摘要
      抗生素耐药性的增长激发了人们对了解抗生素耐药性基因（ARG）动员机制的兴趣。其中，分析环境、食品和人类样本的病毒部分中ARG的存在，并报告噬菌体是ARG传播的媒介的研究，一直是越来越多研究的重点。然而，有人认为，在这些研究中，由于未包装的细菌DNA或其他元素（如外膜囊泡（OMV））的实验污染，携带ARGs的噬菌体的丰度被高估了。这项研究旨在阐明噬菌体、OMV或污染的非包装DNA在多大程度上作为ARGs在病毒中的载体。选择三种食物（鸡、鱼和贻贝）的病毒级分作为携带ARG的噬菌体颗粒的来源，分离后证实了其在大肠杆菌宿主中感染和繁殖的能力。通过CsCl密度梯度离心进一步纯化含有ARG的级分，并在去除衣壳外的DNA后，确认颗粒内的ARG。用SYBR Gold对纯化的部分进行染色，这使得附着在大肠杆菌细胞上并感染大肠杆菌细胞的噬菌体衣壳可见。通过电子显微镜观察具有Myoviridae和Siphoviridae形态的噬菌体。纯化部分中的蛋白质主要属于噬菌体（鱼类中为71.8%，贻贝中为52.9%，鸡样品1中为78.7%，鸡样品2中为64.1%），主要对应于尾部、衣壳和其他结构蛋白，而如果存在OMV，预计膜蛋白会很丰富，仅占蛋白质含量的3.8-21.4%。噬菌体颗粒在病毒体中的优势支持了本研究中使用的方案的可靠性，以及最近关于携带ARG的噬菌体颗粒丰度的发现。 
Abstract
The growth of antibiotic resistance has stimulated interest in understanding the mechanisms by which antibiotic resistance genes (ARG) are mobilized. Among them, studies analyzing the presence of ARGs in the viral fraction of environmental, food and human samples, and reporting bacteriophages as vehicles of ARG transmission, have been the focus of increasing research. However, it has been argued that in these studies the abundance of phages carrying ARGs has been overestimated due to experimental contamination with non-packaged bacterial DNA or other elements such as outer membrane vesicles (OMVs). This study aims to shed light on the extent to which phages, OMVs or contaminating non-packaged DNA contribute as carriers of ARGs in the viromes. The viral fractions of three types of food (chicken, fish, and mussels) were selected as sources of ARG-carrying phage particles, whose ability to infect and propagate in an Escherichia coli host was confirmed after isolation. The ARG-containing fraction was further purified by CsCl density gradient centrifugation and, after removal of DNA outside the capsids, ARGs inside the particles were confirmed. The purified fraction was stained with SYBR Gold, which allowed the visualization of phage capsids attached to and infecting E. coli cells. Phages with Myoviridae and Siphoviridae morphology were observed by electron microscopy. The proteins in the purified fraction belonged predominantly to phages (71.8% in fish, 52.9% in mussels, 78.7% in chicken sample 1, and 64.1% in chicken sample 2), mainly corresponding to tail, capsid, and other structural proteins, whereas membrane proteins, expected to be abundant if OMVs were present, accounted for only 3.8–21.4% of the protein content. The predominance of phage particles in the viromes supports the reliability of the protocols used in this study and in recent findings on the abundance of ARG-carrying phage particles.

https://www.nature.com/articles/s41396-022-01338-0