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。     

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.     

无乳链球菌临床株对四环素类的敏感性研究

为探讨无乳链球菌对四环素类的敏感性及其与耐药基因、多位点序列分型(multilocus sequence typing,MLST)之间的关系,本研究收集2014-2017年深圳市南山区人民医院分离自患者的136株无乳链球菌临床分离株,采用琼脂平板稀释法分析四环素类最低抑菌浓度(minimum inhibitory concentration,MIC)。采用聚合酶链反应检测菌株四环素类耐药基因(tetM、tetK、tetO),MLST测定ST型别,并分析四环素类敏感性与其耐药基因及ST型别之间的关系。结果显示,无乳链球菌对四环素类的耐药率为46.32%,耐四环素类菌株主要携带tetM基因,主要为ST17型和ST19型,且ST17型菌株对四环素类的耐药率显著高于ST19型(P<0.05)。结果提示,无乳链球菌的主要四环素类耐药基因为tetM,ST17型、ST19型是主要流行型别,且ST17型菌株对四环素类的敏感性低于ST19型。     

大肠杆菌四环素耐药基因表达与耐药水平的相关性

为了探索大肠杆菌耐药基因的表达与耐药水平的关系,本研究通过微量肉汤稀释法检测240株大肠杆菌对土霉素(terramycin, OTC)、金霉素(aureomycin, CTC)、四环素(tetracycline, TC)、多西还素(doxycycline,CAS)和米诺环素(minocycline, MINO) 5种四环素类抗菌药物的敏感性,采用PCR方法检测四环素类耐药基因tet B和tet X的携带情况,通过荧光定量PCR法考察不同四环素浓度对tet B表达的影响。研究结果表明:240株猪源大肠杆菌对土霉素、金霉素、四环素、多西还素和米诺环素的耐药率分别为93.33%、92.92%、92.50%、80.42%和37.08%,兽用四环素类药物的治疗效果难以保证;四环素耐药基因的携带率较高,tet B和tet X检出率分别为22.92%和85.00%;Tet B基因mRNA的表达量随着四环素浓度升高而逐步增强,说明tet B属于诱导表达基因,耐药菌难以通过停止用药来恢复对四环素的敏感性,研究人员建议兽医临床应将四环素类药物作为二线药物使用。     

一株四环素高效降解菌的分离及降解特性

四环素类抗生素在养殖业中广泛使用引起药物残留,给环境及人类健康带来了深远影响。从利用四环素类抗生素作为饲料添加剂的养猪厂底泥中,筛选获得一株能够高效降解四环素的细菌XY-1,经革兰氏染色、形态学观察、生理生化及16S rDNA序列分析等,确定该菌株属于拉乌尔菌属,命名为Raoultella sp.XY-1。采用高效液相色谱(HPLC)测定了菌株XY-1对四环素的降解能力,结果表明,该菌株最佳降解条件是温度为25℃,初始pH值为7.0,在此最佳条件下,XY-1在第8天时生长量最大且四环素降解率最高达到70.68%。对XY-1的四环素耐药基因进行了初步探究,首次在该菌株中检测到两种四环素耐药基因tet(D)和tet(E)。     

抗四环素单链抗体基因的构建与序列测定

目的：构建抗盐酸四环素的单链抗体（scFv）基因。方法：以抗盐酸四环素单克隆抗体杂交瘤细胞株的总RNA为模板,用RT-PCR法扩增全套抗体轻、重链基因;经重叠延伸反应,以编码柔性多肽（GIy4Ser）3的基因为接头,将轻、重链基因组装为完整的seFv基因,并克隆到pGEMT-Easy载体中进行测序分析。结果：所克隆的四环素scFV基因全长为735 bp,为VH-Linker-VL结构,VH基因为354bp,Linker为（Gly4Ser）3多肽的核酸序列,VL基因为336 bp。结论：构建了抗四环素的单链抗体基因,为进一步用于四环素的残留枪测奠审某础．     

家养及大型养殖场鸡肠道微生物菌群及四环素耐药菌多样性的比较研究

本研究分析比较了农家家养鸡与大型养殖场鸡肠道菌群组成及抗四环素抗性基因在肠道菌群中的分布情况。通过454焦磷酸法对细菌16S rRNA V3区进行测序来分析肠道菌群的组成;用平板琼脂法筛选四环素耐药菌株,对其16S rRNA基因全长测序,并与RDP (Ribosomal Database Project)数据库比对进行菌种鉴定;聚合酶链反应(polymerase chain reaction,PCR)检测常见四环素耐药基因。家养鸡粪便中菌群香农多样性指数为5.321±0.590,养殖场鸡为4.398±0.440,前者显著大于后者(Mann-Whitney U test,P＝0.008)。从家养鸡和养殖场鸡粪便中随机分离到69株和65株四环素耐药菌株,后者四环素耐药菌的种类多于前者。家养鸡较养殖场鸡的肠道菌群更具多样性,而抗生素抗性基因在养殖场鸡的肠道菌群中分布更广泛。结果表明,不同饲养方式对鸡的肠道菌群有影响,对抗生素抗性基因的分布也有一定影响。     

云南土壤宏基因组文库中替加环素耐药基因的筛选与分析

【背景】随着抗生素的广泛应用以及滥用,出现了多重耐药的"超级细菌",并且在临床上已出现对治疗"超级细菌"感染的最后一线抗生素——替加环素耐药的细菌。【目的】对土壤环境中存在的替加环素耐药基因进行筛选调查,探讨替加环素耐药机制,为临床抗生素治疗提供借鉴。【方法】利用功能宏基因组学技术对土壤宏基因组文库进行替加环素耐药阳性克隆的筛选和耐药亚克隆的构建,对构建成功的亚克隆进行测序和生物信息学分析探知其功能,同时进行最低抑菌浓度(Minimum Inhibitory Concentration,MIC)的测定。【结果】筛选得到一个四环素抗性Major Facilitator Superfamily(MFS)外排泵基因,携带该基因的菌株对四环素类抗生素均耐药,对替加环素和四环素的MIC值分别为16μg/mL和32μg/mL。当MFS外排泵抑制剂羰基氰化物间氯苯腙(CarbonylCyanide3-Chlorophenylhydrazone,CCCP)浓度为4μg/mL时可以抑制其对四环素类抗生素的外排作用。生物信息学结果表明该基因编码483个氨基酸,编码的蛋白质属于疏水稳定蛋白;其含有的14个跨膜区构成MFS外排泵蛋白典型的14次跨膜螺旋保守结构域;系统进化树分析表明其与其他替加环素耐药基因编码的蛋白质位于不同的分支上,相似性较低。【结论】利用功能宏基因组学技术筛选得到一个有替加环素抗性的MFS外排泵基因,编码的蛋白质属于14次跨膜螺旋MFS外排泵家族,为今后研究替加环素耐药机制提供参考。     

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。     

四川省16家大型禽类养殖场抗生素残留调查和细菌耐药性分析

为评估禽类养殖场粪便中抗生素的残留及细菌耐药性情况,本研究以四川省16家大型规模化禽类养殖场为监测点,重点分析粪便中抗生素残留特征和肠杆菌耐药特征,并对粪便中抗生素的选择性耐药风险熵进行评估。16份粪便样品中共检出18种抗生素,抗生素检出浓度0.54～2 780.00μg·kg^-1,其中,ZJZX中多西环素检出浓度最高,达到2 780.00μg·kg^-1;抗生素的选择性耐药风险熵为0.004 0～10 561.13,呈现出高选择性耐药风险;其次,从粪便中共筛选鉴定出14株肠杆菌科Enterobacteriaceae细菌,均表现出多重耐药性,且有33种耐药基因被不同程度检出。畜禽粪便中磺胺类、四环素类、氯霉素类和喹诺酮类抗生素残留较为严重,且养殖环境中肠杆菌科细菌表现出多重耐药性,建议有关部门加强畜禽养殖中多西环素、氟苯尼考和恩诺沙星等抗生素的管控,同时应加强对畜禽养殖企业中肠杆菌耐药性的监控。     

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.     

Occurrence and Persistence of Erythromycin Resistance Genes (erm) and Tetracycline Resistance Genes (tet) in Waste Treatment Systems on Swine Farms

Animal manure from modern animal agriculture constitutes the single largest source of antibiotic resistance (AR) owing to the use of large quantities of antibiotics. After animal manure enters the environment, the AR disseminates into the environment and can pose a potentially serious threat to the health and well-being of both humans and animals. In this study, we evaluated the efficiency of three different on-farm waste treatment systems in reducing AR. Three classes of erythromycin resistance genes (erm) genes (B, F, and X) conferring resistances to macrolide–lincosamides–streptogramin B (MLS_B) and one class of tetracycline resistance genes (tet) gene (G) conferring resistance to tetracyclines were used as models. Real-time polymerase chain reaction assays were used to determine the reservoir sizes of these AR genes present in the entire microbiome. These classes of AR genes varied considerably in abundance, with erm(B) being more predominant than erm(F), erm(X), and tet(G). These AR genes also varied in persistence in different waste treatment systems. Aerobic biofiltration reduced erm(X) more effectively than other AR genes, while mesophilic anaerobic digestion and lagoon storage did not appreciably reduce any of these AR genes. Unlike chemical pollutants, some AR genes could increase after reduction in a preceding stage of the treatment processes. Season might also affect the persistence of AR. These results indicate that AR arising from swine-feeding operations can survive typical swine waste treatment processes and thus treatments that are more effective in destructing AR on farms are required.     

Persistence of Resistance to Erythromycin and Tetracycline in Swine Manure During Simulated Composting and Lagoon Treatments

The use of antimicrobials in food animal production leads to the development of antimicrobial resistance (AMR), and animal manure constitutes the largest reservoir of such AMR. In previous studies, composted swine manure was found to contain substantially lower abundance of AMR genes that encode resistance to tetracyclines (tet genes) and macrolide–lincosamide–streptogramin B (MLS_B) superfamily (erm genes), than manures that were treated by lagoons or biofilters. In this study, temporal changes in AMR carried by both cultivated and uncultivated bacteria present in swine manure during simulated composting and lagoon storage were analyzed. Treatments were designed to simulate the environmental conditions of composting (55°C with modest aeration) and lagoon storage (ambient temperature with modest aeration). As determined by selective plate counting, over a 48-day period, cultivated aerobic heterotrophic erythromycin-resistant bacteria and tetracycline-resistant bacteria decreased by more than 4 and 7 logs, respectively, in the simulated composting treatment while only 1 to 2 logs for both resistant bacterial groups in the simulated lagoon treatment. Among six classes each of erm and tet genes quantified by class-specific real-time PCR assays, the abundance of erm(A), erm(C), erm(F), erm(T), erm(X), tet(G), tet(M), tet(O), tet(T), and tet(W) declined marginally during the first 17 days, but dramatically thereafter within 31 days of the composting treatment. No appreciable reduction of any of the erm or tet genes analyzed was observed during the simulated lagoon treatment. Correlation analysis showed that most of the AMR gene classes had similar persistence pattern over the course of the treatments, though not all AMR genes were destructed at the same rate during the treatments.     

Comparison of treatment efficacy and stability of microbial populations between raw and anaerobically treated liquid pig manure, using PCR-DGGE and 16S sequencing

The effects of adding an adapted inoculum to liquid pig manure (LPM) prior to anaerobic digestion were evaluated by standard analytical methods. In parallel, the phylogenetic diversity of the microbial community of raw and anaerobically digested pig manure was studied by both denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA fragments amplified by polymerase chain reaction. Gas production, volative fatty acid production, removal of soluble chemical oxygen demand, and removal of volatile soluble solids were measured on raw and on inoculated liquid pig manure subjected to anaerobic digestion. DGGE profiles of 16S rRNA genes were used to compare the major elements of the bacterial community composition in raw LPM with those present under various incubation conditions. Major bands were excised and sequenced to gain insight into the identities of the bacterial populations from LPM treated under different conditions. The results show that the addition of an adapted inoculum did not have a major impact on the conversion of pig manure into soluble organic matter and did not significantly change the microbial populations present during anaerobic digestion of LPM. Bacterial composition also indicated that Clostridium species are important constituents of the LPM community.     

Successive rapid reductive dehalogenation and mineralization of pentachlorophenol by the indigenous microflora of farmyard manure compost

AIMS: To determine whether composting with animal manure can be used to effectively remediate soil from a pentachlorophenol (PCP)-contaminated site, and to establish the fate of the degraded xenobiotic. METHODS AND RESULTS: Contaminated soil from a sawmill site was mixed with farm animal manure and composted in a 0.5 m3 silo under fully aerobic conditions. The disappearance and fate of PCP was monitored by gas chromatography (GC-ECD) and extensive mineralization confirmed in experiments with 14C-radiolabelled PCP. The disappearance of PCP was rapid and virtually complete within 6 days, prior to the onset of thermophilic conditions. Dechlorination of the PCP was found to be both reductive and sequential. CONCLUSIONS: PCP removal from contaminated soil by aerobic composting with animal manure is efficient and proceeds via reductive dechlorination to virtually complete mineralization. This contrasts with other chlorophenol composting regimes in which mineralization is achieved but dechlorination intermediates do not accumulate to detectable levels. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study demonstrate that anaerobic reductive dechlorination can proceed in an aerobic composting environment and contribute to efficient pentachlorophenol removal. Farmyard manure composts may represent a rapid, low-cost, low-technology option for treatment of chlorophenol-contaminated soils.     

Challenges and innovations on biological treatment of livestock effluents

Intensification of animal production led to high amounts of manure to be managed. Biological processes can contribute to a sustainable manure management. This paper presents the biological treatments available for the treatment of animal manure, mainly focusing on swine manure, including aerobic processes (nitrification, denitrification, enhanced biological phosphorus removal) and anaerobic digestion. These processes are discussed in terms of pollution removal, ammonia and greenhouse gas emissions (methane and nitrous oxide) and pathogen removal. Application of emerging processes such as partial nitrification and anaerobic ammonium oxidation (anammox) applied to animal manure is also considered. Finally, perspectives and future challenges for the research concerning biological treatments are highlighted in this paper.     

Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment. To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators - including inorganic ions, antibiotics, and antibiotic resistance genes - were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 microg/L. Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes - tet(M), tet(O), tet(Q), and tet(W) - were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.     

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.     

A survey of ammonia-assimilating micro-organisms in cattle manure composting

AIMS: To evaluate the ammonia-assimilating abilities of micro-organisms isolated from cattle manure composting processes and to determine the distribution of cultivable species of ammonia-assimilating micro-organisms in microbial communities during the composting processes. METHODS AND RESULTS: Compost samples were collected from four stages of treatment. Trypto soya agar was used for the isolation of ammonia-assimilating aerobes. Many of the isolates showed high ammonia-assimilating ability in a medium containing basal components and a compost extract. Partial 16S ribosomal DNA sequencing showed that the cultivable species of highly efficient ammonia-assimilating isolates changed during the composting process. The community structure of micro-organisms and actinomycetes was analysed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Two species of actinomycetes identified by PCR-DGGE coincided with those found among the cultured isolates. CONCLUSIONS: Ammonia-assimilating micro-organisms obtained by the cultivation method were not predominant in the microbial community during the composting process: however certain cultured actinomycetes were members of predominant species in the actinomycetes community. SIGNIFICANCE AND IMPACT OF THE STUDY: Ammonia assimilation by micro-organisms is one of the important mechanisms for ammonia retention in the composting process. Cultivable actinomycetes are a means for preventing ammonia emission from the composting process.