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Abstract：
BACKGROUND: Anthropogenic activities significantly contribute to the dissemination of antibiotic resistance genes (ARGs), posing a substantial threat to humankind. The development of methods that allow robust ARG surveillance is a long-standing challenge. Here, we use city-scale monitoring of ARGs by using two of the most promising cutting-edge technologies, digital PCR (dPCR) and metagenomics.
METHODS: ARG hot-spots were sampled from the urban water and wastewater distribution systems. Metagenomics was used to provide a broad view of ARG relative abundance and richness in the prokaryotic and viral fractions. From the city-core ARGs in all samples, the worldwide dispersed sul2 and tetW conferring resistance to sulfonamide and tetracycline, respectively, were monitored by dPCR and metagenomics.
RESULTS: The largest relative overall ARG abundance and richness were detected in the hospital wastewater and the WWTP inlet (up to ≈6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. The abundance of ARGs in DNA and RNA contigs classified as viruses was notably lower, demonstrating a reduction of up to three orders of magnitude compared to contigs associated to prokaryotes. By metagenomics and dPCR, a similar abundance tendency of sul2 and tetW was obtained, with higher abundances in hospital wastewater and WWTP input (≈125-225 ARGs/Gb metagenome). dPCR absolute abundances were between 6,000 and 18,600 copies per ng of sewage DNA (≈105-7 copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point.
CONCLUSIONS: dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative abundance units was not obtained here (r2 < 0.4) suggesting methodological factors that introduce variability. Evolutionary pressure does not significantly select the targeted ARGs in natural aquatic environments.
METHODS: ARG hot-spots were sampled from the urban water and wastewater distribution systems. Metagenomics was used to provide a broad view of ARG relative abundance and richness in the prokaryotic and viral fractions. From the city-core ARGs in all samples, the worldwide dispersed sul2 and tetW conferring resistance to sulfonamide and tetracycline, respectively, were monitored by dPCR and metagenomics.
RESULTS: The largest relative overall ARG abundance and richness were detected in the hospital wastewater and the WWTP inlet (up to ≈6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. The abundance of ARGs in DNA and RNA contigs classified as viruses was notably lower, demonstrating a reduction of up to three orders of magnitude compared to contigs associated to prokaryotes. By metagenomics and dPCR, a similar abundance tendency of sul2 and tetW was obtained, with higher abundances in hospital wastewater and WWTP input (≈125-225 ARGs/Gb metagenome). dPCR absolute abundances were between 6,000 and 18,600 copies per ng of sewage DNA (≈105-7 copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point.
CONCLUSIONS: dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative abundance units was not obtained here (r2 < 0.4) suggesting methodological factors that introduce variability. Evolutionary pressure does not significantly select the targeted ARGs in natural aquatic environments.
摘要：
背景：人为活动极大地促进了抗生素抗性基因（ARGs）的传播，对人类构成重大威胁。开发允许稳健的ARG监测的方法是一个长期的挑战。这里,我们通过使用两种最有前途的尖端技术来使用城市规模的ARG监测，数字PCR(dPCR)和宏基因组学。
方法：从城市水和废水分配系统中采样ARG热点。宏基因组学用于提供原核和病毒部分中ARG相对丰度和丰富度的广泛视图。从所有样本的城市核心ARG中，全球分散的sul2和tetW赋予对磺胺类和四环素的抗性，分别,通过dPCR和宏基因组学进行监测。
结果：在医院废水和WWTP入口（高达约6,000ARG/Gb宏基因组）中检测到最大的相对总体ARG丰度和丰富度，其中大部分未分类的耐药细菌。归类为病毒的DNA和RNA重叠群中ARG的丰度明显较低，与与原核生物相关的重叠群相比，显示出高达三个数量级的减少。通过宏基因组学和dPCR，获得了相似的sul2和tetW丰度趋势，医院废水和污水处理厂输入的丰度较高(≈125-225ARGs/Gb宏基因组)。dPCR绝对丰度在污水处理厂排放点附近的海水中，每ng污水DNA为6,000至18,600个拷贝（约105-7个拷贝/mL）和6.8个拷贝/mL。
结论：dPCR更灵敏、准确，而宏基因组学提供了更广泛的ARG检测覆盖范围。虽然可取，本文未获得dPCR绝对丰度单位与宏基因组相对丰度单位的可靠相关性(r2<0.4),提示引入变异性的方法学因素.在自然水生环境中，进化压力不会显着选择目标ARG。
方法：从城市水和废水分配系统中采样ARG热点。宏基因组学用于提供原核和病毒部分中ARG相对丰度和丰富度的广泛视图。从所有样本的城市核心ARG中，全球分散的sul2和tetW赋予对磺胺类和四环素的抗性，分别,通过dPCR和宏基因组学进行监测。
结果：在医院废水和WWTP入口（高达约6,000ARG/Gb宏基因组）中检测到最大的相对总体ARG丰度和丰富度，其中大部分未分类的耐药细菌。归类为病毒的DNA和RNA重叠群中ARG的丰度明显较低，与与原核生物相关的重叠群相比，显示出高达三个数量级的减少。通过宏基因组学和dPCR，获得了相似的sul2和tetW丰度趋势，医院废水和污水处理厂输入的丰度较高(≈125-225ARGs/Gb宏基因组)。dPCR绝对丰度在污水处理厂排放点附近的海水中，每ng污水DNA为6,000至18,600个拷贝（约105-7个拷贝/mL）和6.8个拷贝/mL。
结论：dPCR更灵敏、准确，而宏基因组学提供了更广泛的ARG检测覆盖范围。虽然可取，本文未获得dPCR绝对丰度单位与宏基因组相对丰度单位的可靠相关性(r2<0.4),提示引入变异性的方法学因素.在自然水生环境中，进化压力不会显着选择目标ARG。
