畜禽粪便中抗生素抗性基因的分布特征及消减技术研究进展

随着集约化畜禽养殖业的不断发展,兽用抗生素的长期使用导致畜禽粪便抗生素抗性基因污染日益严重,对生态环境和人类健康造成严重危害。如何有效消减畜禽粪便中的抗生素抗性基因成为当前研究热点。本文系统总结了畜禽粪便中抗生素抗性基因的产生途径、分布和影响因素,并阐述了好氧堆肥、厌氧消化及其强化工艺消减畜禽粪便抗生素抗性基因的研究进展,根据现有工艺研究存在的问题展望了今后的重点研究方向,为畜禽粪便中抗生素抗性基因的消减提供理论基础和技术支撑。     

基于CiteSpace文献计量的畜禽粪便抗生素抗性基因研究进展与热点分析

自20世纪40年代抗生素被发现以来,抗生素广泛应用于畜禽养殖系统,极大地促进了畜禽健康和生长。由于大多数抗生素的性质都是水溶性的,有30%−90%的兽用抗生素会以母体化合物的形式通过尿液或粪便释放到环境中。抗生素的不当使用会对环境产生选择性压力,促使抗生素抗性基因(antibiotic resistance gene,ARG)快速传播,对人类健康和生态系统构成潜在风险。因此,认识和回顾近20年国内外关于畜禽粪便ARG的研究热点、前沿和趋势,可为畜禽粪便ARG污染管控提供重要参考,也为今后的研究提供思路。本研究应用CiteSpace可视化软件对2002–2022年发表在Web of Science (WoS)核心合集和中国知网(China National Knowledge Infrastructure,CNKI)的文献通过关键词共现分析、关键词聚类分析、关键词突现分析、发文数量分析、国家分布机构合作共现等方式对畜禽养殖系统中ARG研究现状及新兴趋势进行分析,最终共筛选出896篇有效文献。自2011年以来,畜禽粪便ARG国内外文献发表数量逐年增加,2014年以后增速明显加快,其中英文文献的发文量要多于中文文献;中国是发文量最多的国家,而中国科学院是发文量最多的机构。热门的研究领域包括ARG的产生和来源、在环境中的传播及其对人类病原体的潜在暴露途径及消减机制。今后研究工作可利用宏基组学技术、高通量定量基因芯片手段和单细胞拉曼光谱等新兴分子生物学技术进行更为深入的机理性探索,同时加强畜禽粪便ARG消减技术策略的发展和应用。本文以可视化的方式阐释了畜禽粪便ARG研究发展态势,提出了未来重点研究方向,为管控畜禽粪便ARG提供了理论基础。     

高风险四环素抗性基因在人工湿地中分布和去除的季节变化

畜禽养殖废水是抗生素抗性基因(ARGs)的重要贮存库,环境风险不容忽视。本研究考察了夏、冬两季养猪废水中高环境风险四环素抗性基因(TRGs)在水平潜流人工湿地中的分布和去除情况,通过外源添加四环素(TC)和铜离子(Cu²⁺)探究了养猪废水中抗生素与重金属单一和复合污染对湿地出水中TRGs丰度的影响。结果表明: 养猪废水中3种高环境风险TRGs(tetM、tetO、tetW)均有检出,人工湿地可有效消减废水中的TRGs,夏、冬两季时出水中TRGs的绝对丰度较进水分别降低1.1～2.4和1.7～2.9个数量级。TRGs在湿地土壤中的丰度呈现出水端低于进水端、植物非根际低于根际、冬季低于夏季的特征。与对照相比,养猪废水中TC、Cu²⁺单一和复合污染在夏、冬两季均会导致湿地出水中TRGs相对丰度的增加。人工湿地作为一种生态处理工艺可用于控制畜禽养殖废水中的ARGs向环境中扩散。     

污水处理厂中四环素和磺胺类抗生素抗性基因的分布、传播及去除

近年来,抗生素滥用造成的抗性基因(ARGs)污染问题引起了人们的关注.四环素及磺胺类抗生素由于价格低廉被广泛使用,大量残留的四环素和磺胺通过各种途径进入污水处理厂,并进一步导致ARGs的污染.为深入了解四环素和磺胺类ARGs的污染及治理现状,本研究对污水处理厂中四环素和磺胺类ARGs的分布情况及传播机制进行了综述,并重点讨论了不同污水处理工艺对ARGs的去除效果.在此基础上,从加大污水处理厂ARGs污染调查、改进污水处理工艺以及探讨ARGs传播机制等方面进行了展望.     

抗生素耐药基因在畜禽粪便-土壤系统中的分布、扩散及检测方法

抗生素耐药基因作为一种新型的环境污染物已引起研究者的高度关注。畜禽养殖业长期将抗生素添加到饲料中,在促进动物生长、预防和治疗动物疾病等方面起了重要作用。这些抗生素大多数不能被动物完全吸收,在动物肠道中诱导出耐抗生素细菌和抗生素耐药基因,并随着粪便排出体外。畜禽粪便作为重要的抗生素、耐抗生素细菌和抗生素耐药基因储存库,通过堆粪、施肥等农业活动进入土壤环境中,可刺激土壤中耐抗生素细菌和抗生素耐药基因的富集。耐药基因借助于基因水平转移等方式在土壤介质中进一步传播扩散,甚至进入植物中随食物链传播,对生态环境和人类健康造成极大的威胁。为了正确评估抗生素耐药基因的生态风险,本文结合国内外相关研究,系统阐述了畜禽粪便-土壤系统中抗生素耐药基因的来源、分布及扩散机制,同时探讨了细菌耐药性的主要研究方法,指出堆肥化处理仍是目前去除抗生素耐药基因的主要手段,并对今后的研究方向进行展望。     

畜禽粪便堆肥过程中酶活性及微生物数量的变化研究

实验选取鸡粪和猪粪进行好氧堆肥发酵,研究畜禽粪便腐熟过程中酶活性和微生物的变化趋势以及相互联系。结果表明:过氧化氢酶活性和纤维素酶活性在堆肥初期较高,随后迅速降低,最终过氧化氢酶维持在9~12ml/g之间,纤维素酶维持在12.37~15.07mg/(kg·h)之间,而脲酶活性变化趋势为\"升高-降低-升高\"。细菌数量变化趋势为\"低-高-低\";放线菌为\"高-低\";真菌为\"高-低-高\"。通过相关分析发现,放线菌可能是影响堆肥中过氧化氢酶和纤维素酶的关键因素。鸡粪中放线菌与过氧化氢酶呈极显著正相关;猪粪中放线菌与过氧化氢酶和纤维素酶呈显著正相关;鸡粪+猪粪中放线菌与过氧化氢酶和纤维素酶呈极显著正相关。     

不同畜禽粪便浸出液对青钱柳种子发芽特性的影响研究

本文选取猪粪、牛粪、羊粪、鸡粪等4种畜禽粪便堆肥为供试样品,分别采用不同浓度的浸提液,对青钱柳种子进行育苗,统计其发芽率、根数、叶片数并测量根长。结果表明,猪粪、羊粪、鸡粪粪便浸提液在1∶40浸提比浓度处理下对青钱柳种子的相对发芽率、根数、叶片数有明显的促进作用,鸡粪浸提液的促进作用最明显,且鸡粪在1∶50和1∶60浸提比浓度下处理对相对发芽率也有一定的促进作用,因此鸡粪能显著提高青钱柳种子的相对发芽率、根数、叶片数。而猪粪、羊粪、鸡粪粪便浸提液1∶20浸提比浓度对青钱柳相对根长具有显著的促进作用,其余浸提比对青钱柳相对根长则有一定的抑制作用,且随着浓度的下降其抑制作用越显著。因此,用鸡粪粪便浸提液在1∶40浸提比浓度处理下对青钱柳种子育苗是有积极作用的,为今后对青钱柳种子育苗提供参考。     

四环素与3,4-苯并芘复合污染对抗性基因tetA(C)产生高抗性突变的影响

【目的】探究环境中同时存在低浓度四环素(tetracycline,TC)和3,4-苯并芘(benzo [a] pyrene,Bap)对抗性基因tetA(C)产生高抗性突变的影响。【方法】以大肠杆菌(Escherichia coli,E. coli)为宿主菌株,pACYC184质粒作为载体,四环素抗性基因tetA(C)作为研究对象,采用易错PCR构建基因文库的方法,建立基因突变位点对应高抗性的关系密码表。同时设置添加低浓度TC且添加0–30 mg/L Bap以及仅添加0–30 mg/L Bap的处理组,培养携带pACYC184质粒的大肠杆菌14 d,每组中随机挑选10株获得高抗性的菌株,对其中的tetA(C)基因片段进行测序,再结合突变位点密码表,计算高抗性菌株中由基因突变产生高抗性菌株的比例。【结果】测序结果显示在低浓度TC选择压力下,Bap浓度越高时,高抗性基因突变株占的比例也越高(P≤0.01),而不添加TC时,Bap浓度与高抗性基因突变株占比之间无变化规律(P0.05)。【结论】当环境中同时存在Bap和低浓度TC时,高抗性突变基因易于通过选择压力保存下来。     

抗生素抗性基因的生态风险研究进展

自抗生素被发现和使用以来,其在人类和动物疾病预防与治疗、提高动物生产等方面均发挥了重要作用。但抗生素的批量生产及大量应用,特别是在养殖业和临床医疗上的滥用,导致抗生素抗性基因(ARGs)在环境中普遍存在,其借助质粒、转座子、整合子等可移动元件通过接合、转座、转化等方式在环境中广泛传播,导致微生物药性不断增强,对人类健康和生态安全造成严重威胁。当前,ARGs对人类健康的影响已受到高度关注,但有关ARGs在环境中的生态风险研究还相对薄弱。本文综述了ARGs污染的现状及其生态风险,并对该领域中未来研究重点进行了展望,以期为今后抗性基因的研究和生态防控提供参考。     

不同畜禽粪便所含金霉素在土壤中的动态变化及降解途径

采用室内模拟培养试验,研究了畜禽粪便中所含金霉素在土壤中含量的动态变化及消解途径.结果表明:畜禽粪便中金霉素在土壤中的降解呈\"L\"型,但不同粪肥种类和用量处理的变化速率和减少率有显著差异(P＜0.05);180 d时,鸡粪处理土壤中金霉素减少率低于猪粪处理,低浓度鸡粪和猪粪处理土壤中金霉素减少率最大,可达85.4%和92.3%;减少率与畜禽粪便用量呈负相关,与时间呈正相关;畜禽粪便中的金霉素在土壤中的降解提高主要是外源微生物降解,占总减少量的75.7%,光降解和其他降解所占比例较小;随着培养时间的延长,微生物的降解作用增强,光解作用减弱.综上所述,随着时间的延长,畜禽粪便中的金霉素随自身分解和微生物等作用降解而逐渐减少,但短期内可能产生环境危害.     

Composting of animal manures and chemical criteria for compost maturity assessment. A review

New livestock production systems, based on intensification in large farms, produce huge amount of manures and slurries without enough agricultural land for their direct application as fertilisers. Composting is increasingly considered a good way for recycling the surplus of manure as a stabilised and sanitised end-product for agriculture, and much research work has been carried out in the last decade. However, high quality compost should be produced to overcome the cost of composting.     

Resistance to tetracycline, macrolide-lincosamide-streptogramin, trimethoprim, and sulfonamide drug classes

The discovery and use of antimicrobial agents in the last 50 yr has been one of medicine’s greatest achievements. These agents have reduced morbidity and mortality of humans and animals and have directly contributed to human’s increased life span. However, bacteria are becoming increasingly resistant to these agents by mutations, which alter existing bacterial proteins, and/or acquisition of new genes, which provide new proteins. The latter are often associated with mobile elements that can be exchanged quickly across bacterial populations and may carry multiple antibiotic genes fo resistance. In some case, virulence factors are also found on these same mobile elements. There is mounting evidence that antimicrobial use in agriculture, both plant and animal, and for environmental purposes does influence the antimicrobial resistant development in bacteria important in humans and in reverse. In this article, we will examine the genes which confer resistance to tetracycline, macrolide-lincosamide-streptogramin (MLS), trimethoprim, and sulfonamide.     

Effect of bioaugmentation and nitrogen supplementation on composting of paddy straw

A composting experiment was conducted to evaluate the effect of a hyperlignocellulolytic fungal consortium and different nitrogen amendments on paddy straw composting in terms of changes in physicochemical and biological parameters. A fungal consortium comprising four lignocellulolytic mesophilic fungal cultures was used as inoculum for bioaugmentation of paddy straw in perforated pits. The comparative effect of farmyard manure (FYM), soybean trash, poultry litter and urea on the composting process was evaluated at monthly intervals in terms of physicochemical (pH, EC, available P, C:N ratio and humus content) and biological (enzymatic and microbial activity) parameters. The compost prepared from bioaugmented paddy straw composting mixture, with poultry manure as nitrogen supplement attained desirable C:N ratio in 1 month and displayed least phytotoxicity levels along with higher production of β-1,4-Exoglucanase. The combined activity of the autochthonous composting microbiota as well as the externally applied fungal inoculum accelerated the composting process of paddy straw. Supplementation of paddy straw with poultry manure in 8:1 ratio was identified as the best treatment to hasten the composting process. This study highlights the importance of application of fungal inoculum and an appropriate N-amendment such as poultry manure for preparation of compost using a substrate having high C:N ratio, such as paddy straw.     

The behavior of tetracyclines and their degradation products during swine manure composting

The purposes of this study were to investigate the behavior of three tetracyclines including chlortetracycline (CTC), oxytetracycline (OTC) and tetracycline (TC) and their degradation products in a pilot scale swine manure composting, and also to study the degradation kinetics of CTC, OTC and TC. During the pilot scale composting, CTC, OTC and TC were degraded by 74%, 92% and 70%, respectively. Several degradation products were found like 4-epitetracycline (ETC), 4-epioxytetracycline (EOTC), 4-epichlortetracycline (ECTC), demeclocycline (DMCTC) and anhydrotetracycline (ATC). Both the simple and the adjusted first-order kinetic models successfully fit the degradation process of CTC, OTC and TC during the composting, but the adjusted first-order kinetic model fit much better with the calculated half-lives of 8.2, 1.1 and 10.0 days, respectively.     

Characterization of organic matter degradation during composting of manure-straw mixtures spiked with tetracyclines

The objective of this study was to investigate humification and mineralization of manure-straw mixtures contaminated by tetracyclines during composting. Hen manure, pig manure and rice straw were used as the raw materials. The manure-straw mixtures were spiked with tetracycline, chlortetracycline, and oxytetracycline at the concentration of 60 mg/kg dry matter. The results show that tetracyclines had no obvious influence on the composting process and more than 93% of the tetracyclines was decreased during a 45-day composting. The Fourier transform infrared (FTIR) spectra indicated that easily biodegradable components such as aliphatic substrates, carbohydrates and polysaccharides were decomposed and the contents of aromatic components relatively rose during the composting. The X-ray diffraction (XRD) spectra confirmed the natural formation of struvite, the degradation of easily biodegradable components, and the mineralization of organic matter during the composting. Therefore, FTIR and XRD analysis can be useful tools for monitoring the composting process.     

Composting of goat dung with various additives for improved fertilizer capacity

Composting of goat dung mixed with lime, soil, Napier grass or urea was followed by monitoring the C:N (w/w) ratio of the substrate, temperature, pH, moisture content, water-holding capacity, weight loss and mineral content. Equilibration to 24°C took between 90 and 120 days, the dung with Napier grass or urea composting fastest. Napier grass addition was also beneficial in decreasing loss of C and PO_inf4^sup-3and increasing N content. The most active region of the compost was 10 to 30 cm from the base of the heap. Depletion of C, PO_inf4^sup-3and K⁺ occurred during all compostings but varied among the treatments. The moisture content in the substrate remained between 34% and 73% (w/w) for all treatments, while the water-holding capacity averaged 100% to 150%. The average weight loss of substrate was between 55 to 84%. The compost could be used as manure for Napier grass production, although effluent from a palm oil mill was found to be superior.The author is with the Institute of Advanced Studies, University of Malaya, 59100 Kuala Lumpur, Malaysia     

Mutational and sequence analysis of transmembrane segment 6 orientation in TetA proteins

The packing orientations of the 8 transmembrane (TM) segments that line the central, aqueous transport channel within tetracycline resistance proteins (TetA) have been established. However, the orientations of the remaining 4 segments, TMs 3, 6, 9, and 12, located at the periphery, and away from the transport channel, have not yet been determined. In this study, the packing orientation of TM6 within the class C TetA protein encoded by plasmid pBR322 was evaluated by substitution mutagenesis and analysis of sequence conservation and amphipathicity. The combined data support a model in which the conserved and polar face of the TM6 alpha-helix containing Asn170 and Asn173 orients towards channel-lining TM segments, and the relatively non-conserved and hydrophobic face of TM6 points towards membrane lipids.