摘要

       牲畜废物管理结构中的污泥隔间是细菌间抗菌素耐药性出现和扩散的潜在温床。关于污泥中细胞内和细胞外 DNA 库之间的抗菌素抗性基因 (ARG) 的分布知之甚少。本研究的总体目标是评估细胞外 ARGs 对牲畜废物管理结构污泥中总 ARGs 的重要性。在这项研究中，从四个牛粪储存池和三个猪粪处理泻湖收集了污泥样本，并分析了抗性的遗传指标。使用优化方案分别提取污泥中的细胞内 DNA (iDNA) 和细胞外 DNA (eDNA)。 iDNA 和 eDNA 提取物中的 ARG [sul(I)、sul(II)、tet(O)、tet(Q) 和 tet(X)] 使用定量聚合酶链反应 (qPCR) 和抗菌剂进行量化，包括磺胺类药物和四环素类药物使用液相色谱串联质谱法测定。结果表明，eDNA 占污泥总 DNA 的比例不到 1.5%。在几乎所有 eDNA 和 iDNA 样本中都检测到所有测试的 ARG。此外，每克干污泥含有 1.7 × 103 至 4.2 × 108 份细胞外 ARG 和 3.2 × 107 至 3.2 × 1010 份细胞内 ARG。金霉素浓度范围在 187 和 2674 微克/克污泥湿重 (ww) 之间，而磺胺浓度低于 6.3 微克/克污泥湿重 (ww)。 eDNA 提取物中 ARG 的检测表明，转化是牲畜废物管理结构污泥中 ARG 增殖的潜在机制。

       The sludge compartment in livestock waste management structures is a potential hotbed for the emergence and proliferation of antimicrobial resistance among bacteria. Little is known about the distribution of antimicrobial resistance genes (ARGs) between the intracellular and extracellular DNA pools in the sludge. The overall objective of this study was to assess the significance of extracellular ARGs to the total ARGs in the sludge of livestock waste management structures. In this study, sludge samples were collected from four cattle manure storage ponds and three swine waste treatment lagoons and analyzed for genetic indicators of resistance. Intracellular DNA (iDNA) and extracellular DNA (eDNA) in the sludge were separately extracted using an optimized protocol. ARGs [sul(I), sul(II), tet(O), tet(Q), and tet(X)] in both the iDNA and eDNA extracts were quantified using quantitative polymerase chain reaction (qPCR), and antimicrobials, including sulfonamides and tetracyclines, were measured using liquid chromatography tandem mass spectrometry. Results showed that eDNA constituted less than 1.5% of the total DNA in sludge. All ARGs tested were detected in nearly all eDNA and iDNA samples. Furthermore, every gram of dry sludge contained from 1.7 × 103 to 4.2 × 108 copies of extracellular ARG and from 3.2 × 107 to 3.2 × 1010 copies of intracellular ARG. Chlortetracycline concentrations ranged between 187 and 2674 μg/g of sludge wet weight (ww), while sulfonamide concentrations were lower than 6.3 μg/g of sludge ww. The detection of ARGs in eDNA extracts suggests that transformation is a potential mechanism in ARG proliferation in the sludge of livestock waste management structures.

https://pubs.acs.org/doi/abs/10.1021/es401964s