摘要

      由于目前提取方法的局限性，细胞外DNA（eDNA）与细胞内DNA（iDNA）虽然对抗生素耐药基因（ARGs）的繁殖有着独特的贡献，但却很少被区分出来。此外，eDNA可以是游离的（f-eDNA），也可以吸附在悬浮固体上，包括细胞（a-eDNA），这会影响ARG的持久性和透射率。我们开发了一种利用磁珠分离iDNA、a-eDNA和f-eDNA的新方法，以评估废水处理厂中ARGs的物理状态如何变化。该方法有效地提取了eDNA（>85.3%），回收率高于现有的醇沉法、CTAB提取法和DNA提取试剂盒（<10%）。生物处理和紫外线消毒降低了细胞内ARGs（iARGs）和吸附的细胞外ARGs（a-eARGs）的浓度，导致释放的游离细胞外ARGs（f-eARGs）增加。通过废弃生物固体排放的arg多于废水；iARGs和a-eARGs主要分布在废弃生物固体中（分别占总arg的73.9%、23.4%、15.3%），f-eARGs主要分布在废水中（90.3%、16.5%）。细菌群落分析表明，特定属与ARGs之间存在显著的相关性（如气单胞菌、假单胞菌和不动杆菌与多药耐药基因blaTEM呈强相关）。该处理系统减少了iARGs向接收环境的排放，但污水中eARG浓度增加，可能有助于环境抵抗。

      Due to the limitations of current extraction methods, extracellular DNA (eDNA) is rarely discerned from intracellular DNA (iDNA) despite having unique contributions to antibiotic resistance genes (ARGs) propagation. Furthermore, eDNA may be free (f-eDNA) or adsorbed to or suspended solids, including cells (a-eDNA), which affects ARG persistence and transmissivity. We developed a novel method using magnetic beads to separate iDNA, a-eDNA, and f-eDNA to assess how these physical states of ARGs change across a wastewater treatment plant. This method efficiently extracted eDNA (>85.3%) with higher recovery than current methods such as alcohol precipitation, CTAB-based extraction, and DNA extraction kits (<10%). Biological treatment and UV disinfection decreased the concentration of intracellular ARGs (iARGs) and adsorbed extracellular ARGs (a-eARGs), causing an increase of released free extracellular ARGs (f-eARGs). More ARGs were discharged through the wasted biosolids than in the effluent; iARGs and a-eARGs are prevalent in wasted biosolids ((73.9 ± 22.5) % and (23.4 ± 15.3) % of total ARGs respectively), while f-eARGs were prevalent in the effluent ((90.3 ± 16.5) %). Bacterial community analysis showed significant correlations between specific genera and ARGs (e.g., Aeromonas, Pseudomonas and Acinetobacter were strongly correlated with multidrug-resistance gene bla_TEM). This treatment system decreased the discharge of iARGs to receiving environments, however, increased eARG concentrations were present in the effluent, which may contribute to the environmental resistome.

       https://www.sciencedirect.com/science/article/pii/S0160412019311614?via%3Dihub