基于宏基因组测序揭示马肠道微生物组成及其耐药性

Revealing the composition and antibiotic resistance of equine gut microbiota based on metagenomic sequencing

  • 摘要: 【目的】 基于宏基因组测序揭示阿哈尔捷金马与云南矮马肠道微生物组成及其耐药性,为明确马肠道微生态的维持机制与开发针对性的微生态调控策略提供理论依据。【方法】 分别采集新鲜阿哈尔捷金马与云南矮马新鲜粪便各10份,提取微生物总DNA并进行宏基因组测序。通过序列组装与分箱获得宏基因组组装基因组(MAGs),并进行物种注释及系统发育树构建。基于CARD数据库鉴定抗生素耐药基因(ARGs)的类型、相对丰度及分布规律。利用VIBRANT完成对噬菌体辅助代谢基因(AMGs)功能分析,并结合CRISPR-spacer匹配技术预测噬菌体—宿主互作关系。【结果】 阿哈尔捷金马与云南矮马肠道优势菌门均为厚壁菌门和拟杆菌门,另有螺旋体门、纤维杆菌门和疣微菌门等,属分类水平上物种组成存在品种差异。门分类水平上的LEfSe分析结果显示,纤维杆菌门在云南矮马肠道菌群中显著富集(P<0.05,下同),产甲烷古菌门在阿哈尔捷金马肠道菌群中显著富集。阿哈尔捷金马与云南矮马部分MAGs的相对丰度存在明显差异,肠道菌群整体结构呈现明显分离。阿哈尔捷金马和云南矮马的ARGs谱明显分离,耐药基因lnuCtet_44ACl-1CfxACfxA6在云南矮马中相对丰度较高,OXA-347tetOpoxtAtet32在阿哈尔捷金马中相对丰度较高。头霉素类抗生素和四环素类抗生素抗性基因在两品种马肠道中普遍存在。【结论】 阿哈尔捷金马与云南矮马肠道微生物组成存在明显的品种特异性,表现为核心菌群厚壁菌门和拟杆菌门的保守性与品种特异性微生物类群、噬菌体及耐药基因的并存,宿主遗传背景与共同饲养环境共同塑造了阿哈尔捷金马与云南矮马肠道微生态的结构与功能。

     

    Abstract: 【Objective】 Metagenomic sequencing was adopted to reveal the gut microbiota composition and antibiotic resistance of Akhal-Teke horses and Yunnan dwarf horses, which could provide theoretical basis for clarifying the maintenance mechanism of equine gut microbiota and developing targeted microecology regulation strategies.【Method】 Ten fresh fecal samples each from Akhal-Teke horses and Yunnan dwarf horses were collected. Total microbial DNA was extracted and metagenomic sequencing was performed. Metagenomic assembled genomes (MAGs) were obtained through sequence assembly and binning, followed by species annotation and phylogenetic construction. The types, relative abundance, and distribution patterns of antibiotic resistance genes (ARGs) were identified using the CARD database. Functional analysis of phage accessory metabolic genes (AMGs) was performed using VIBRANT, and phage-host interaction relationships were predicted using CRISPR-spacer matching technology.【Result】 The dominant gut floral phyla of both Akhal-Teke horses and Yunnan dwarf horses belonged to Firmicutes and Bacteroidetes, with additional phyla such as Spirochetes, Fibrobacteria, and Verruciformis. At the genus level, there were differences in species composition between different breeds. LEfSe analysis at the phylum level showed that Fibrobacteria was significantly enriched in the gut flora of Yunnan dwarf horses (P<0.05, the same below), and Methanogenic Archaea was also significantly enriched in the gut flora of Akhal-Teke horses. Obvious relative abundance differences were observed in some MAGs between Akhal-Teke horses and Yunnan dwarf horses, indicating a clear separation in the overall gut flora structure. The ARGs profiles of Akhal-Teke horses and Yunnan dwarf horses were clearly separated. Antibiotic resistance genes lnuCtet_44ACl-1CfxA, and CfxA6 were relatively abundant in Yunnan dwarf horses, while OXA-347tetOpoxtA, and tet32 were relatively abundant in Akhal-Teke horses. The genes contained resistance to cephalosporins and tetracyclines were commonly found in the gut of both breeds of horses.【Conclusion】 The gut microbiota composition of Akhal-Teke horses and Yunnan dwarf horses exhibits obvious breed specificity, characterized by the conservation of the core flora of Firmicutes and Bacteroidetes, the coexistence of breed-specific microbial groups, phages, and antibiotic resistance genes. The host genetic background and shared rearing environment jointly shape the structure and function of the gut microecology of Akhal-Teke horses and Yunnan dwarf horses.

     

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