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With the growing application of high-throughput sequencing-based metagenomics for profiling antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs), comparison of sample pretreatment and DNA extraction methods are needed to move toward standardized comparisons among laboratories. Three widely employed DNA extraction methods (FastDNA® Spin Kit for Soil, PowerSoil® DNA Isolation Kit and ZR Fecal DNA MiniPrep), with and without preservation in 50% ethanol and freezing, were applied to the influent, activated sludge and effluent of two WWTPs, in Hong Kong and in the USA. Annotated sequences obtained from the DNA extracted using the three kits shared similar taxonomy and ARG profiles. Overall, it was found that the DNA yield and purity, and diversity of ARGs captured were all highest when applying the FastDNA SPIN Kit for Soil for all three WWTP sample types investigated here (influent, activated sludge, effluent). Quantitative polymerase chain reaction of 16S rRNA genes confirmed the same trend as DNA extraction yields and similar recovery of a representative Gram-negative bacterium (Escherichia coli). Moreover, sample fixation in ethanol, deep-freezing and overseas shipment had no discernable effect on ARG profiles, as compared to fresh samples. This approach serves to inform future efforts toward global comparisons of ARG distributions in WWTPs.

Next-generation DNA sequencing and metagenomic analysis provide powerful tools for the environmentally friendly design of nanoparticles. Herein we demonstrate this approach using a model community of environmental microbes (that is, wastewater-activated sludge) dosed with gold nanoparticles of varying surface coatings and morphologies. Metagenomic analysis was highly sensitive in detecting the microbial community response to gold nanospheres and nanorods with either cetyltrimethylammonium bromide or polyacrylic acid surface coatings. We observed that the gold-nanoparticle morphology imposes a stronger force in shaping the microbial community structure than does the surface coating. Trends were consistent in terms of the compositions of both taxonomic and functional genes, which include antibiotic resistance genes, metal resistance genes and gene-transfer elements associated with cell stress that are relevant to public health. Given that nanoparticle morphology remained constant, the potential influence of gold dissolution was minimal. Surface coating governed the nanoparticle partitioning between the bioparticulate and aqueous phases.

This study investigated the response of antibiotic resistance genes (ARGs) to nanosilver (Ag) in lab-scale nitrifying sequencing batch reactors (SBRs), compared to Ag(+)-dosed and undosed controls. Quantitative polymerase chain reaction (q-PCR) targeting sul1, tet(O), ermB and the class I integron gene intI1 and corresponding RNA expression did not indicate measureable effects of nanoAg or Ag(+) on abundance or expression of these genes. However, high-throughput sequencing based metagenomic analysis provided a much broader profile of gene responses and revealed a greater abundance of aminoglycoside resistance genes (mainly strA) in reactors dosed with nanoAg. In contrast, bacitracin and macrolide-lincosamide-streptogramin (MLS) resistance genes were more abundant in the SBRs dosed with Ag(+). The distinct ARG profiles associated with nanoAg and Ag(+) were correlated with the taxonomic composition of the microbial communities. This study indicates that nanoAg may interact with bacteria differently from Ag(+) during biological wastewater treatment. Therefore, it cannot necessarily be assumed that nanosilver behaves identically as Ag(+) when conducting a risk assessment for release into the environment.