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。     

食品动物养殖环境中细菌耐药性研究进展

抗生素耐药性被世界卫生组织认为是21世纪人类面临的最大的公共卫生安全问题之一。近年来,抗生素耐药基因作为一种新型污染物而受到广泛关注。养殖场现已成为耐药基因的一个重要储库,耐药菌及耐药基因随着动物排泄物进入环境,从而加速了耐药基因在环境中的传播。畜禽养殖环境中耐药基因和耐药菌可能经食物链、空气等途径传至人类,给人类健康带来巨大威胁。文中结合最新文献,主要介绍了动物养殖场抗菌药物耐药菌和耐药基因的分布特点、耐药基因的持留和传播扩散、研究方法等方面的研究进展,为食品动物养殖环境的抗菌药物耐药性风险评估提供一定支持。     

Antibiotic resistance genes in water environment

The use of antibiotics may accelerate the development of antibiotic resistance genes (ARGs) and bacteria which shade health risks to humans and animals. The emerging of ARGs in the water environment is becoming an increasing worldwide concern. Hundreds of various ARGs encoding resistance to a broad range of antibiotics have been found in microorganisms distributed not only in hospital wastewaters and animal production wastewaters, but also in sewage, wastewater treatment plants, surface water, groundwater, and even in drinking water. This review summarizes recently published information on the types, distributions, and horizontal transfer of ARGs in various aquatic environments, as well as the molecular methods used to detect environmental ARGs, including specific and multiplex PCR (polymerase chain reaction), real-time PCR, DNA sequencing, and hybridization based techniques.     

Antibiotic resistance in surface water ecosystems: Presence in the aquatic environment,prevention strategies,and risk assessment

Antibiotic-resistant bacteria (ARB) have gained increased notoriety due to their continued detection in environmental media and consequently their threat to human and animal health. The continuing spread of antibiotic resistance throughout the environment is of growing environmental and public health concern, making it difficult to treat harmful resistant diseases. This paper examines the presence of antibiotics, ARB, and antibiotic-resistant genes (ARGs) in aquatic environments; the effectiveness of current water treatment strategies to remove them; and risk assessment methods available that can be used to evaluate the risk from antibiotic resistance. Antibiotics, ARB, and ARGs have been reported at varying levels in wastewater treatment plants, hospital wastewater, irrigation water, recreational water, and drinking water. There are many different water treatments capable of reducing antibiotic resistance (including chlorination, UV, and ozone); however, no one method can fully eliminate it with much variation in the reported effects. Risk assessment models can be used for interpreting field data into the risk to human health from antibiotic resistance. Currently, there is no gold standard risk assessment method for evaluating antibiotic resistance. Methods in this area need further development to reflect evolving risk assessment methodologies and dynamic data as it emerges.     

土壤噬菌体及其介导的抗生素抗性基因水平转移研究进展

土壤中抗生素耐药性的扩散对全球的公共卫生和食品安全造成威胁,严重挑战人类感染类疾病的预防与治疗.噬菌体介导的抗生素抗性基因(ARGs)的水平转移是环境中抗性基因扩散的重要机制.但是,噬菌体对土壤环境中抗性基因传播的贡献尚未见报道.本文综述了土壤环境中噬菌体的分布特征与影响因子,总结了纯化和富集土壤噬菌体的主要研究方法;...     

气溶胶中抗生素抗性基因研究进展

抗生素的不合理使用导致细菌耐药问题日趋严峻,给人类健康造成巨大威胁。学者们对抗生素抗性菌和抗生素抗性基因(antibiotic resistance genes, ARGs)在多种环境介质中的环境行为开展了大量研究。气溶胶作为ARGs的潜在储存库,是抗生素抗性基因在环境中的重要传播途径之一。目前缺乏对其来源、传播、人类接触和健康风险系统性的梳理。本文针对人类生活功能场所、养殖场、城市污水处理厂和医院等4类气溶胶研究的典型场所,重点综述了上述4类典型场所中气溶胶ARGs的来源、传播途径及对人体的暴露和对健康的危害,为气溶胶中ARGs的预防和控制提供参考。     

水平基因转移介导抗菌素耐药性传播机制的研究进展

近几十年来,病原菌耐药性的出现和蔓延已上升为严峻的公共卫生问题。越来越多研究表明,抗菌素抗性基因(antibiotic resistance genes,ARGs)不仅仅见于临床所分离的病原体,而是包括所有的致病菌、共生菌以及环境中的细菌,它们都能在可移动遗传元件和噬菌体的作用下,通过水平基因转移(horizontal gene transfer,HGT)途径获得耐药性,进而形成抗菌素耐药基因簇(耐药基因组)。HGT可导致抗菌素的耐药性在环境共生菌和病原菌之间传播扩散,这可通过临床上一些重要的抗菌素耐药基因的传播证实。传统观念认为HGT的三种机制中,接合对ARGs的传播影响最大,最近研究表明转化和转导对ARGs播散起到不可忽视的作用。通过深入了解耐药基因组的传播及其在动员病原菌耐药中发挥的作用,对于控制这些基因的播散是至关重要的。将讨论耐药基因组的概念,提供临床相关的抗菌素抗性基因水平基因转移的例子,对当前已研究的促使抗菌素耐药性传播的各种HGT机制进行回顾。     

环境中胞内胞外抗性基因的分离检测、分布与传播

抗生素抗性基因(ARGs)传播对人类健康具有潜在的风险。胞内抗性基因(iARGs)和胞外抗性基因(eARGs)是抗生素抗性基因的两种存在形式,其在不同环境介质中的分布与传播过程具有截然不同的特征。本文首先基于ARGs赋存形态的差异,对染色体抗性基因、质粒抗性基因、噬菌体抗性基因等ARGs的胞内/胞外分类给予明确界定,并根据环境样品来源归纳了现有分离检测技术的应用场景,总结了iARGs和eARGs在养殖场、污水处理厂、河道、海洋、大气等环境中的分布特征,同时比较了其在传播方式和传播能力上的差异,以期为ARGs的分类阻控和健康风险评估提供理论参考。     

Research progress on distribution,migration, transformation of antibiotics and antibiotic resistance genes (ARGs) in aquatic environment

Antimicrobial and antibiotics resistance caused by misuse or overuse of antibiotics exposure is a growing and significant threat to global public health. The spread and horizontal transfer of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) by the selective pressure of antibiotics in an aquatic environment is a major public health issue. To develop a better understanding of potential ecological risks die to antibiotics and ARGs, this study mainly summarizes research progress about: (i) the occurrence, concentration, fate, and potential ecological effects of antibiotics and ARGs in various aquatic environments, (ii) the threat, spread, and horizontal gene transfer (HGT) of ARGs, and (iii) the relationship between antibiotics, ARGs, and ARB. Finally, this review also proposes future research direction on antibiotics and ARGs.     

A review of the emergence of antibiotic resistance in bioaerosols and its monitoring methods

Despite significant public health concerns regarding infectious diseases in air environments, potentially harmful microbiological indicators, such as antibiotic resistance genes (ARGs) in bioaerosols, have not received significant attention. Traditionally, bioaerosol studies have focused on the characterization of microbial communities; however, a more serious problem has recently arisen due to the presence of ARGs in bioaerosols, leading to an increased prevalence of horizontal gene transfer (HGT). This constitutes a process by which bacteria transfer genes to other environmental media and consequently cause infectious disease. Antibiotic resistance in water and soil environments has been extensively investigated in the past few years by applying advanced molecular and biotechnological methods. However, ARGs in bioaerosols have not received much attention. In addition, ARG and HGT profiling in air environments is greatly limited in field studies due to the absence of suitable methodological approaches. Therefore, this study comprehensively describes recent findings from published studies and some of the appropriate molecular and biotechnological methods for monitoring antibiotic resistance in bioaerosols. In addition, this review discusses the main knowledge gaps regarding current methodological issues and future research directions.

     

Occurrence and Abundance of Antibiotics and Resistance Genes in Rivers,Canal and near Drug Formulation Facilities – A Study in Pakistan

Antibiotic resistance (AR) is a global phenomenon that has severe epidemiological ramifications world-wide. It has been suggested that antibiotics that have been discharged into the natural aquatic environments after usage or manufacture can promote the occurrence of antibiotic resistance genes (ARG). These environmental ARGs could serve as a reservoir and be horizontally transferred to human-associated bacteria and thus contribute to AR proliferation. The aim of this study was to investigate the anthropogenic load of antibiotics in Northern Pakistan and study the occurrence of ARGs in selected samples from this region. 19 sampling sites were selected; including six rivers, one dam, one canal, one sewage drain and four drug formulation facilities. Our results show that five of the rivers have antibiotic levels comparable to surface water measurements in unpolluted sites in Europe and the US. However, high levels of antibiotics could be detected in the downstream river in close vicinity of the 10 million city Lahore, 1100, 1700 and 2700 ng L⁻¹ for oxytetracycline, trimethoprim, and sulfamethoxazole respectively. Highest detected levels were at one of the drug formulation facilities, with the measured levels of 1100, 4100, 6200, 7300, 8000, 27000, 28000 and 49000 ng L⁻¹ of erythromycin, lincomycin, ciprofloxacin, ofloxacin, levofloxacin, oxytetracycline, trimethoprim and sulfamethoxazole respectively. ARGs were also detected at the sites and the highest levels of ARGs detected, sulI and dfrA1, were directly associated with the antibiotics detected at the highest concentrations, sulfamethoxazole and trimethoprim. Highest levels of both antibiotics and ARGs were seen at a drug formulation facility, within an industrial estate with a low number of local residents and no hospitals in the vicinity, which indicates that the levels of ARGs at this site were associated with the environmental levels of antibiotics.     

新型抗生素抗性基因传播元件ISCR及其生态风险

抗生素抗性基因(antibiotic resistance genes, ARGs)作为一种新型的环境污染物,成为多个学科关注的焦点.其在不同环境介质中的扩散和传播具有极大的环境危害性,对人类健康造成严重威胁.插入序列共同区(insertion sequence common region, ISCR),是一种新发现的抗性基因传播元件,因其特殊的遗传结构,能够通过滚环复制及同源重组等机制移动邻近的任何DNA序列,是ARGs在不同DNA分子或不同种属细菌间水平传播的高效媒介.目前世界上发现了27种ISCR元件.大量间接证据表明,ISCR可能与许多耐药基因的移动和扩散有关,特别是多重耐药性(multiple drug resistance, MDR)形成与传播.因此,ISCR很可能是抗生素抗性基因在环境中扩散传播的关键因子.本文就ARGs水平传播、ISCR结构特征、ISCR种类及其相关ARGs及其研究方法等进行综述,并揭示ISCR元件可能的生态风险,提出了今后的研究重点,以期为今后深入开展相关研究打下基础.     

Targeted (PCR-based) screening of antibiotic resistance genes’ prevalence in the gut microbiota of tribal people of Nabarangpur,Odisha, India

Antibiotic resistance is a major public health concern worldwide. The gut microbiota harbours multiple antibiotic resistant genes (ARGs) that contribute to the existing and future microbial population in a community or ecosystem. This study aimed to investigate the prevalence of 35 antibiotic resistance genes (ARGs) in the gut microbiota of the tribal people of Nabarangpur, Odisha, India. A total of 83 faecal samples were collected from three different tribes (Bhatra, Gond, and Paraja). Total faecal DNA was extracted, and the simplex polymerase chain reaction was performed to detect selected ARGs. Further analysis was done to estimate the incidence of these ARGs across these tribes based on alcohol consumption habits. We identified a higher prevalence of tetracycline resistance genes (tetW, tetQ and tetM) in the gut microbiota among three populations. Furthermore, a significant (P = 0·024) difference in ARG prevalence against vancomycin in individuals with and without alcohol consumption habits was noticed. The overall distribution of ARGs among the three major tribes of this location was found to be very similar. Together, irrespective of the tribes, the people of this location have gut microbiota harbouring different kinds of ARGs and tetracycline-resistant genes are the most commonly found ARGs.     

Distribution and Quantification of Antibiotic Resistant Genes and Bacteria across Agricultural and Non-Agricultural Metagenomes

There is concern that antibiotic resistance can potentially be transferred from animals to humans through the food chain. The relationship between specific antibiotic resistant bacteria and the genes they carry remains to be described. Few details are known about the ecology of antibiotic resistant genes and bacteria in food production systems, or how antibiotic resistance genes in food animals compare to antibiotic resistance genes in other ecosystems. Here we report the distribution of antibiotic resistant genes in publicly available agricultural and non-agricultural metagenomic samples and identify which bacteria are likely to be carrying those genes. Antibiotic resistance, as coded for in the genes used in this study, is a process that was associated with all natural, agricultural, and human-impacted ecosystems examined, with between 0.7 to 4.4% of all classified genes in each habitat coding for resistance to antibiotic and toxic compounds (RATC). Agricultural, human, and coastal-marine metagenomes have characteristic distributions of antibiotic resistance genes, and different bacteria that carry the genes. There is a larger percentage of the total genome associated with antibiotic resistance in gastrointestinal-associated and agricultural metagenomes compared to marine and Antarctic samples. Since antibiotic resistance genes are a natural part of both human-impacted and pristine habitats, presence of these resistance genes in any specific habitat is therefore not sufficient to indicate or determine impact of anthropogenic antibiotic use. We recommend that baseline studies and control samples be taken in order to determine natural background levels of antibiotic resistant bacteria and/or antibiotic resistance genes when investigating the impacts of veterinary use of antibiotics on human health. We raise questions regarding whether the underlying biology of each type of bacteria contributes to the likelihood of transfer via the food chain.     

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.     

Nonnutritive sweeteners can promote the dissemination of antibiotic resistance through conjugative gene transfer

Antimicrobial resistance (AMR) poses a worldwide threat to human health and biosecurity. The spread of antibiotic resistance genes (ARGs) via conjugative plasmid transfer is a major contributor to the evolution of this resistance. Although permitted as safe food additives, compounds such as saccharine, sucralose, aspartame, and acesulfame potassium that are commonly used as nonnutritive sweeteners have recently been associated with shifts in the gut microbiota similar to those caused by antibiotics. As antibiotics can promote the spread of antibiotic resistance genes (ARGs), we hypothesize that these nonnutritive sweeteners could have a similar effect. Here, we demonstrate for the first time that saccharine, sucralose, aspartame, and acesulfame potassium could promote plasmid-mediated conjugative transfer in three established conjugation models between the same and different phylogenetic strains. The real-time dynamic conjugation process was visualized at the single-cell level. Bacteria exposed to the tested compounds exhibited increased reactive oxygen species (ROS) production, the SOS response, and gene transfer. In addition, cell membrane permeability increased in both parental bacteria under exposure to the tested compounds. The expression of genes involved in ROS detoxification, the SOS response, and cell membrane permeability was significantly upregulated under sweetener treatment. In conclusion, exposure to nonnutritive sweeteners enhances conjugation in bacteria. Our findings provide insight into AMR spread and indicate the potential risk associated with the presence of nonnutritive sweeteners.Subject terms: Microbial ecology, Water microbiology     

Zoonosis: An Emerging Link to Antibiotic Resistance Under “One Health Approach”

Current scenario in communicable diseases has generated new era that identifies the “One health” approach to understand the sharing and management of etiological agents with its impact on ecosystem. Under this context the relevance of zoonotic diseases generates major concern. The indiscriminate and higher use of antibiotics in animal husbandry creates substantial pressure on the gut microbiome for development of resistance due to shorter generation time and high density. Thus, gut works as a bioreactor for the breeding of ARBs in this scenario and are continuously released in different niches. These ARBs transfer resistance genes among native flora through horizontal gene transfer events, vectors and quorum sensing. About 60% of infectious diseases in human are caused by zoonotic pathogens have potential to carry ARGs which could be transmitted to humans. The well documented zoonotic diseases are anthrax cause by Bacillus anthracis, bovine tuberculosis by Mycobacterium tuberculosis, brucellosis by Brucella abortus, and hemorrhagic colitis by Escherichia coli. Similarly, most of the antibiotics are not completely metabolized and released in unmetabolized forms which enters the food chain and affect various ecological niches through bioaccumulation. The persistence period of antibiotics ranges from?<?1 to 3466 days in environment. The consequences of misusing the antibiotic in livestock and their fate in various ecological niches have been discussed in this review. Further the light sheds on antibiotics persistence and it biodegradation through different abiotic and biotic approaches in environment. The knowledge on personnel hygiene and strong surveillance system for zoonotic disease including ARBs transmission, prevention and control measures should be established to regulate the spread of AMR in the environment and subsequently to the human being through a food web.

     

宏基因组分析城市休闲水域九山湖的微生物群落结构和抗性基因

【背景】城市水环境正面临着严峻的抗生素抗性基因(antibiotic resistance genes,ARGs)污染。然而关于城市休闲水域中ARGs的研究较少。【目的】对城市休闲水域夏冬季节的微生物群落和抗性基因组成进行研究分析,促进对休闲水域水生生态系统的认识。【方法】基于高通量测序技术,对休闲湖泊九山湖夏季和冬季的微生物及ARGs组成进行分析。【结果】在夏季和冬季样本中分别检测到148门和152门。变形菌门(Proteobacteria)和放线菌门(Actinobacteria)是两个季节样本的主要菌门。夏季优势属为聚球藻属(Synechococcus),冬季优势属为Liminohabitans。此外,共鉴定出449种抗性基因型(304种是两个季节所共有的抗性基因,66种是夏季特有的抗性基因,79种是冬季特有的抗性基因)。夏季样本中ARGs的相对丰度显著高于冬季。MCR-1.2和BcI分别是夏季和冬季水样的主要ARGs。九山湖样本检测到的抗性基因的耐药机制主要是抗生素外排、抗生素失活或抗生素靶位改变。冗余分析(redundancy analysis,RDA)和典型对应分析(canonical correspondence analysis,CCA)结果表明,环境因子与微生物群落和抗性基因的分布显著相关。【结论】九山湖冬季和夏季的微生物群落结构和抗性基因组成存在明显差异,为进一步认识和了解城市休闲水生生态系统的结构提供了有用的信息,并强调了水体的ARGs污染可能造成的健康危害。     

土壤中抗生素的环境风险及污染土壤的生物修复技术

抗生素的广泛使用导致其在环境中普遍存在,所引发的抗性基因问题已对全球公共卫生构成重大威胁。土壤是环境中抗生素的重要汇,抗生素暴露会对土壤生物带来危害,甚至会间接对人体健康造成潜在风险,因此需采取有效手段修复抗生素污染的土壤。文中综述了抗生素对土壤植物表型生长指标、土壤动物生理特征及群落分布、微生物群落组成与功能的影响,以及抗生素抗性基因在土壤生物间的传播风险等;总结了利用耐受土壤植物、动物、微生物以及其互作关系修复抗生素污染土壤的潜力与前景,指出了已有土壤中抗生素环境风险和生物修复研究中尚存在的问题,展望了未来的研究方向。     

Status of pathogens,antibiotic resistance genes and antibiotic residues in wastewater treatment systems

Pathogens are becoming nearly untreatable due to the rise in gaining new resistance against standard antibiotics. Coexistence of microbial pathogens, antibiotics and antibiotic resistant genes (ARGs) in wastewater treatment plants (WWTP) provide favourable conditions for the development of new antibiotic resistant bacteria (ARB); facilitate horizontal gene transfer among pathogens and may also serve as a hotspot for the spread of ARB and genes into the environment. In this study, the current status of wastewater treatment systems in the removal of pathogens, ARGs, and antibiotic residues are discussed. WWTP are efficient in removing pathogens and antibiotic residues to a greater extend during secondary and tertiary treatment processes. Recent studies, however, have shown high variations in the presence of pathogens including ARB as well as antibiotic resistance genes (ARG) in the final effluent. Prolonged sludge retention time (SRT) and hydraulic retention time (HRT) during secondary treatment will facilitate antibiotic removal by adsorption and biodegradation. However, the above conditions can also lead to the enhancement of antibiotic resistance process in microbes. Therefore, optimum conditions for the operation of conventional WWTP for the efficient removal of antibiotics are yet to be established. The removal of antibiotic residues can be accelerated by combining conventional activated sludge (CAS) process with an additional treatment technology involving dosing with ozone. The advanced biological treatment method using membrane bioreactors (MBR) in combination with coagulation reportedly has the best ARG removal efficiency, and removes both ARB and extracellular ARGs. While studies have predicted the fate for ARGs in wastewater treatment plants, the mechanisms of ARGs acquisition remains to be conclusively established. Thus, strategies to investigate the underlying mechanism of acquisition of ARGs within the WWTP are also provided in this review.