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。     

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。     

Antibiotic resistance gene spread due to manure application on agricultural fields

The usage of antibiotics in animal husbandry has promoted the development and abundance of antibiotic resistance in farm environments. Manure has become a reservoir of resistant bacteria and antibiotic compounds, and its application to agricultural soils is assumed to significantly increase antibiotic resistance genes and selection of resistant bacterial populations in soil. The genome location of resistance genes is likely to shift towards mobile genetic elements such as broad-host-range plasmids, integrons, and transposable elements. Horizontal transfer of these elements to bacteria adapted to soil or other habitats supports their environmental transmission independent of the original host. The human exposure to soil-borne resistance has yet to be determined, but is likely to be severely underestimated.     

抗生素抗性基因在环境中的传播扩散及抗性研究方法

抗生素在医药、畜牧和水产养殖业的大量使用造成了环境中抗性耐药菌和抗性基因日益增加,抗生素抗性基因作为一种新型环境污染物引起人们的广泛关注.本文综述了近年来国内外有关抗生素抗性基因的研究进展,其在水、土壤、空气等环境介质中和动,植物体内的传播扩散,以及开展环境中抗生素抗性基因研究的必要性,重点介绍了有关抗生素抗性（包括抗性细菌和抗性基因）的研究方法,指出抗性基因研究中存在的问题,并对未来的相关研究进行了展望.     

抗生素在环境中的转归及其生态毒性

抗生索被长期大量地用于人和动物的疾病治疗,并以亚治疗剂量添加于动物饲料长期用于动物疾病的预防和促进生长,大部分抗生紊不能完全被机体吸收,而有高达85％以上抗生素以原形或代谢物形式经由病人和畜禽粪尿排人环境,经不同途径对土壤和水体造成污染。最近研究显示在城市废水和表面水中检测到了抗生索的存在。但关于抗生素在环境中的分布、迁移和稳定性等的研究资料很少。环境中的抗生素会对环境生态系统包括细菌、水生生物、土壤生物和植物等产生危害,并产生大量耐药菌,对人类健康构成威胁。为评估抗生素在环境中潜在的危害,就抗生素在环境中的分布、转归及对环境和人的危害等方面进行综述,并对今后的研究方向作了探索。     

两种外源抗生素在农业土壤中的吸附与迁移特性

长期施用禽畜排泄物可导致抗生素在土壤中的积累,对生态环境产生不良影响.为了解长江三角洲和珠江三角洲农业土壤中长期施用含抗生素的有机肥可能产生的环境影响,选择了7个典型农业土壤,用室内模拟方法研究了我国常用的2种抗生素(泰乐菌素和土霉素)在这些土壤中吸附和迁移行为.研究表明,抗生素在土壤中的吸附、迁移行为与抗生素种类和土壤性质有关,土壤对土霉素的吸附能力明显高于泰乐菌素.土壤对抗生素的吸附能力主要与土壤粘粒含量、有机质和氧化铁呈正相关,与其它土壤性质的相关性较小.这2类抗生素在粘质农业土壤剖面中的迁移能力较弱;但泰乐菌素在砂质土壤中较易迁移,有较大的环境风险.     

Recent investigations and updated criteria for the assessment of antibiotic resistance in food lactic acid bacteria

The worldwide use, and misuse, of antibiotics for about sixty years in the so-called antibiotic era, has been estimated in some one to ten million tons, a relevant part of which destined for non-therapeutic purposes such as growth promoting treatments for livestock or crop protection. As highly adaptable organisms, bacteria have reacted to this dramatic change in their environment by developing several well-known mechanisms of antibiotic resistance and are becoming increasingly resistant to conventional antibiotics. In recent years, commensal bacteria have become a cause of concern since they may act as reservoirs for the antibiotic resistance genes found in human pathogens. In particular, the food chain has been considered the main route for the introduction of animal and environment associated antibiotic resistant bacteria into the human gastrointestinal tract (GIT) where these genes may be transferred to pathogenic and opportunistic bacteria. As fundamental microbial communities in a large variety of fermented foods and feed, the anaerobe facultative, aerotolerant lactic acid bacteria (LAB) are likely to play a pivotal role in the resistance gene exchange occurring in the environment, food, feed and animal and human GIT. Therefore their antibiotic resistance features and their genetic basis have recently received increasing attention. The present article summarises the results of the latest studies on the most typical genera belonging to the low G + C branch of LAB. The evolution of the criteria established by European regulatory bodies to ensure a safe use of microorganisms in food and feed, including the assessment of their antibiotic resistance is also reviewed.     

Impact of Manure Fertilization on the Abundance of Antibiotic-Resistant Bacteria and Frequency of Detection of Antibiotic Resistance Genes in Soil and on Vegetables at Harvest

Consumption of vegetables represents a route of direct human exposure to bacteria found in soil. The present study evaluated the complement of bacteria resistant to various antibiotics on vegetables often eaten raw (tomato, cucumber, pepper, carrot, radish, lettuce) and how this might vary with growth in soil fertilized inorganically or with dairy or swine manure. Vegetables were sown into field plots immediately following fertilization and harvested when of marketable quality. Vegetable and soil samples were evaluated for viable antibiotic-resistant bacteria by plate count on Chromocult medium supplemented with antibiotics at clinical breakpoint concentrations. DNA was extracted from soil and vegetables and evaluated by PCR for the presence of 46 gene targets associated with plasmid incompatibility groups, integrons, or antibiotic resistance genes. Soil receiving manure was enriched in antibiotic-resistant bacteria and various antibiotic resistance determinants. There was no coherent corresponding increase in the abundance of antibiotic-resistant bacteria enumerated from any vegetable grown in manure-fertilized soil. Numerous antibiotic resistance determinants were detected in DNA extracted from vegetables grown in unmanured soil. A smaller number of determinants were additionally detected on vegetables grown only in manured and not in unmanured soil. Overall, consumption of raw vegetables represents a route of human exposure to antibiotic-resistant bacteria and resistance determinants naturally present in soil. However, the detection of some determinants on vegetables grown only in freshly manured soil reinforces the advisability of pretreating manure through composting or other stabilization processes or mandating offset times between manuring and harvesting vegetables for human consumption.     

Bacteriophage applications: where are we now?

Bacteriophages are bacterial viruses and have been used for almost a century as antimicrobial agents. In the West, their use diminished when chemical antibiotics were introduced, but they remain a common therapeutic approach in parts of eastern Europe. Increasing antibiotic resistance in bacteria has driven the demand for novel therapies to control infections and led to the replacement of antibiotics in animal husbandry. Alongside this, increased pressure to improve food safety has created a need for faster detection of pathogenic bacteria. Hence, there has been a resurgence of interest in bacteriophage applications, and this has encouraged the emergence of a large number of biotech companies hoping to commercialize their use. Research in Europe and the United States has increased steadily, leading to the development of a range of applications for bacteriophage agents for the healthcare, veterinary and agricultural sectors. This article will attempt to answer the question of whether bacteriophages are now delivering on their potential.     

Antimicrobial resistance in humans,livestock and the wider environment

Antimicrobial resistance (AMR) in humans is inter-linked with AMR in other populations, especially farm animals, and in the wider environment. The relatively few bacterial species that cause disease in humans, and are the targets of antibiotic treatment, constitute a tiny subset of the overall diversity of bacteria that includes the gut microbiota and vast numbers in the soil. However, resistance can pass between these different populations; and homologous resistance genes have been found in pathogens, normal flora and soil bacteria. Farm animals are an important component of this complex system: they are exposed to enormous quantities of antibiotics (despite attempts at reduction) and act as another reservoir of resistance genes. Whole genome sequencing is revealing and beginning to quantify the two-way traffic of AMR bacteria between the farm and the clinic. Surveillance of bacterial disease, drug usage and resistance in livestock is still relatively poor, though improving, but achieving better antimicrobial stewardship on the farm is challenging: antibiotics are an integral part of industrial agriculture and there are very few alternatives. Human production and use of antibiotics either on the farm or in the clinic is but a recent addition to the natural and ancient process of antibiotic production and resistance evolution that occurs on a global scale in the soil. Viewed in this way, AMR is somewhat analogous to climate change, and that suggests that an intergovernmental panel, akin to the Intergovernmental Panel on Climate Change, could be an appropriate vehicle to actively address the problem.     

Antimicrobial resistance of bacteria isolated from slaughtered and retail chickens in South Africa

Animal feed is increasingly being supplemented with antibiotics to decrease the risk of epidemics in animal husbandry. This practice could lead to the selection for antibiotic resistant micro-organisms. The aim of this study was to determine the level of antibiotic resistant bacteria present on retail and abattoir chicken. Staphylococci, Enterobacteriaceae, Salmonella and isolates from total aerobic plate count were tested for resistance to vancomycin, streptomycin, methicillin, tetracycline and gentamicin using the disc diffusion susceptibility test; resistance to penicillin was determined using oxacillin. Results from the antibiotic code profile indicated that many of the bacterial strains were displaying multiple antibiotic resistance (MAR). A larger proportion of resistance to most antibiotics, except for vancomycin, was displayed by the abattoir samples, therefore suggesting that the incidence of MAR pathogenic bacteria was also higher in the abattoir samples. This resistance spectrum of abattoir samples is a result of farmers adding low doses of antibiotics to livestock feed to improve feeding efficiency so that the animals need less food to reach marketable weight. The lower incidence of MAR pathogenic bacteria in the retail samples is a result of resistance genes being lost due to lack of selective pressure, or to the fact that the resistant flora are being replaced by more sensitive flora during processing. The use of subtherapeutic levels of antibiotics for prophylaxis and as growth promoters remains a concern as the laws of evolution dictate that microbes will eventually develop resistance to practically any antibiotic. Selective pressure exerted by widespread antimicrobial use is therefore the driving force in the development of antibiotic resistance. This study indicated that a large proportion of the bacterial flora on fresh chicken is resistant to a variety of antibiotics, and that resultant food-related infections will be more difficult to treat.     

Insects Represent a Link between Food Animal Farms and the Urban Environment for Antibiotic Resistance Traits

Antibiotic-resistant bacterial infections result in higher patient mortality rates, prolonged hospitalizations, and increased health care costs. Extensive use of antibiotics as growth promoters in the animal industry represents great pressure for evolution and selection of antibiotic-resistant bacteria on farms. Despite growing evidence showing that antibiotic use and bacterial resistance in food animals correlate with resistance in human pathogens, the proof for direct transmission of antibiotic resistance is difficult to provide. In this review, we make a case that insects commonly associated with food animals likely represent a direct and important link between animal farms and urban communities for antibiotic resistance traits. Houseflies and cockroaches have been shown to carry multidrug-resistant clonal lineages of bacteria identical to those found in animal manure. Furthermore, several studies have demonstrated proliferation of bacteria and horizontal transfer of resistance genes in the insect digestive tract as well as transmission of resistant bacteria by insects to new substrates. We propose that insect management should be an integral part of pre- and postharvest food safety strategies to minimize spread of zoonotic pathogens and antibiotic resistance traits from animal farms. Furthermore, the insect link between the agricultural and urban environment presents an additional argument for adopting prudent use of antibiotics in the food animal industry.     

耐药微生物和抗生素耐药基因与全健康

因人类的各种活动,耐药微生物和抗生素耐药基因在“人-动物-环境”界面发生跨物种和跨生境的传播。将人类、动物和环境视作有机整体的“全健康”（One Health）理念有望成为解决这种传播的有效策略。抗生素及其代谢活性产物在环境中富集,再经动物及动物制品传播到人,产生耐药微生物并造成耐药基因的传播。本文综述了人-动物-环境界面耐药菌和抗生素耐药基因传播的流动与循环,总结了我国和其他国家应对抗生素耐药性问题的政策,倡导更多的国家和地区将“全健康”理念和方法用于控制抗生素耐药性传播;通过医疗卫生部门、食品药品监督管理部门、农林渔牧部门与教育、财政等多部门合作来应对抗生素耐药性的全球挑战。     

Bacteriophages as antibiotic resistance genes carriers in agro-food systems

Antibiotic resistance genes (ARGs) are a global health concern. Antibiotic resistance occurs naturally, but misuse of antibiotics in humans and animals is accelerating the process of antibiotic resistance emergency, which has been aggravated by exposure to molecules of antibiotics present in clinical and agricultural settings and the engagement of many countries in water reuse especially in Middle East and North Africa region. Bacteriophages have the potential to be significant actors in ARGs transmission through the transduction process. These viruses have been detected along with ARGs in non impacted habitats and in anthropogenic impacted environments like wastewater, reclaimed water and manure amended soil as well as minimally processed food and ready to eat vegetables. The ubiquity of bacteriophages and their persistence in the environment raises concern about their involvement in ARGs transmission among different biomes and the generation of pathogenic-resistant bacteria that pose a great threat to human health. The aim of this review is to give an overview of the potential role of bacteriophages in the dissemination and the transfer of ARGs to pathogens in food production and processing and the consequent contribution to antibiotic resistance transmission through faecal oral route carrying ARGs to our dishes.     

禽畜养殖粪便中多重抗生素抗性细菌研究

通过对新乡地区8家养猪场和11家养鸡场饲喂抗生素情况的调研,发现头孢氨苄、阿莫西林、卡那霉素、庆大霉素等4种抗生素是该地区被普遍使用的兽药抗生素。通过多点取样法和微生物培养技术对3家养鸡场和3家养猪场不同养殖时期的粪便进行单一抗生素和多重抗生素抗性细菌的检测,结果表明养鸡场堆置1周的粪便中抗头孢氨苄的细菌比例最高,达到65.90%,对所研究的3种和4种抗生素同时抗性的比例高达8.60%—12.51%和9.73%,明显高于饲喂中药的对照养鸡场样本检测结果(0.02%—2.73%和0.12%)。养猪场堆置1周的粪便中检测到抗头孢氨苄的细菌比例也是最高,达到49.12%上,但养猪场粪便中多重抗生素抗性细菌的比例明显低于养鸡场。同时研究发现,在两种养殖场中,幼龄期粪便中检测到的多重抗性细菌比例明显高于成熟期粪便,这可能与养殖过程中鸡、猪在幼龄期由于防病和促生长等因素而同时大剂量使用多种抗生素有关。     

Abundance of Antibiotic Resistance Genes in Bacteriophage following Soil Fertilization with Dairy Manure or Municipal Biosolids,and Evidence for Potential Transduction

Animal manures and municipal biosolids recycled onto crop production land carry antibiotic-resistant bacteria that can influence the antibiotic resistome of agricultural soils, but little is known about the contribution of bacteriophage to the dissemination of antibiotic resistance genes (ARGs) in this context. In this work, we quantified a set of ARGs in the bacterial and bacteriophage fractions of agricultural soil by quantitative PCR. All tested ARGs were present in both the bacterial and phage fractions. We demonstrate that fertilization of soil with dairy manure or human biosolids increases ARG abundance in the bacterial fraction but not the bacteriophage fraction and further show that pretreatment of dairy manure can impact ARG abundance in the bacterial fraction. Finally, we show that purified bacteriophage can confer increased antibiotic resistance to soil bacteria when combined with selective pressure. The results indicate that soilborne bacteriophage represents a substantial reservoir of antibiotic resistance and that bacteriophage could play a significant role in the horizontal transfer of resistance genes in the context of an agricultural soil microbiome. Overall, our work reinforces the advisability of composting or digesting fecal material prior to field application and suggests that application of some antibiotics at subclinical concentrations can promote bacteriophage-mediated horizontal transfer of ARGs in agricultural soil microbiomes.     

Biochar from animal manure: A critical assessment on technical feasibility,economic viability,and ecological impact

Animal manure has been used to manage soil fertility since the dawn of agriculture. It provides plant nutrients and improves soil fertility. In the last decades, animal husbandry has been significantly expanded globally. Its economics were optimized via the (international) trade of feed, resulting in a surplus of animal manure in areas with intensive livestock farming. Potentially toxic elements (PTEs), pathogenic microorganisms, antibiotic residues, biocides, and other micropollutants in manure threaten animal, human, and environmental health. Hence, manure application in crop fields is increasingly restricted, especially in hotspot regions with intensive livestock activities. Furthermore, ammonia volatilization and greenhouse gas (GHG) emissions during manure storage, field application, and decomposition contribute to air pollution and climate change. Conventional manure management scenarios such as composting and anaerobic digestion partially improve the system but cannot guarantee to eliminate sanitary and contamination risks and only marginally reducing its climate burden. Hence, this review discusses the potential of pyrolysis, the thermochemical conversion under oxygen-limited conditions as an alternative treatment for animal manure providing energy and biochar. Manure pyrolysis reduces the bioavailability of PTEs, eliminates pathogenic microorganisms and organic micropollutants, and reduces GHG emissions. Pyrolysis also results in the loss of nitrogen, which can be minimized by pretreatment, that is, after removing soluble nitrogen fraction of manure, for example, by digestion and stripping of ammonia–nitrogen or liquid–solid separation. However, conclusions on the effect of manure pyrolysis on crop yield and fertilization efficiencies are hampered by a lack of nutrient mass balances based on livestock unit equivalent comparisons of manure and manure biochar applications. Hence, it is essential to design and conduct experiments in more practically relevant scenarios and depict the observations based on the amount of manure used to produce a certain amount of biochar.     

Geographical variation in cloacal microflora and bacterial antibiotic resistance in a threatened avian scavenger in relation to diet and livestock farming practices

The impact on wildlife health of the increase in the use of antimicrobial agents with the intensification of livestock production remains unknown. The composition, richness and prevalence of cloacal microflora as well as bacterial resistance to antibiotics in nestlings and full-grown Egyptian vultures Neophron percnopterus were assessed in four areas of Spain in which the degree of farming intensification differs. Differences in diet composition, especially the role of stabled livestock carrion, appear to govern the similarities of bacterial flora composition among continental populations, while the insular vulture population (Fuerteventura, Canary Islands) showed differences attributed to isolation. Evidence of a positive relationship between the consumption of stabled livestock carrion and bacterial resistance to multiple antibiotics was found. Bacterial resistance was high for semisynthetic penicillins and enrofloxacin, especially in the area with the most intensive stabled livestock production. The pattern of antibiotic resistance was similar for the different bacterial species within each area. Bacterial resistance to antibiotics may be determined by resistance of bacteria present in the livestock meat remains that constituted the food of this species, as indicated by the fact that resistance to each antibiotic was correlated in Escherichia coli isolated from swine carrion and Egyptian vulture nestlings. In addition, resistance in normal faecal bacteria (present in the microflora of both livestock and vultures) was higher than in Staphylococcus epidermidis, a species indicator of the transient flora acquired presumably through the consumption of wild rabbits. Potential negative effects of the use of antimicrobials in livestock farming included the direct ingestion of these drug residues and the effects of bacterial antibiotic resistance on the health of scavengers.     

养殖动物及其相关环境耐药组的研究进展

畜牧养殖业中大量抗生素的使用,导致养殖动物及其相关环境中存在大量的耐药基因/耐药细菌。这些耐药基因可以借助基因水平转移等方式在环境中进一步扩散,甚至进入食品动物随食物链传播,对生态环境、食品安全和人类健康造成极大的威胁。随着基因组学研究手段的不断进步,养殖动物及其相关环境中耐药基因的多样性和生态学分布规律被广泛揭示。文中综述了相关领域耐药基因的研究进展,探讨了其对人体健康的潜在影响,并对未来的研究方向进行了展望。     

土霉素暴露对小麦根际抗生素抗性细菌及土壤酶活性的影响

通过培养的方法研究了土霉素暴露和小麦根际抗性细菌的数量、种类、分布特征及土壤酶活性之间的剂量效应关系。结果表明,土霉素暴露下小麦根际单一抗生素抗性细菌数量和抗土霉素—链霉素双重抗性细菌数都明显增加,且与暴露剂量呈正效应关系;同时,土壤磷酸酶、脱氢酶活性下降,但与土霉素的剂量效应关系不明显。从土霉素暴露的土壤中分离到50株抗性细菌,经形态观察、RFLP分组和16S rDNA序列测定与分析,将它们聚集在Actinobacteria、Bacilli、Alphaproteobacteria、Gammaproteobacteria 和Sphingobacteria类群。其中放线菌最多（15株）,占抗性菌总数的30 %;其次是Bacillus属细菌（9株）和Pseudomonas属细菌（8株）,分别占18 %和16 %。同时,具有抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌在土霉素暴露的样品中均被分离到,分别占抗性菌株总数的16 %、8 %和4 %。值得注意的是,随着土霉素暴露剂量的增加,小麦根际优势促生菌Bacillus属细菌的抗性检出率逐步降低;但具有抗生素抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌的检出率却明显增加,提示可能会进一步增大其机会致病性。