摘要

       微生物生态系统中自由漂浮的细胞外 DNA (exDNA) 部分含有抗生素抗性基因 (ARG) 和移动遗传元件 (MGE)。这些异种元素的自然转化可以产生对一种或多种抗生素具有抗性的微生物细胞。由于 exDNA 在废水环境中的浓度较低，因此分离并获得高产量的 exDNA 具有挑战性。分析 exDNA 对于解开自由漂浮的 ARG 和 MGE 的生态学及其对水平基因转移的贡献至关重要。我们开发了一种使用色谱分离和富集 exDNA 的方法，而不会从复杂的废水基质中引起细胞裂解，例如进水（1 L 中的 9 µg exDNA）、活性污泥（1 L 中的 5.6 µg）和处理过的流出物（4.3 µg 中的1 升）。 ARGs 和 MGEs 对活性污泥的 exDNA 和细胞内 DNA (iDNA) 进行宏基因组分析，并通过 qPCR 在流出水中进行量化。 qPCR 显示 ARGs 和 MGEs 在 iDNA 部分中更丰富，同时对流出水中的 exDNA（100-1000 个基因拷贝 mL-1）仍然很重要。宏基因组强调 exDNA 主要由 MGEs (65%) 组成。根据它们在 exDNA 的抗性组中相对较低的丰度，通过自然转化吸收 ARG 可能不是主要的转移机制。尽管 ARGs 在 exDNA 中的含量不是很高，但 exDNA 部分中 MGEs 的流行可以间接促进抗生素耐药性的发展。这种方法与功能宏基因组学的结合有助于阐明微生物群落中耐药性的转移和发展。对不同 DNA 部分的系统分析将促进跨水系统的微生物风险评估，支持水务部门制定保护环境和公共健康的措施。

       The free-floating extracellular DNA (exDNA) fraction of microbial ecosystems harbors antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Natural transformation of these xenogenetic elements can generate microbial cells resistant to one or more antibiotics. Isolating and obtaining a high yield of exDNA is challenging due to its low concentration in wastewater environments. Profiling exDNA is crucial to unravel the ecology of free-floating ARGs and MGEs and their contribution to horizontal genetransfer. We developed a method using chromatography to isolate and enrich exDNA without causing cell lysis from complex wastewater matrices like influent (9 µg exDNA out of 1 L), activated sludge (5.6 µg out of 1 L), and treated effluent (4.3 µg out of 1 L). ARGs and MGEs were metagenomically profiled for both the exDNA and intracellular DNA (iDNA) of activated sludge, and quantified by qPCR in effluent water. qPCR revealed that ARGs and MGEs are more abundant in the iDNA fraction while still significant on exDNA (100–1000 gene copies mL−1) in effluent water. The metagenome highlighted that exDNA is mainly composed of MGEs (65%). According to their relatively low abundance in the resistome of exDNA, ARGs uptake by natural transformation is likely not the main transfer mechanism. Although ARGs are not highly abundant in exDNA, the prevalence of MGEs in the exDNA fraction can indirectly promote antibiotic resistance development. The combination of this method with functional metagenomics can help to elucidate the transfer and development of resistances in microbial communities. A systematic profiling of the different DNA fractions will foster microbial risk assessments across water systems, supporting water authorities to delineate measures to safeguard environmental and public health.

https://www.sciencedirect.com/science/article/pii/S0043135420311271