Transmission of antibiotic resistance genes in agroecosystems: an overview

The use of antibiotics in human medicine and animal husbandry has resulted in the continuous release of antibiotics into the environment, which imposes high selection pressure on bacteria to develop antibiotic resistance. The spread and aggregation of antibiotic resistance genes (ARGs) in multidrug-resistant pathogens is one of the most intractable clinical challenges. Numerous studies have been conducted to profile the patterns of ARGs in agricultural ecosystems, as this is closely related to human health and wellbeing. This paper provides an overview of the transmission of ARGs in agricultural ecosystems resulting from the application of animal manures and other organic amendments. The future need to control and mitigate the spread of antibiotic resistance in agricultural ecosystems is also discussed, particularly from a holistic perspective, and requires multiple sector efforts to translate fundamental knowledge into effective strategies.     

慢性阻塞性肺疾病急性加重患者痰病原菌分布及耐药性分析

目的 分析本院慢性阻塞性肺疾病急性加重期（acute exacerbation of chronic obstructive pulmonary disease,AECOPD）患者痰菌分布及耐药特点,更好的指导早期临床用药。方法 采集2014年8月到2016年7月本院呼吸病房的613例行痰培养的AECOPD患者的临床资料及痰培养结果,分析其痰菌分布及耐药特点。结果 送检痰标本共检出病菌406株,其中革兰阴性菌、真菌、革兰阳性菌各247株（60.8%）、103株（25.4%）、56株（13.8%）,常见的病菌包括铜绿假单胞菌（17.0%）、白假丝酵母菌（12.8%）、金黄色葡萄球菌（9.9%）、鲍氏/溶血不动杆菌（8.1%）、肺炎克雷伯菌（7.9%）、大肠埃希氏菌（6.9%）、嗜麦芽窄食单胞菌（6.9%）。大肠埃希菌及肺炎克雷伯杆菌产超广谱β-内酰胺酶（ESBLs）阳性率分别为71.4%和34.4%,二者对大部分抗生素有不同程度耐药,铜绿假单胞菌、鲍氏/溶血不动杆菌、嗜麦芽窄食单胞菌耐药率均较高。结论 革兰阴性杆菌为AECOPD患者痰培养的主要菌群,总体耐药性高,结合当地病菌分布及耐药特点选用抗菌药物尤为重要。     

Antibiotics and antibiotic resistance genes in natural environments

The large majority of antibiotics currently used for treating infections and the antibiotic resistance genes acquired by human pathogens each have an environmental origin. Recent work indicates that the function of these elements in their environmental reservoirs may be very distinct from the "weapon-shield" role they play in clinical settings. Changes in natural ecosystems, including the release of large amounts of antimicrobials, might alter the population dynamics of microorganisms, including selection of resistance, with consequences for human health that are difficult to predict.     

重金属协同选择环境细菌抗生素抗性及其机制研究进展

抗生素的长期滥用,引起环境细菌耐药性不断增强,加速了抗生素抗性基因(Antibiotic resistance genes,ARGs)在环境中的传播扩散。在重金属污染的环境中,细菌不仅具备重金属抗性,并且具备多种抗生素抗性,抗生素抗性基因的污染水平也随之升高。在介绍重金属与抗生素抗性最新研究进展的基础上,阐述了环境细菌的抗生素抗性、重金属抗性及其相关抗性机制,并着重论述重金属和抗生素协同选择环境细菌耐药性及其机制。     

TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification

Host protection from infection relies on the recognition of pathogens by innate pattern-recognition receptors such as Toll-like receptors (TLRs). Here, we show that the orphan receptor TLR13 in mice recognizes a conserved 23S ribosomal RNA (rRNA) sequence that is the binding site of macrolide, lincosamide, and streptogramin group (MLS) antibiotics (including erythromycin) in bacteria. Notably, 23S rRNA from clinical isolates of erythromycin-resistant Staphylococcus aureus and synthetic oligoribonucleotides carrying methylated adenosine or a guanosine mimicking a MLS resistance-causing modification failed to stimulate TLR13. Thus, our results reveal both a natural TLR13 ligand and specific mechanisms of antibiotic resistance as potent bacterial immune evasion strategy, avoiding recognition via TLR13.     

Bacteria subsisting on antibiotics

Antibiotics are a crucial line of defense against bacterial infections. Nevertheless, several antibiotics are natural products of microorganisms that have as yet poorly appreciated ecological roles in the wider environment. We isolated hundreds of soil bacteria with the capacity to grow on antibiotics as a sole carbon source. Of 18 antibiotics tested, representing eight major classes of natural and synthetic origin, 13 to 17 supported the growth of clonal bacteria from each of 11 diverse soils. Bacteria subsisting on antibiotics are surprisingly phylogenetically diverse, and many are closely related to human pathogens. Furthermore, each antibiotic-consuming isolate was resistant to multiple antibiotics at clinically relevant concentrations. This phenomenon suggests that this unappreciated reservoir of antibiotic-resistance determinants can contribute to the increasing levels of multiple antibiotic resistance in pathogenic bacteria.     

斜带髭鲷感染鱼肠道弧菌的分离及分析

[目的]对患病斜带髭鲷(Hapalogenys nitens)幼鱼进行病原菌的分离及鉴定。[方法]对2013年1月福州罗源湾某海水网箱养殖场斜带髭鲷幼鱼死亡病例进行了发病情况、临床表现及病原学方面的检验;对其中5尾患病斜带紫鲷的肝脏、脾脏、肾脏及鳃丝进行细菌分离与纯培养,对病原菌进行形态、生理生化测定和16S rRNA基因序列测定及系统发育学分析,并用30种抗菌类药物对5株菌进行了药敏试验。[结果]斜带髭鲷幼鱼死亡病例为细菌性感染症,分离纯培养的菌株(fzlyw201301071和fzlyw201301072)与弧菌属(Vibrio)的鱼肠道弧菌(Vibrio ichthyoenteri)可认定为同一种属。药敏试验结果显示,在不同菌株间无明显的敏感与耐药性差异。[结论]为水产养殖动物的疾病防治提供了理论依据。     

Acceleration of emergence of bacterial antibiotic resistance in connected microenvironments

The emergence of bacterial antibiotic resistance is a growing problem, yet the variables that influence the rate of emergence of resistance are not well understood. In a microfluidic device designed to mimic naturally occurring bacterial niches, resistance of Escherichia coli to the antibiotic ciprofloxacin developed within 10 hours. Resistance emerged with as few as 100 bacteria in the initial inoculation. Whole-genome sequencing of the resistant organisms revealed that four functional single-nucleotide polymorphisms attained fixation. Knowledge about the rapid emergence of antibiotic resistance in the heterogeneous conditions within the mammalian body may be helpful in understanding the emergence of drug resistance during cancer chemotherapy.     

临床型奶牛乳腺炎致病菌分析及药敏试验

对佳木斯市4所大型奶牛场17头患有临床型乳腺炎的奶牛的不同乳区的20份乳汁样品进行致病菌的分离鉴定,并对分离得到的致病菌进行8种氟喹诺酮类药物和3组中药方剂的敏感性试验。结果表明,从被检乳汁样品中共分离得到致病菌508株,包括金黄色葡萄球菌、表皮葡萄球菌、无乳链球菌、乳房链球菌和化脓链球菌,分别占致病菌总数的35.43%、10.63%、39.96%、11.22%、2.76%。药敏试验表明,分离得到的致病菌对8种氟喹诺酮类药物均敏感,其中金黄色葡萄球菌对恩诺沙星最敏感,无乳链球菌对加替沙星最敏感;3组中药方剂中以方剂Ⅰ的抑菌效果最好。     

奶牛乳房炎病原菌的分离鉴定及耐药性分析

为弄清中国不同地区奶牛乳房炎主要病原菌种类、分布情况及其药物敏感特性,并为奶牛乳房炎的综合防治提供依据,对在内蒙古、甘肃、四川和上海4地部分奶牛场采集的472份临床型或亚临床型乳房炎乳样进行细菌分离鉴定,并对主要病原菌进行药敏试验。结果表明,检测奶样中检出细菌482株,其中金黄色葡萄球菌149株,占30.91%;表皮葡萄球菌91株,占18.88%;无乳链球菌60株,占12.45%;停乳链球菌60株,占12.45%;乳房链球菌35株,占7.26%;大肠杆菌33株,占6.85%。各个地区优势菌群存在差异,药敏试验结果也不相同,分离出的细菌绝大部分对环丙沙星、左氟沙星、氧氟沙星和先锋霉素V高度敏感,但对青霉素、红霉素和万古霉素有较强耐药性。     

山西省猪致病性大肠杆菌血清型调查及耐药性监测

从山西省50多个规模化养猪场无菌采集疑似大肠杆菌病死仔猪心、肝、脾、肠系膜淋巴结以及可疑病猪的直肠内容物等病料,进行病原菌分离、生化试验、致病性试验、血清型鉴定及药物敏感试验。结果表明,从50多个养猪场中分离出69株致病性大肠杆菌,分属30个血清型,其中,主要流行血清型为O138,O101,O9,O119,O141。药敏试验结果表明,大肠杆菌致病菌株对头孢呋辛钠、头孢哌酮、头孢噻肟、磷霉素这4种抗生素高度敏感,而对青霉素、链霉素、磺胺嘧啶、利福平、痢菌净、红霉素、诺氟沙星、阿莫西林、氨苄青霉素、氯霉素、庆大霉素、强力霉素、罗红霉素、环丙沙星等药物的耐药性菌株比例在65%以上,说明山西地区猪致病性大肠杆菌血清型多样、耐药性强、耐药谱广。     

土霉素诱导土壤可培养细菌产生耐药性的研究

通过在土壤中反复添加不同浓度的土霉素进行预处理,采用平板稀释法测定了土壤细菌在含土霉素培养基及普通培养基上的生长情况,用耐药菌发生率作为指标评价了土霉素污染浓度和污染次数对土壤抗土霉素可培养细菌的影响。结果表明,土霉素污染可诱导土壤土霉素抗性细菌的产生,耐药菌发生率与土霉素添加浓度和添加次数有关。当土霉素添加浓度大于4 mg/kg时,一次处理就可诱导土壤可培养细菌产生耐药性;当土霉素浓度为3 mg/kg时,二次处理可诱导土壤可培养细菌产生耐药性;当土霉素浓度为2 mg/kg时,需要5次处理才可诱导土壤可培养细菌产生耐药性;而当土霉素浓度为1 mg/kg时,5次处理均没有明显诱导土壤可培养细菌产生耐药性。研究结果表明,长期施用含土霉素的畜禽粪可导致土壤土霉素抗性细菌的形成和发展。     

家养禽类肠道可培养细菌抗生素抗性的种类、数量和分布

采用微生物培养和影印法对家养禽类鸡、鸭和肉鸽肠道可培养细菌抗生素抗性的种类、数量和分布进行了调查.结果表明:在调查的鸡、鸭和肉鸽3种禽类中,肠道可培养细菌抗生素抗性的分布非常普遍,所有测试细菌至少可以抗一种抗生素,抗5种以上抗生素的细菌占测试细菌的比例分别为75.0%、58.9%和97.4%;个别细菌对测试的10种抗生素均具有抗性.在抗生素方面,肠道可培养细菌对萘啶酮酸、盐酸四环素、盐酸克林霉素、磺胺嘧啶和红霉素的抗性分布比较广泛.提取细菌基因组并扩增16S rRNA基因,并对这些细菌进行分子鉴定的结果表明,分离的肠道可培养细菌为埃希氏大肠杆菌.在美国国家生物技术信息中心数据库进一步搜索的结果显示:与肠道可培养细菌同源性大于99%的细菌广泛分布于不同的环境、宿主和病原菌中,表明这些肠道可培养细菌抗生素抗性是一个潜在的环境微生物以及人体病原菌抗生素抗性的传播来源.     

畜禽粪便中抗生素抗性基因(ARGs)污染问题及环境调控

抗生素在畜禽养殖业的大量使用造成抗生素抗性基因(ARGs)污染日益严重。动物体内诱导出的抗性菌株随粪便排出后,通过基因水平转移进入土壤进而污染土壤和地下水环境。堆肥作为一种将粪便资源化的优良传统方法,能否有效去除畜禽粪便中的ARGs而防止环境污染值得探讨。通过总结畜禽粪便ARGs污染现状,粪便堆肥过程中微生物群落结构变化与影响微生物变化的因素以及堆肥可能对粪便中ARGs造成的影响,提出将堆肥作为去除畜禽粪便中ARGs的一种有效手段,利用堆肥产生的高温去除抗性菌株和抗性质粒等,并且考虑加入能直接灭杀肠道微生物的化学抑制剂(如石灰氮、胺类、吲哚等),实现降低畜禽粪便ARGs丰度的可能。据此强调开展畜禽粪便中ARGs研究的必要性,认为将堆肥和ARGs研究结合起来,可以有效地降低这种新型污染物的污染水平。     

浙江省主要奶牛养殖区乳房炎致病菌金黄色葡萄球菌的耐药性

为了解浙江省奶牛乳房炎致病菌的耐药情况,评估其耐药表型与基因型的相关性,本研究从浙江省主要奶牛养殖区奶牛乳房炎病例分离到70株金黄色葡萄球菌,测定它们对13种常用抗菌药物的药敏性,并采用聚合酶链式反应(PCR)检测这些细菌耐青霉素G、庆大霉素、红霉素和四环素的耐药基因mecA、aac(6')/aph(2″)、erm和tetM的携带情况.结果表明,奶牛乳房炎金黄色葡萄球菌对常用抗生素的耐药情况严重,耐药表型和耐药基因型的符合率较高.70株金黄色葡萄球菌主要对青霉素G、庆大霉素、红霉素和四环素耐药,表型耐药率分别为90.0%、67.1%、50.0%和60.0%,耐药基因mecA、aac(6')/aph(2″)、erm和tetM的检出率分别是92.9%、77.1%、48.6%和61.4%,耐药表型与耐药基因型的符合率分别为97.1%、70.0%、87.1%和81.4%.     

生防菌株PF-1基于全基因组数据的分类鉴定及拮抗能力分析

基于全基因组数据,确定生防菌株PF-1的分类地位,验证其对植物病原菌的拮抗作用以及挖掘其潜在的生防功能。通过全基因组中16S rRNA基因序列的系统发育分析及基因组分析方法确定生防菌株PF-1的分类地位,通过平板对峙方法研究其对马铃薯枯萎病菌和棉花黄萎病菌的拮抗能力;利用antiSMASH软件分析和预测菌株PF-1的抗生素相关基因并挖掘其生防潜力。基于16S rRNA基因序列的系统发育分析及基因组分析结果,PF-1被鉴定为防御假单胞菌(Pseudomonas protegens),同时发现PF-1基因组中存在15种次生代谢产物基因簇,具有良好的抑制病原菌生长和提高植物抗病性的能力,在农业中具有良好的应用前景。     

湛江东海岛对虾育苗场水体细菌的抗生素耐药性

利用K-B纸片扩散法研究分离自湛江东海岛6个对虾育苗场(分别编号为YF,JD,DC,HM,SD,XR)水体中的217株细菌菌株对12种抗生素的耐药性。研究结果表明,参试的12种抗生素之间的细菌耐药性差异显著(ANOVA,P0.05),6个育苗场内细菌对多粘菌素B、左氟沙星和环丙沙星高度敏感,对氨苄西林、甲氧苄胺嘧啶、复方新诺明和四环素则高度耐药;多重耐药菌株在每个育苗场中均有发现,52.07%的菌株呈现多重耐药性,未发现对参试的12种抗生素100%耐药的菌株。统计分析结果显示,尽管HM和XR育苗场的细菌耐药性比其他育苗场高,但6个对虾育苗场之间水体细菌的抗生素耐药性无显著差异(ANOVA,P0.05)。     

鸡肠道大肠杆菌多抗菌株C20耐药性基因的预测和分析

为揭示动物源大肠杆菌耐药性机制,追踪潜在的抗性基因传播机制及其来源,在先前公布的分离自鸡肠道的多抗耐药性菌株大肠杆菌Escherichia coli C20基因组基础上,对E.coli C20抗生素抗性基因种类、数量、来源及其耐药性机制进行分析。通过与已知的抗生素抗性基因数据库进行对比和注释,一共预测和注释了80个潜在的耐药性基因,包括与先前筛选的7种抗生素抗性对应的抗性基因。其中,喹诺酮类抗生素抗性基因位于C20基因组上,四环素、磺胺类、氯霉素、氨基糖苷类以及大环内酯类抗生素的抗性基因则位于C20环状质粒分子上。基因组聚类分析表明,菌株C20与大肠杆菌K-12菌株DH1、BW2952和MG1655亲缘关系较近,序列相似性97%~99%。3株K-12菌株基因组中没有发现与C20质粒类似的序列,表明C20菌株的抗性基因主要是通过质粒介导的水平基因转移方式从其他环境微生物中获得。     

肉牛溶血性曼氏杆菌的分离鉴定及药敏试验

目的分离鉴定四川某规模化山羊场引起山羊大规模呼吸道疾病和死亡的病原菌,并分析其致病和耐药机制。方法将病原分离纯化后,依次进行生化试验、小鼠致病性试验、药敏试验、16S rRNA 序列分析及荚膜血清分型,并通过全基因组测序对分离菌株进行物种分型以及基因功能分析。结果从病死山羊鼻液、气管、肺脏和肝脏中分离出同一种菌,经鉴定为荚膜血清2型的溶血性曼氏杆菌,命名为MHLB002;对小鼠的LD₅₀值为4.4×10⁷ CFU/mL;药敏试验结果表明：该溶血性曼氏杆菌对头孢类、β-内酰胺类、磺胺类和喹诺酮类药物敏感,对部分大环内酯类和氨基糖苷类药物耐药;全基因组序列分析显示该菌基因组大小为2 580 488 bp,序列类型为ST-43,与MH1475菌株同源性最近,且2菌株基因组ANI 值达98.60%;MHLB002菌株全基因组中共有19个ORF编码与致病性相关的基因,9个ORF编码耐药基因。结论本试验所分离出的荚膜血清2型溶血性曼氏杆菌,是引起该养殖场山羊大规模呼吸道疾病和死亡的致病菌,同时为预防和治疗山羊溶血性曼氏杆菌病提供理论依据。     

How Specific is Non-Hypersensitive Host and Nonhost Resistance of Barley to Rust and Mildew Fungi？

Full nonhost resistance can be defined as immunity, displayed by an entire plant species against all genotypes of a plant pathogen. Interesting biological questions are, whether the genes responsible for the nonhost status of a plant species have a general or a specific effectiveness to heterologous （＂nonhost＂） pathogens？ Is the nonhost resistance to pathogens of plant species that are related to the nonhost based on R-genes or on other types of genes？ We study this question in barley （Hordeum vulgare L.）, which is a near-nonhost to several rusts （Puccinia） of cereals and grasses. By crosses and selection we accumulated susceptibility and developed an experimental line, SusPtrit, with high susceptibility to at least nine different heterologous rust taxa such as the wheat and Agropyron leaf rusts （P. triticina and P. persistens, respectively）. At the microscopic level there is also some variation among barley accessions in the degree that the heterologous wheat powdery mildew （Blumeria graminis f.sp. tritici） is able to form haustoria in epidermal cells. So, also the genetics of the variation in level of nonhost resistance to heterologous mildew fungi can be studied in barley. Our data obtained on mapping populations involving three regular nonhost-immune accessions （Vada, Cebada Capa and Golden Promise） suggest that nonhost resistance is the joined effect of multiple, quantitative genes （QTLs） and very occasionally a major gene （R-gene？） is involved. Most QTLs have effect to only one or two heterologous rusts, but some have a wider spectrum. This was confirmed in a set of QTL-NILs. Those QTL-NILs are used to fine-map the effective genes. In some cases, a QTL region with effectiveness to several heterologous rusts might be a cluster of genes with a more narrow spectrum of effectiveness. Our evidence suggests that nonhost resistance in barley to rust and powdery mildew fungi of related Gramineae is not due to R-genes, but to pathogen species-specific quantitative resistance genes.