High Throughput Analysis of Integron Gene Cassettes in Wastewater Environments

Abstract

Integrons are extensively targeted as a proxy for anthropogenic impact in the environment. We developed a novel high-throughput amplicon sequencing pipeline that enables characterization of thousands of integron gene cassette-associated reads, and applied it to acquire a comprehensive overview of gene cassette composition in effluents from wastewater treatment facilities across Europe. Between 38 100 and 172 995 reads per-sample were generated and functionally characterized by screening against nr, SEED, ARDB and β-lactamase databases. Over 75% of the reads were characterized as hypothetical, but thousands were associated with toxin-antitoxin systems, DNA repair, cell membrane function, detoxification and aminoglycoside and β-lactam resistance. Among the reads characterized as β-lactamases, the carbapenemase bla was dominant in most of the effluents, except for Cyprus and Israel where bla was also abundant. Quantitative PCR assessment of bla and bla genes in the European effluents revealed similar trends to those displayed in the integron amplicon sequencing pipeline described above, corroborating the robustness of this method and suggesting that these integron-associated genes may be excellent targets for source tracking of effluents in downstream environments. Further application of the above analyses revealed several order-of-magnitude reductions in effluent-associated β-lactamase genes in effluent-saturated soils, suggesting marginal persistence in the soil microbiome.

Impact of Wastewater Treatment on the Prevalence of Integrons and the Genetic Diversity of Integron Gene Cassettes

“…The differences in microbial biomass might be mainly responsible for the higher abundance of integrons in sludge. Samples from different treatment stages harbored distinct microbes with varied profiles of integron gene cassettes (24), and the bacteria that proliferate in activated sludge do not potentially harbor integrons to the same extent as influent microbiota (30,33). The concentration of integrons in effluents was 1 to 2 orders of magnitude lower than that in influents, suggesting that the wastewater treatment process could effectively reduce the concentration of integrons.…”

“…Both approaches have their advantages and limitations. High-throughput sequencing can provide a high-throughput pipeline for tracking gene cassettes in integrons (24) and cover the whole picture of gene cassette distribution owing to greater sequencing depth while avoiding the bias caused by cloning. Clone library analysis may overcome the drawbacks of highthroughput sequencing caused by assembling bias of short reads derived from amplified sequences with high similarity.…”

“…However, these studies have frequently focused on quantities of integrons using quantitative PCR (qPCR) and limited numbers of host bacteria based on culturedependent methods. High-throughput sequencing analyses have been applied for characterizing class 1 integron gene cassettes, revealing a wide distribution of ARGcarrying class 1 integrons in different environmental samples (e.g., water environments, sediment, and feces) (24,25). A recent study revealed that hospital effluents (EFFs) are important sources of integrons in sewage treatment plants (26).…”

Reusing Treated Wastewater: Consideration of the Safety Aspects Associated with Antibiotic-Resistant Bacteria and Antibiotic Resistance Genes

“…The recent advancement of metagenomics-based approaches have provided a powerful means to give a snapshot of the diversity of ARGs and mobile genetic elements (MGEs) that facilitate HGT (e.g., plasmids, integron gene cassettes) in wastewater and environmental samples [36,37]. Metagenomics also makes it possible to assess the roles of other microbes besides bacteria, such as phages, in propagating resistance in WWTPs, as elucidated in a recent review paper [38].…”

Antibiotic resistance associated with air quality and transferred by airborne particulate matter

“…Antibiotic resistance is a serious threat to public health [1]. Diverse and abundant antibiotic resistance genes (ARGs) are widespread in most environments (e.g., air, water, soil, remote and pristine glacier and caves, urban sewage, and drinking water) [2][3][4][5][6][7][8][9][10][11]. Horizontal gene transfer (HGT) is a major driver for disseminating ARGs within and across bacterial genera, via mobile genetic elements (MGEs), such as plasmids, integrons, transposes, and prophages [12].…”