摘要

       由于当前提取方法的局限性，尽管对抗生素抗性基因 (ARG) 的传播有独特的贡献，但细胞外 DNA (eDNA) 很少与细胞内 DNA (iDNA) 区分开来。此外，eDNA 可能是游离的 (f-eDNA) 或吸附在或悬浮的固体上，包括细胞 (a-eDNA)，这会影响 ARG 的持久性和透射率。我们开发了一种使用磁珠分离 iDNA、a-eDNA 和 f-eDNA 的新方法，以评估 ARG 的这些物理状态如何在废水处理厂中发生变化。这种方法能有效提取 eDNA (>85.3%)，回收率高于目前的方法，如酒精沉淀、基于 CTAB 的提取和 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。通过废弃的生物固体排放的 ARG 多于流出液； iARG 和 a-eARG 在废弃的生物固体中普遍存在（分别占总 ARG 的 (73.9±22.5) % 和 (23.4±15.3) %），而 f-eARG 在流出物中普遍存在（(90.3±16.5) %）。细菌群落分析显示特定属与 ARG 之间存在显着相关性（例如，气单胞菌、假单胞菌和不动杆菌与多药耐药基因 blaTEM 密切相关）。该处理系统减少了 iARG 向接收环境的排放，但是，流出物中存在的 eARG 浓度增加，这可能会导致环境抵抗。

       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 blaTEM). 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