosphere is an important reservoir of antibiotic resistance genes(ARGs), but the transfer mechanism of ARGs from soil and air to phyllosphere remains unclear. This study demonstrated that soil-air-phyllosphere was the dominant ARG transfer pathway, and blocking it by film mulching can reduce typical phyllosphere ARGs in lettuce by 80.7%-98.7%(89.5% on average). To further eliminate phyllosphere ARGs in lettuce grown with film mulching, the internal soil-endosphere-phyllosphere transfer pathw...     

铜对蚯蚓转化牛粪过程抗生素抗性基因的影响

以赤子爱胜蚓（Eisenia fetida）为试验蚓种,设定不同Cu浓度梯度,研究蚯蚓转化牛粪过程中Cu对ARGs消长的影响.结果表明,不同Cu浓度处理下,ARGs累积总丰度呈现出CF （未添加Cu）-1降低至4.33×10^-1copies/16S copies.低、中浓度Cu的添加显著提高了样品中ARGs的总丰度水平（P<0.05）.通过研究不同类型ARGs在不同时间阶段的变化,发现低、高浓度Cu处理组（T1、T3）在蚯蚓转化过程中提高了tet-ARGs、erm-ARGs和str-ARGs的丰度,而中浓度Cu处理组（T2）中tet-ARGs和str-ARGs丰度降低.冗余分析（RDA）得出pH值显著影响蚯蚓转化过程中ARGs相对丰度的变化（P<0.05）,且TOC、TN和Cu对ARGs的消长也发挥着重要作用.蚯蚓转化可加速牛粪中ARGs丰度的削减,一定程度上能够减少其环境风险,但牛粪中Cu的残留可导致蚯蚓转化过程中部分ARGs的富集,因此亟需进一步优化蚯蚓转化处理工艺.     

氯和紫外消毒过程中胞外抗性基因的产生特征

考察了城市污水氯和紫外消毒过程中不同物理形态的胞外抗性基因的产生行为与及微生物群落的关联特征.结果表明,氯消毒尽管使胞内抗性基因丰度下降,但使结合型胞外抗性基因丰度明显上升（0.7±0.1）log,而游离型胞外抗性基因丰度下降（0.2±0.1）log.紫外消毒也使胞内抗性基因下降,但使游离型胞外抗性基因显著上升（0.4±0.2）log,而结合型胞外抗性基因丰度下降（0.3±0.1）log.氯消毒后,结合型胞外DNA（a-eDNA）中变形菌门丰度下降而其他菌门的丰度上升,细菌多样性指数由4.2上升到4.7;而游离型胞外DNA（f-eDNA）中变形菌门上升了6.6%,多样性指数则从3.5降低到2.8.紫外消毒后,a-eDNA中变形菌门丰度下降了36.6%,多样性则上升到4.8,而f-eDNA中细菌丰度变化较小.分子生态网络分析揭示了抗性基因与细菌间广泛的寄存关系,tetA、tetX、sulI和sulII分别与17、15、15和5种菌属间存在共现性,表明抗性基因潜在宿主的变化是导致消毒后胞外抗性基因产生的关键原因.本研究表明氯和紫外消毒不能消除抗性基因风险,反而通过导致不同胞外抗性基因的大量产生,使风险的形式发生变化.     

梅花鹿养殖场抗生素抗性基因分布特征

梅花鹿养殖是近年来我国南方地区出现的新兴养殖业.为探究梅花鹿养殖对养殖环境抗生素抗性基因的影响,采用高通量荧光定量PCR研究了抗生素抗性基因在养殖场区对照土壤、梅花鹿新鲜粪便、梅花鹿粪便堆肥产物和施用堆肥菜地土壤中的分布、丰度和多样性.结果表明,抗性基因的绝对丰度梅花鹿粪便堆肥产物梅花鹿新鲜粪便施用堆肥菜地土壤养殖场区对照土壤,抗生素抗性基因在4种环境样品中具有不同分布格局;梅花鹿养殖场环境中抗性基因丰度与可移动基因元件丰度显著相关(P0.05),表明可移动基因元件可能加快了抗性基因的水平转移过程,促进了抗性基因的迁移、传播和富集,加剧了养殖环境抗生素抗性基因污染.     

死菌DNA对厌氧消化污泥中抗生素抗性基因及微生物群落分析的干扰

为评估死菌DNA对厌氧消化污泥抗生素抗性基因(ARGs)和微生物群落分析的潜在干扰,本研究对3种不同类型厌氧消化污泥进行叠氮溴化丙锭(PMA)处理,比较在PMA屏蔽死菌DNA PCR扩增情况下污泥ARGs和微生物群落分析结果与未经PMA处理情况下的差异.结果表明,经PMA处理后,剩余污泥自厌氧消化样品和高含固厌氧消化污泥样品中的ARGs丰度分别下降了41%～86%和74%～98%;污泥水解液厌氧消化15 d后的污泥样品中ARGs下降幅度相对较小,但降幅最高也达到34%.PMA处理对3个来源不同的厌氧消化污泥微生物群落组成分析结果呈现不同程度的影响,对高含固厌氧消化污泥的微生物群落结构分析影响最为显著.在经PMA处理与未经PMA处理两种情况下,厌氧消化污泥ARGs与微生物群落组成相关性分析的结果也截然不同.研究证明了死菌DNA对厌氧消化污泥ARGs和微生物群落分析的潜在干扰,采用PMA预处理能够更准确地反映厌氧消化污泥中的微生物群落及菌体携带ARGs的特征.     

不同预处理方式对剩余污泥中活菌菌群及ARGs的影响

剩余污泥作为抗生素抗性基因（ARGs）的储库,对其进行预处理有助于后续厌氧消化（AD）,同时控制ARGs进入AD后的传播风险.本文采用叠氮碘化丙锭（PMA）处理手段,来研究不同预处理方式对活菌菌群及携带ARGs的影响.研究结果显示,热碱、热水解和微波预处理对污泥ARGs去除效果较优,分别为3.32log、3.13log和2.95log;超声波预处理对活菌ARGs仅去除0.58log.在热水解预处理时间延长过程中,菌群大量死亡出现在1~2h间,4h后去除率达2.42log.变形菌门是污泥中最主要的优势菌门,厚壁菌门、绿弯菌门在微波、热水解预处理后实现较大提升.相关性结果显示,sulI与tetC高度相似,且同时与多个科属的微生物成显著正相关,推测其宿主范围较广,且sulI与tetC在预处理中较好的去除率说明其宿主菌对预处理耐受性差;ermB、tetA与占比较高的菌群没有正相关.多数ARGs携带菌对热水解、热碱和微波不耐受,污泥预处理能够降低ARGs丰度,适当延长低温热水解预处理时间,才能有效削减污泥中的ARGs.     

施肥对农田土壤抗生素抗性基因影响的整合分析

探讨施肥措施对农田土壤中抗生素抗性基因(ARGs)的影响,为了解和掌握农田土壤中ARGs的来源与积累特征,制定保障生态环境安全和人类健康的管理策略提供科学依据.通过收集近20年(2000～2020年)来已发表的文献,获得独立实验条件下农田土壤ARGs数量和相对丰度匹配数据215组和201组.结果表明,与不施肥相比,配施...