摘要
      随着环境中抗生素耐药性基因的丰度和多样性的增加，对公众和生态系统健康的威胁也随之增加。细胞外抗生素耐药性基因（eARGs）是一种无细胞DNA，可通过感受态细菌细胞的转化促进抗生素耐药性的发展。尽管有这种作用，eARG在不同的环境水域中并没有得到很好的表征。它们体积小，在一些水生环境中浓度低，因此很难提取。本研究的目的是修改eARG提取方法，以确定同一水样中eARGs和细胞内ARG（iARGs）的丰度。改进的方法包括顺序过滤以将iARGs从eARGs中分离出来，用氢氧化铝涂布的硅胶吸附洗脱和沉淀，提取eARGs和iARGs，回收率在79.5%-99.0%之间。然后利用新方法提取四种ARG的细胞外和细胞内部分，一种可移动的遗传元件，以及自来水、河流地表水、湖泊地表水、雨水和废水中的16S rRNA。这是首次报道了雨水和湖面水中16S rRNA、intI1、blaTEM、ermF、sul1和tetC基因的细胞外和细胞内部分。此外，这种改进的方法首次能够定量环境水中红霉素抗性基因ermF的细胞外浓度；该基因的丰度在1.26×105至8.82×106个基因拷贝/L之间。此外，首次在自来水（7.00×104个基因拷贝/L）中定量了可移动遗传元件intI1的细胞外丰度。该方法在不同环境基质中的验证和应用应允许进行进一步的研究，以更好地了解eARGs在抗生素耐药性传播中的作用。
Abstract
As the abundance and diversity of antibiotic resistance genes increases in the environment, there is a concurrent increase in the threat to public and ecosystem health. Extracellular antibiotic resistance genes (eARGs) are cell-free DNA that can promote the development of antibiotic resistance via transformation by competent bacterial cells. Despite this role, eARGs have not been well characterized in different environmental waters. Their small size and low concentrations in some aquatic environments render them difficult to extract. The aim of this research was to modify an eARG extraction method to determine the abundance of both eARGs and intracellular ARGs (iARGs) in the same water sample. The modified method, consisting of sequential filtration to separate iARGs from eARGs, adsorption-elution with aluminum hydroxide–coated silica gel, and precipitation, extracted eARGs and iARGs with a recovery rate between 79.5% and 99.0%. The novel method was then utilized for the extraction of the extracellular and intracellular fractions of four ARGs, one mobile genetic element, and the 16S rRNA in tap water, river surface water, lake surface water, stormwater, and wastewater effluent. This is the first instance in which the extracellular and intracellular fractions of the 16S rRNA, intI1, blaTEM, ermF, sul1, and tetC genes in stormwater and lake surface water are reported. In addition, this modified method enabled the quantification of the extracellular concentration of the erythromycin resistance gene ermF in environmental waters for the first time; the gene’s abundance ranged from 1.26×105 to 8.82×106 gene copies/L across the aquatic waters sampled. The extracellular abundance of the mobile genetic element intI1, moreover, was quantified in tap water (7.00×104 gene copies/L) for the first time. The validation and application of this method to diverse environmental matrices should allow for further research to be conducted to better understand the role of eARGs in the spread of antibiotic resistance.

https://ascelibrary.org/doi/abs/10.1061/%28ASCE%29EE.1943-7870.0001993