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.     

环境微生物的抗生素抗性和抗性组

环境和医疗实践中广泛存在的细菌抗生素抗性已经成为食品安全和人类健康领域的主要威胁。近年来的研究表明,病原菌主要通过水平基因转移而不是基因突变获得抗性,大量的研究支持病原微生物抗生素抗性基因的环境来源。系统论述了环境微生物抗生素抗性起源、进化及病原菌抗性基因与环境抗生素抗性组相互之间的交叉传播和水平转移机制,介绍了近年来环境微生物抗生素抗性组生态和进化生物学研究的最新进展和方法学应用。     

土壤中抗性基因的产生,扩散传播以及消减的研究进展

近年来,土壤中残留的大量抗生素不可避免的导致耐药微生物和抗性基因的增加和扩散,引起一系列土壤污染和生态风险。作为一类新兴污染物,抗性基因的污染水平已经远远超出我们的预想,因此对土壤中抗性基因的分布水平、扩散传播及消减技术的研究刻不容缓。本文对国内外土壤中抗生素和抗性基因残留水平进行了总结分析,探讨了土壤中抗性基因的产生、扩散的内在动力和机制。同时,分析了土壤中抗性基因分布和扩散的影响因素,如:抗生素残留水平,土壤理化性质和环境条件等。在此基础上,探讨了土壤抗性基因阻隔和消减技术,包括传统降解方法:高温,光照催化、微波-H2O2-微生物联合处理技术等,并提出新型消解技术:取代活性基团、靶位修饰以及改变外排泵的通透性等。讨论未来在控制抗性基因生态风险,降低其在土壤中的丰度,有效阻截技术的发展趋势。     

环境抗生素抗性基因研究进展

抗生素耐药性及其在全球范围内的传播已成为国际关注的热点。本文结合最新文献,综述了抗生素抗性基因在环境中的来源、传播、分布以及新型抗性基因的发现等方面的研究进展。环境中抗生素抗性基因的来源主要是环境中细菌的内在抗性基因及随人或动物粪便排到体外的抗性细菌。功能宏基因组学技术的应用极大地丰富了人们对抗生素抗性组学的认知,并已从环境中筛选到多种新型抗性基因。近年来,由于抗生素在医疗以及养殖业中的大量使用,增加了抗性基因在环境中的丰度和多样性,加速了抗性基因在环境中的传播,在多种环境介质(如养殖水域、污水处理厂、河流、沉积物和土壤等)均检测到多种高丰度的抗生素抗性基因。我们建议今后在以下方面开展深入研究:(1)抗性基因传播和扩散的机制;(2)新型抗性基因筛选和抗性机制;(3)抗生素和抗性基因环境风险评估体系等。     

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。     

不同施肥处理农田土壤中噬菌体与细菌携带抗生素抗性基因的比较北大核心CSCD

为了揭示有机肥和化肥长期施用对农田土壤噬菌体携带的抗生素抗性基因(antibiotic resistance genes,ARGs)多样性和丰度的影响,并与土壤细菌携带的ARGs进行对比,本文将土壤抗生素抗性分为细菌和噬菌体两个部分,利用微滴数字PCR(droplet digital PCR,ddPCR)技术定量分析了土壤噬菌体和细菌DNA中25种ARGs亚型和I类整合子(intl1)的丰度。结果表明,土壤噬菌体中ARGs的检出率和总丰度以及intl1丰度均低于土壤细菌,其中噬菌体中检测到20种ARGs亚型,在不施肥、单施化肥和单施有机肥土壤的噬菌体中,目标ARGs的检出率分别为68%、72%和76%。土壤噬菌体中ARGs的总丰度在有机肥施用土壤中显著高于不施肥和化肥施用土壤(P<0.05),其中多耐药类、大环内酯-林肯酰胺-链阳性菌素B(MLSB)类和β-内酰胺类抗性基因丰度占显著优势。除了β-内酰胺类抗性基因blaTEM,噬菌体中其他ARGs亚型的丰度均显著低于细菌(P<0.05)。噬菌体与细菌携带ARGs在不同施肥处理中均存在显著正相关(P<0.05)。冗余分析结果显示,施肥可能通过改变土壤pH、重金属和营养因子水平来影响细菌和噬菌体中ARGs的赋存特征。本研究结果表明,噬菌体是除细菌之外的农田土壤另一个重要ARGs储存库,施用有机肥能同时显著提高土壤细菌和噬菌体中ARGs的多样性和丰度。     

细菌遗传元件水平转移与抗生素抗性研究进展

细菌可移动遗传元件包括噬菌体、质粒、转座子、插入序列、整合子、基因组岛（genomic island and genomic islet）等,其中接合性质粒、转座子、整合子及基因组岛等是与抗生素抗性有关的元件,可以在向种甚至于不同种菌株间水平转移,加速了临床上耐药及多重耐药菌株的产生。综述了细菌与抗生素抗性有关的可移动遗传元件的种类、特征及转移机制的研究进展。     

武夷菌素对土壤微生物群落及抗生素抗性基因的影响

抗生素抗性基因(antibiotic resistance genes, ARGs)作为一种新型环境污染物近年来受到广泛关注。目前关于抗生素的环境污染研究主要集中于医疗和养殖业,对植物保护领域的农用抗生素环境污染研究很少。武夷菌素是一种环保、高效、广谱的农用抗生素,在农业生产中得到了广泛应用,对农作物真菌性病害具有良好的防治效果。本研究分别选取了未使用武夷菌素和使用武夷菌素的蔬菜大棚中的土壤,通过高通量测序分析了土壤中微生物群落结构,发现两份土壤中主要的微生物群落种类没有发生明显改变,但是优势菌群的丰度有显著差异。通过荧光定量PCR技术,对18个典型的抗生素抗性基因进行了检测,发现aadA、aac(3)-Ⅱ、strA、strB、aacA4、tetX、sulI和intI1 8个基因在两份土壤中的绝对含量和丰度均有显著差异,表明武夷菌素对土壤中微生物的群落结构及抗生素抗性基因的绝对含量和丰度均会造成一定影响。本研究为评估武夷菌素的环境安全性及合理正确使用武夷菌素提供了理论依据,也为其它农用抗生素的相关研究提供了借鉴。     

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。     

细菌遗传元件水平转移与抗生素抗性研究进展*

细菌可移动遗传元件包括噬菌体、质粒、转座子、插入序列、整合子、基因组岛(genomic island and genomic islet)等,其中接合性质粒、转座子、整合子及基因组岛等是与抗生素抗性有关的元件,可以在同种甚至于不同种菌株间水平转移,加速了临床上耐药及多重耐药菌株的产生。综述了细菌与抗生素抗性有关的可移动遗传元件的种类、特征及转移机制的研究进展。     

Triclosan - an update

The discovery in 1998 that triclosan has a site-specific action in the bacterial cell as an inhibitor of NADH- or NADPH-dependent enoyl-acyl carrier protein reductase led to a lively debate in the scientific press. The thesis of this debate was that such a mode of action may allow triclosan to induce resistance and cross-resistance in bacterial cells. The debate last saw review in 2004, and this paper aims at updating our knowledge in this area, given recent research on the topic.     

Managing resistance with multiple pesticide tactics: theory, evidence, and recommendations

Sequences, mixtures, rotations, and mosaics are potential strategies for using more than one pesticide to manage pest populations and for slowing the evolution of pesticide resistance. Results from theoretical models suggest that, under certain conditions, mixtures might be especially effective for resistance management. The assumptions of such models, however, are probably not widely applicable. Potential disadvantages associated with mixtures that are usually not considered in modeling studies include disruption of biological control, promotion of resistance in secondary pests, and intense selection for cross-resistance. Results from limited experimental work suggest that pesticide combinations do not consistently suppress resistance development. More thorough evaluation of tactics that seek to optimize benefits of more than one insecticide will require rigorous experiments with the particular pest and pesticide combinations. Because of the difficulty in generalizing results across systems and the potential negative effects of multiple insecticide use, emphasis on minimizing insecticide use is recommended.     

Stability of bacterial populations in tropical soil upon exposure to Lindane

The effect of the pesticide Lindane on microbial populations was analyzed in soil with a history of contamination with various chemicals, including this pesticide. Soil microcosms were amended with 100 mg Lindane/kg soil and microbial populations were monitored for 70 days. Bacterial cell concentrations, metabolic versatility (whole community Biolog), and genetic diversity (16S rDNA/denaturing gradient gel electrophoresis) were used to monitor microbial communities. Results show the persistence of Lindane in the soil environment; at the end of the experiment, 70% of the added Lindane remained undegraded. A reduction of 50% in bacterial cell concentration was observed in Lindane-amended microcosms during the 2nd week of the experiment. This reduction was correlated with a reduction in the rate of substrate utilization as observed by Biolog. Overall, no effect of Lindane was observed on the metabolic versatility and genetic diversity in these soils, demonstrating the ability of these bacterial populations to tolerate the pressure caused by the addition of pesticides.     

Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metal-contaminated soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

Current status and future perspectives on natural enemies for pest control in Taiwan

ABSTRACT

In Taiwan, the agricultural policy, ‘Reduce the consumption of pesticide to half in the next 10 years’, was launched in 2017. Pesticide application, which results in contamination of food by chemical residues, pest resistance, and other adverse ecological effects, is a growing public and environmental concern. Pest control by natural predators is, thus, the best alternative. Biological control methods implemented based on insights obtained from studies on pest behaviour, rearing, and various crop management modes, increase the possibility of controlling pests in modern organic agricultural systems. More than a decade has passed since the first introduction of a predatory insect in Taiwan for pest control (in the 1990s). Predatory and parasitic natural enemies, including lacewing, predatory stink bugs, Orius, and parasitic wasps, were initially used for controlling thrips, aphids, spider mites, whiteflies, and lepidopteran pests. At present, there exists a wide range of integrated pest management (IPM) methods incorporating other non-chemical, biological, and agricultural methods. However, recently, there has been an increase in research and development on the utilisation of natural enemies of insects and the associated food safety issues. Mass production and release, storage, and handling techniques of insect predators and parasitoids have been successful in recent years. The final goal of present day research is to develop natural enemy products and provide an IPM-based model to farmers for using natural enemies in agricultural production systems, thereby reducing pesticide application and ensuring food security.     

Aminoacyl-tRNA synthetases and their inhibitors as a novel family of antibiotics

The emergence of multidrug-resistant strains of pathogenic microorganisms and the slow progress in new antibiotic development has led in recent years to a resurgence of infectious diseases that threaten the well-being of humans. The result of many microorganisms becoming immune to major antibiotics means that fighting off infection by these pathogens is more difficult. The best strategy to get around drug resistance is to discover new drug targets, taking advantage of the abundant information that was recently obtained from genomic and proteomic research, and explore them for drug development. In this regard, aminoacyl-tRNA synthetases (ARSs) provide a promising platform to develop novel antibiotics that show no cross-resistance to other classical antibiotics. During the last few years there has been a comprehensive attempt to find the compounds that can specifically target ARSs and inhibit bacterial growth. In this review, the current status in the development of ARS inhibitors will be briefly summarized, based on their chemical structures and working mechanisms.     

非编码RNA与细菌耐药

细菌非编码RNA是一类新发现的基因表达调控因子,通过与靶mRNA配对,导致mRNA翻译和稳定性的变化,从而影响细胞的各种生理功能,如个体发育、翻译激活与抑制、细菌毒性等,而且一个单独的非编码RNA就能调控大量基因并对细胞生理产生深远影响。近年来诸多研究证实,非编码RNA与细菌耐药性也存在一定的关系。我们对此进行简要综述,为细菌耐药性的研究奠定基础。     

Impact of Oil-Spill Contamination on a Soil Bacterial Community: A 40-Year History of Rehabilitation in the Arava Valley

The present study evaluated the effect of crude oil contamination on a microbial community in hyper-arid soils. The Evrona Nature Reserve of Israel, situated in the Arava, was exposed twice in the last 40 years to petroleum-hydrocarbon-spill pollution. The first pollution event took place 40 years ago and was never treated, presenting a unique future time-point perspective to the second (2014) contamination event. Soil samples were collected after the second spill, and abiotic properties and bacterial diversity in the sampled soil were analyzed. The results showed that there is a significant decrease over time in the number of observed bacterial species in the contaminated samples, coupled together with bacterial species replacement toward species capable of using source oil as the sole carbon and energy source. The presence of petroleum in soil significantly changed the composition and functional diversity of a microbial community, and the Evrona Nature Reserve is still in the middle of a bioremediation process even 40 years after the crude oil contamination.