Department of Civil Engineering, University of Nebraska-Lincoln , 844 North 16th Street, Lincoln, Nebraska 68588-6105, United States.
Environ Sci Technol. 2013 Sep 17;47(18):10206-13. doi: 10.1021/es401964s. Epub 2013 Sep 4.
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 × 10(3) to 4.2 × 10(8) copies of extracellular ARG and from 3.2 × 10(7) to 3.2 × 10(10) 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.
在牲畜废物管理结构中，污泥隔室是细菌产生和抗药性扩散的潜在温床。关于污泥中细胞内和细胞外 DNA 池之间的抗药性基因（ARGs）分布，人们知之甚少。本研究的总体目标是评估细胞外 ARGs 对牲畜废物管理结构中污泥总 ARGs 的重要性。在这项研究中，从四个牛粪便储存池和三个猪废物处理池收集了污泥样本，并分析了抗性的遗传指标。使用优化的方案分别从污泥中提取细胞内 DNA（iDNA）和细胞外 DNA（eDNA）。使用定量聚合酶链反应（qPCR）定量测定 iDNA 和 eDNA 提取物中的 ARGs [sul（I），sul（II），tet（O），tet（Q）和 tet（X）]，并使用液相色谱串联质谱法测量抗生素，包括磺胺类和四环素。结果表明，eDNA 在污泥中的总 DNA 中不到 1.5％。几乎所有的 eDNA 和 iDNA 样品中都检测到所有测试的 ARGs。此外，每克干污泥中含有 1.7×10(3)至 4.2×10(8)个拷贝的细胞外 ARG 和 3.2×10(7)至 3.2×10(10)个拷贝的细胞内 ARG。金霉素浓度在污泥湿重（ww）的 187 至 2674μg/g 之间，而磺胺类浓度低于污泥 ww 的 6.3μg/g。在 eDNA 提取物中检测到 ARGs 表明转化是 ARG 在牲畜废物管理结构的污泥中增殖的潜在机制。
