通过宏基因组学研究影响抗生素耐药基因在肉鸡肠道菌群中持久存在的关键因素

Antibiotic resistance genes in gut microbiota of livestock and poultry seriously harm human health. However, the factors influencing the persistence of resistance genes in gut microbiota of livestock and poultry still remain uncertain. In our previous study, we observed that resistance genes had high background levels and persisted in gut microbiota of chickens; chlortetracycline disturbed normal structure of gut microbiota to change bacterial resistome. In this study, we will deeply analyze the spectrum of resistance genes and their bacterial hosts in gut microbiota of broiler chickens by metagenomics, and will analyze the key factors influencing the persistence of these resistance genes in three aspects of antimicrobials, mobile genetic elements and bacterial community structure, by (1) analyzing the cross-selection of resistance genes imposed by antimicrobials after broiler chickens are cross-fed by amoxicillin, chlortetracycline and florfenicol; (2) analyzing sequence arrangement of plasmids, integrons and phages, and the relevance to the persistence of resistance genes combination with quantitative PCR technology; (3) analyzing the co-occurrence network relationships between bacterial community structure, resistance genes and their bacterial hosts by high-throughput sequencing of amplicon. This study will deeply recognize the unknown resistance genes to firstly establish antibiotic resistance gene pool in gut microbiota of chickens, and will specify the key factors influencing the persistence of these resistance genes in gut microbiota of broiler chickens through metagenomics in order to provide a theoretical basis for prevention and control of drug resistance from animal farming in the next step.

畜禽肠道菌群抗生素耐药基因严重危害人类健康。然而影响耐药基因在畜禽肠道菌群中持久存在的因素并未明确。我们前期试验发现雏鸡肠道菌群耐药基因背景值高且持久存在；金霉素干扰肠道正常菌群结构，改变菌群染色体耐药性。本研究通过宏基因组学深入分析肉鸡肠道菌群耐药基因谱及其宿主菌谱，从抗菌药物、可移动遗传元件和菌群结构三个方面研究影响这些耐药基因持久存在的关键因素：(1) 给肉鸡交叉饲喂阿莫西林、金霉素和氟苯尼考，分析抗菌药物对耐药基因的交叉选择作用；(2) 结合定量PCR技术，研究质粒、整合子和噬菌体的序列排布特征及其与耐药基因持久存在的关联性；(3) 通过扩增子高通量测序，分析肠道菌群结构、耐药基因及其宿主菌之间的共存网络关系。本研究通过宏基因组学深度识别肉鸡肠道菌群中未知的耐药基因，首次建立肉鸡肠道菌群耐药基因池，阐明影响这些耐药基因持久存在的关键因素，为下一步防控养殖源耐药性提供理论依据。

抗生素耐药性已成为当前全球公共卫生领域的重大议题。作为全球最大的肉鸡消费国和生产国，我国在肉鸡生产中普遍使用抗菌药。阿莫西林、金霉素和氟苯尼考由于抗菌效果好且副作用较少，被广泛应用于肉鸡养殖业。探究这些药物对肉鸡肠道菌群耐药基因组持久存在的影响，有助于指导生产实践中合理使用抗菌药、减少细菌耐药性的产生和传播。商品肉鸡随机分成5组，包括空白对照组、阿莫西林组、金霉素组、氟苯尼考组和混合给药组。在给药处理前后不同时间点采集鸡粪，通过分离培养和宏基因组学技术，分析不同处理组中菌群耐药基因组和菌群结构的变化规律。结果发现，金霉素促进了四环素耐药基因tetA和tetW的丰度显著升高；质粒介导的黏菌素耐药基因mcr-1在氟苯尼考组和多药组显著升高；acrAB-tolC及其相关的调控基因MarA、SoxS和SdiA等在阿莫西林的作用下显著升高。在饲喂各抗菌药15天后，四环素耐药基因tetA是氟苯尼考、金霉素、混合给药处理组中丰度最高的耐药基因，而磺胺类耐药基因sul2是阿莫西林处理组中丰度最高的耐药基因。携带耐药基因最多的5个菌属分别为Escherichia（83.7%）、Klebsiella（5.1%）、Bacteroides（3.9%）、Shigella（2.9%）、Clostridium（1.7%）。Escherichia 是大部分耐药基因的宿主。在给药10天后携带耐药基因的宿主发生变化，Escherichia和Bacteroides是变化最显著的宿主菌。Escherichia携带的β-内酰胺类耐药基因、四环素类耐药基因和氯霉素类耐药基因在给药10天后升高。结果表明抗菌药物的使用改变了鸡肠道菌群结构，进而促进特定耐药基因持久性存在。本研究为生产实践中合理使用抗菌药、减少细菌耐药性的产生提供理论依据。