兰州大学机构库 >草地农业科技学院
青贮饲料中抗生素抗性基因的分布特征与传播机制
Alternative TitleDistribution Characteristics and Transmission Mechanism of Antibiotic Resistance Genes in Silage
Zhang xia
Subtype博士
Thesis Advisor郭旭生 ; 王虎成
2023-05-26
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name农学博士
Degree Discipline畜牧学
Keyword抗生素抗性基因 Antibiotic resistance genes 青贮 silage 可移动基因元件 mobile genetic elements 乳酸菌 lactic acid bacteria 甲酸 formic acid
Abstract

抗生素抗性基因(antibiotics resistance genes,ARGs)广泛存在于不同环境介质中,能够导致全球公共卫生和生态环境安全风险,并对人类健康构成了巨大威胁。青贮饲料是草食家畜的优质粗饲料,同时很有可能是动物潜在的ARGs来源之一。迄今为止,研究者对青贮饲料的研究主要集中在不同处理措施(添加剂、贮藏温度、干物质(dry matter,DM)和农艺措施等)对其发酵品质、营养成分变化、微生物互作与演替以及代谢产物相互转化的影响,但对 ARGs 的分布和传播情况知之甚少。为此,本研究采用宏基因组学技术阐释了 不同处理措施(乳酸菌、甲酸、DM、贮藏温度和生长年限)对苜蓿和全株玉米青贮饲料中 ARGs 分布特征和传播机制的响应;同时评估了青贮饲料中 ARGs 转移和传播的驱动因素,并明确了青贮饲料中 ARGs 宿主和临床病原体携带的 ARGs。取得了如下研究结果: 1. 土壤-新鲜苜蓿-苜蓿青贮系统中抗生素抗性基因在不同生长年限的分布 特征和驱动机制研究随着苜蓿生长年限的延长,土壤样本中 ARGs 的多样性增加(P < 0.05), 但没有影响 ARGs 的丰度。苜蓿生长年限越长,新鲜苜蓿和青贮苜蓿中 ARGs 的多样性和丰度均增加( P < 0.05 )。 万古霉素 ( vancomycin ) 和多药 ( multidrug )是土壤和新鲜苜蓿中主要的抗性基因 。 多药和杆菌肽(bacitracin)是青贮苜蓿中主要的抗性基因。转座酶(transposase)是各样本中最丰富的可移动基因元件(mobile genetic element,MGEs)类型。生长年限没有改变土壤和新鲜苜蓿中ARGs水平基因转移的潜力,但青贮苜蓿中ARGs水平基因转移的潜力随着生长年限的延长而减弱(P < 0.05)。土壤和新鲜苜蓿样本中ARGs 的主要携带者是类诺卡氏菌属(Nocardioides)。青贮苜蓿中ARGs 的主要携带者是肠杆菌属(Enterobacter)和肠球菌属(Enterococcus)和双歧杆菌属(Bifidobacterium)。溯源分析表明苜蓿生长年限越长,ARGs 在新鲜苜 蓿表面积累的越多;然而,苜蓿青贮饲料中大部分ARGs是在青贮过程中获得。土壤-苜蓿-苜蓿青贮系统中ARGs变化的主要驱动因素是细菌群落,并且青贮苜蓿中水平基因转移可能加强了 ARGs 的传播。2. 甲酸对高水分苜蓿青贮饲料中ARGs及其潜在宿主命运的影响多药抗性基因是高水分青贮苜蓿中丰度最高的ARGs 类型。自然青贮增加了ARGs的富集(P < 0.05)。然而青贮5天后,甲酸处理的青贮苜蓿降低了ARGs的丰度(P < 0.05)。同时,甲酸能够改善高水分青贮苜蓿的发酵品质。青贮90天后,甲酸处理的青贮苜蓿增加了 ARGs 的丰度(P < 0.05)。值得注意的是 , 几种病原菌 , 如葡萄 球菌属 ( Staphylococcus )、 梭状芽胞杆菌 (Clostridium)和假单胞菌属(Pseudomonas)被注释为高水分苜蓿青贮饲料中 ARGs 的主要潜在宿主,一些临床ARGs(acrB、mdtB、mdtC 和 multidrug transporter)也存在于高水分苜蓿青贮饲料中。细菌群落是高水分青贮苜蓿微生态系统中ARGs传播的主要驱动因素。 3. 乳酸菌接种剂对青贮苜蓿中抗生素抗性基因的生物消减作用研究 多药和杆菌肽抗性基因是苜蓿青贮饲料中主要的ARGs类型。萎蔫过程增加了新鲜苜蓿中ARGs的富集(P < 0.05)。30%DM含量下,自然青贮增加了青贮苜蓿中杆菌肽、β-内酰胺(beta_lactam)和氨基糖苷(aminoglycoside)抗性基因丰度(P < 0.05)。40% DM 含量下,自然青贮增加了万古霉素抗性基因丰度(P < 0.05)。有趣的是,接种植物乳杆菌 MTD/1(Lactiplantibacillus plantarum MTD/1)或布氏乳杆菌40788(Lactiplantibacillus buchneri 40788)降 低了苜蓿青贮饲料中ARGs 总丰度以及多药、大环内酯-林肯酰胺-链霉素 (macrolides-lincoamide-streptomycin,MLS)、万古霉素、氨基糖苷和四环素 (tetracycline)等抗性基因丰度(P < 0.05)。苜蓿青贮饲料中 ARGs 的主要宿主 是有害细菌或病原菌(葡萄球菌属、假单胞菌属、泛菌属(Pantoea)和欧文氏菌属(Erwinia)),并且在苜蓿青贮饲料中存在临床ARGs(ArlR、 EmrB-QacA、cpxR 和 penA)。此外,细菌群落、MGEs 和发酵品质的结合效 应是苜蓿青贮饲料中ARGs转移和传播的主要驱动力,其中细菌群落是关键因素。 4. 乳酸菌接种剂对不同干物质含量青贮全株玉米中抗生素抗性基因分布和传播机制研究大环内酯物(macrolide)和四环素是全株玉米鲜样和青贮料中主要的 ARGs 类型。自然青贮增加了大多数 ARGs 的丰度(P < 0.05)。全株玉米在青贮前后,40% DM含量下的部分 ARGs(多肽(peptide)、氨基糖苷类、糖肽 (glycopeptide)、吖啶(acridine)、林可胺类(lincosamide)等)的丰度均高于 30% DM 含量(P < 0.05),但对 ARGs 的总丰度没有影响。虽然接种布氏乳杆菌 40788 增加了 ARGs 的分布和传播潜力(P < 0.05),但是接种植物乳杆菌 MTD/1 对ARGs 分布无显著影响。在青贮全株玉米中优势菌(明串珠菌属(Leuconostoc)和广布乳杆菌属(Latilactobacillus)是 ARGs 的主要宿主。相 关性分析表明,DM 含量的变化引起的细菌群落的演替是驱动青贮全株玉米 ARGs 分布的最主要因素,乳酸菌接种剂以及 MGEs 驱动的水平基因转移不是主要因素。此外,青贮全株玉米中存在 tetW、mecA、vatE、mdtA 和 mdtM 等高风险 ARGs。有趣的是,接种布氏乳杆菌 40788 并没有增加青贮全株玉米中高风险ARGs 的丰度。 5. 乳酸菌接种剂对不同贮藏温度全株玉米青贮中抗生素抗性基因分布和传播机制研究两个贮藏温度(20℃ vs 30℃)下,自然青贮降低了全株玉米中ARGs的alpha 多样性(P < 0.05),但是增加了ARGs总丰度以及糖肽、林可胺类和恶唑烷酮(oxazolidinone)等抗性基因的丰度(P < 0.05)。相比 20℃贮藏温度, 30℃降低了青贮全株玉米中ARGs 的多样性和总丰度以及氟 喹诺酮 (fluoroquinolone)、吖啶和甘氨酰环素(glycylcycline)等抗性基因丰度(P < 0.05)。值得注意的是,两个贮藏温度下,接种布氏乳杆菌40788 增加了青贮全 株玉米中ARGs总丰度(P < 0.05),而接种植物乳杆菌 MTD/1 对其没有影响。 青贮全株玉米中 ARGs 的主要宿主是优势菌( 明串珠菌属和乳杆菌属 (Lactobacillus))。新鲜全株玉米中整合酶(integrase)和插入序列(insertion sequence)是主要的 MGEs 类型,而青贮全株玉米中质粒(plasmid)和转座酶是主要的 MGEs 类型。相关性分析显示贮藏温度的变化引起的细菌群落演替是 影响 ARGs 变化的主要驱动因素。同样的,在不同贮藏温度下,我们发现接种 布氏乳杆菌 40788 并没有增加青贮全株玉米中高风险 ARGs 的丰度。 本论文研究首次基于宏基因组学技术全面地阐明了苜蓿青贮和全株玉米青贮饲料中 ARGs 的分布特征和潜在风险。同时,探讨了乳酸菌接种剂、DM 含 量、甲酸抑菌剂和生长年限对青贮苜蓿,以及贮藏温度、乳酸菌接种剂和 DM 含量对青贮全株玉米中 ARGs 分布的影响与作用机制。研究结果揭示了全株玉米青贮和苜蓿青贮饲料中优势抗性基因和宿主菌,解析了理化特性指标、发酵品质、MGEs和细菌群落对 ARGs分布贡献以及它们之间相关性;同时,明确 了青贮饲料中由临床病原体携带的高风险ARGs。上述研究结果对青贮饲料和草食畜动物生产系统中 ARGs 的生物安全防控具有重要参考价值。

Other Abstract

Antibiotics resistance genes (ARGs) is found in various environmental media, which can lead to global public health and ecological and environmental security risks, and pose a huge threat to human health. Silage is a high-quality forage for herbivorous livestock, and it may be one of the potential sources of ARGs for animals. So far, studies on silage mainly focuses on the effects of different treatments (additives, temperature, dry matter (DM), and agronomic measure) on the changes of fermentation quality, nutrient composition, microbial interactions and succession, and the mutual transformation of metabolites, but little is known about the distribution and spread of ARGs. Therefore, in the present study, using metagenomics explained the response of different treatments (lactic acid bacteria, formic acid, DM, temperature, and growth years) to the distribution characteristics and transmission mechanism of ARGs in alfalfa and whole-plant corn silages. Meanwhile, the driving factors of ARGs transfer and spread in silage were also evaluated. The ARGs host and ARGs carried by clinical pathogens in silage were identified. The following study results are obtained: 1. Study on the distribution characteristics and driving mechanism of antibiotic resistance genes in soil-fresh alfalfa- alfalfa silage system at different growth years With increasing of alfalfa growth years, the ARGs diversity in the soil samples increased (P < 0.05), but did not affect the ARGs abundance. The diversity and abundance of ARGs in fresh alfalfa and alfalfa silage increased with the growing years of alfalfa (P < 0.05). The vancomycin and multidrug were the main resistance genes in soil and fresh alfalfa. The multidrug and bacitracin were the main resistance genes in alfalfa silage. The transposase was the most abundant mobile genetic elements (MGEs) type in each sample. Growth years did not alter the horizontal gene transfer potential of ARGs in soil and fresh alfalfa, but the horizontal gene transfer potential of ARGs in alfalfa silage decreased with increasing of growth years (P < 0.05). The dominant bacteria in the soil and fresh alfalfa samples were Nocardioides. The dominant bacteria in the alfalfa silages were Enterobacter, Enterococcus and Bifidobacterium. Tracker source analysis showed that the longer the alfalfa grew, the more ARGs accumulated on the surface of fresh alfalfa, however, the source of most ARGs in alfalfa silage was obtained during the silage process. The main driver of ARGs variation in the soil-fresh alfalfa-alfalfa silage system is the bacterial community, and horizontal gene transfer in alfalfa silage might enhance the spread of ARGs. 2. Effects of formic acid on the fate of antibiotic resistance genes and their potential hosts in high-moisture alfalfa silage Multidrug resistance genes were the most abundant ARGs type in high-moisture alfalfa silage. The natural silage process increased the ARGs enrichment (P < 0.05). However, after 5 days of ensiling, formic acid-treated alfalfa silage decreased the ARGs abundance. After 90 days of ensiling, formic acid-treated alfalfa silage increased the ARGs abundance (P < 0.05). Notably, some of pathogenic bacteria, such as Staphylococcus, Clostridium and Pseudomonas were annotated as potential hosts of ARGs in high-moisture alfalfa silage. Some clinical ARGs (acrB, mdtB, mdtC, and multidrug transporter) were also present in high-moisture alfalfa silage. Bacterial communities were the main drivers of ARGs distribution in the high-moisture alfalfa silage microecosystem. 3. Study on biological reduction of antibiotic resistance genes in alfalfa silage inoculated with lactic acid bacteria Multidrug and bacitracin resistance genes were the main ARGs type in alfalfa silage. The wilting process increased the ARGs enrichment in fresh alfalfa (P < 0.05). Naturally ensiling process increased the abundances of bacitracin, beta_lactam and aminoglycoside resistance genes in alfalfa silage at 30% DM content (P < 0.05). Natural ensiling process increased the abundance of vancomycin resistance gene at 40% DM content (P < 0.05). Interestingly, alfalfa silage inoculated with Lactiplantibacillus plantarum MTD/1 or Lactiplantibacillus buchneri 40788 decreased the total ARGs abundance as well as the abundances of multidrug, macrolides-lincoamide-streptomycin (MLS), vancomycin, aminoglycoside, and tetracycline resistance genes (P < 0.05). The main hosts of ARGs in alfalfa silage were harmful bacteria or pathogenic bacteria (Staphylococcus, Pseudomonas, Pantoea, and Erwinia). The clinical ARGs (ArlR, EmrB-QacA, cpxR, and penA) were found in alfalfa silage. In addition, the combined effects of bacterial communities, MGEs and fermentation quality were the main drivers of ARGs transfer and spread in alfalfa silage, in which bacterial communities were the key factor. 4. Study on the distribution and transmission mechanism of antibiotic resistance genes in whole-plant corn silage inoculated with lactic acid bacteria at different dry matter contents Macrolide and tetracycline were the main ARGs type in whole-plant corn silage and fresh whole-plant corn. Natural silage process increased the abundance of most ARGs in whole-plant corn (P < 0.05). Whole-plant corn before and after ensiling, the abundances of some ARGs types (peptide, aminoglycosides, glycopeptide, acridine, and lincosamide)at 40% DM content were higher than that at 30% DM content (P < 0.05), but it had no effect on the total ARGs abundance. Although L. buchneri 40788 inoculant increased the distribution and transmission potential of ARGs in wholeplant corn silage (P < 0.05), L. plantarum MTD/1 inoculant had no significant effect on ARGs distribution. The dominant bacteria (Leuconostoc and Latilactobacillus) were the main ARGs hosts in whole-plant corn silage. Correlation analysis showed that the change of bacterial community succession caused by the change of DM content was the most important factor driving the ARGs distribution in whole-plant corn silage, while lactic acid bacteria inoculant and horizontal gene transfer driven by MGEs were not the main factors. In addition, high-risk ARGs such as tetW, mecA, vatE, mdtA, and mdtM were found in whole-plant corn silage. It was interesting that L. buchneri 40788 inoculant did not increase the abundance of high-risk ARGs in wholeplant corn silage. 5. Study on the distribution and transmission mechanism of antibiotic resistance genes in whole-plant corn silage inoculated with lactic acid bacteria at different storage temperatures The natural silage process reduced the alpha diversity of ARGs in whole-plant corn silage at two storage temperatures (P < 0.05), but the abundances of total ARGs, glycopeptide, lincosamide, and oxazolidinone resistance genes were increased (P < 0.05). Compared with 20℃ storage temperature, 30℃ storage temperature decreased the ARGs diversity and total ARGs abundance as well as the abundances of fluoroquinolone, acridine and glycylcycline resistance genes in whole-plant corn silage (P < 0.05). Notably, L. buchneri 40788 inoculants increased the total ARGs abundance in whole-plant corn silage at both storage temperatures (P < 0.05), while L. plantarum MTD/1 inoculant had no effect on it. The main ARGs hosts in whole-plant corn silage were dominant bacteria (Leuconostoc and Lactobacillus). Integrase and insertion sequence were the main MGEs types in fresh whole-plant corn, while plasmid and transposase were the main MGEs types in whole-plant corn silage. Correlation analysis showed that bacterial community succession caused by the change of storage temperatures were the main driving factors affecting the variation in ARGs. Similarly, L. buchneri 40788 inoculant did not increase the high-risk ARGs abundances in whole-plant corn silage at two storage temperatures. It is the first time to elucidate the distribution characteristics and potential risks of ARGs in alfalfa silage and whole-plant corn silage using metagenomics. Meanwhile, the effects of lactic acid bacteria inoculant, DM content, formic acid and growth years on the distribution of ARGs in alfalfa silage, and effects of storage temperature, lactic acid bacteria inoculant and DM content on the distribution of ARGs in whole-plant corn silage were investigated. The results revealed the dominant resistance genes and host bacteria in whole-plant corn silage and alfalfa silage, and analyzed the contribution of physicochemical characteristics, fermentation quality, MGEs and bacterial communities to ARGs distribution and their correlation. At the same time, high-risk ARGs carried by clinical pathogens in silage were identified. The above results had important reference value for the biosafety control of ARGs in silage and herbivore animal production systems.

Subject Area饲草加工与利用
MOST Discipline Catalogue农学 - 畜牧学 - 动物营养与饲料科学
URL查看原文
Language中文
Other Code262010_120190900370
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/537695
Collection草地农业科技学院
Affiliation
兰州大学草地农业科技学院
Recommended Citation
GB/T 7714
Zhang xia. 青贮饲料中抗生素抗性基因的分布特征与传播机制[D]. 兰州. 兰州大学,2023.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Altmetrics Score
Google Scholar
Similar articles in Google Scholar
[Zhang xia]'s Articles
Baidu academic
Similar articles in Baidu academic
[Zhang xia]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[Zhang xia]'s Articles
Terms of Use
No data!
Social Bookmark/Share
No comment.
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.