Metagenomic Characterization of Antibiotic Resistance Genes in Full-Scale Reclaimed Water Distribution Systems and Corresponding Potable Systems

Water
reclamation provides a valuable resource for meeting nonpotable
water demands. However, little is known about the potential for wastewater
reuse to disseminate antibiotic resistance genes (ARGs). Here, samples
were collected seasonally in 2014–2015 from four U.S. utilities’
reclaimed and potable water distribution systems before treatment,
after treatment, and at five points of use (POU). Shotgun metagenomic
sequencing was used to profile the resistome (i.e., full contingent
of ARGs) of a subset (n = 38) of samples. Four ARGs
(qnrA, blaTEM, vanA, sul1) were quantified by quantitative
polymerase chain reaction. Bacterial community composition (via 16S
rRNA gene amplicon sequencing), horizontal gene transfer (via quantification
of intI1 integrase and plasmid genes), and selection
pressure (via detection of metals and antibiotics) were investigated
as potential factors governing the presence of ARGs. Certain ARGs
were elevated in all (sul1; p ≤
0.0011) or some (blaTEM, qnrA; p ≤ 0.0145) reclaimed POU samples compared
to corresponding potable samples. Bacterial community composition
was weakly correlated with ARGs (Adonis, R2 = 0.1424–0.1734)
and associations were noted between 193 ARGs and plasmid-associated
genes. This study establishes that reclaimed water could convey greater
abundances of certain ARGs than potable waters and provides observations
regarding factors that likely control ARG occurrence in reclaimed
water systems.

水再生回用（water reclamation）可为满足非饮用水需求提供宝贵资源。然而，目前对于废水回用传播抗生素抗性基因（antibiotic resistance genes, ARGs）的潜在可能性尚缺乏充分认知。本研究于2014至2015年按季节采集样本，采样对象为美国4家水务公司的再生水与饮用水输配系统，采样点涵盖处理前、处理后以及5个使用点（points of use, POU）。研究采用鸟枪法宏基因组测序（shotgun metagenomic sequencing）技术，对其中38份样本的抗性组（resistome，即全部抗生素抗性基因集合）进行解析。通过定量聚合酶链式反应（quantitative polymerase chain reaction）对qnrA、blaTEM、vanA、sul1这4种抗生素抗性基因进行定量检测。本研究还针对调控抗生素抗性基因存在的潜在影响因素展开探究，包括细菌群落组成（通过16S rRNA基因扩增子测序（16S rRNA gene amplicon sequencing））、水平基因转移（通过定量检测I型整合酶intI1与质粒基因）以及选择压力（通过检测金属与抗生素残留）。相较于对应的饮用水样本，部分抗生素抗性基因在所有再生水使用点样本（以sul1为例，p≤0.0011）或部分再生水使用点样本（以blaTEM、qnrA为例，p≤0.0145）中丰度显著升高。细菌群落组成与抗生素抗性基因仅存在弱相关性（Adonis分析，R²=0.1424~0.1734），同时研究发现193种抗生素抗性基因与质粒相关基因存在关联。本研究证实，相较于饮用水，再生水中部分抗生素抗性基因的丰度更高，并为揭示再生水系统中抗生素抗性基因的分布调控因素提供了研究依据。