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Use of a Targeted Method to Link the Resistome to Microbial Hosts in Class B Biosolids
Citation:
Gomez-Alvarez, V. AND L. Boczek. Use of a Targeted Method to Link the Resistome to Microbial Hosts in Class B Biosolids. Presented at ASM Microbe, Washington, D.C, June 09 - 13, 2022.
Impact/Purpose:
Biosolids are defined as the treated sewage sludge residuals that are left over after wastewater treatment. The US EPA through the Clean Water Act regulates land application of biosolids (40 CFR Part 503). Biosolids must undergo treatments to be land applied because these raw residuals may contain pathogenic microbes. An increasing amount of antibiotics are released into wastewater through different sources, and ultimately find their way into wastewater treatment plants (WWTP). This research provides evidence of the feasibility of using the next generation sequencing (NGS) technology to characterize the wastewater microbial community. This research has considerable importance in developing a monitoring assay to estimate the risk of dissemination of antimicrobial resistances (AMR) and ultimately helps to safeguard drinking water and human health.
Description:
Biosolids are defined as the treated sewage sludge residuals that are left over after wastewater treatment. The US EPA through the Clean Water Act regulates land application of biosolids. Biosolids must undergo treatments to be land applied because these raw residuals may contain pathogenic microbes. An increasing amount of antibiotics are released into wastewater through different sources, and ultimately find their way into wastewater treatment plants (WWTPs). Studies have demonstrated a correlation between antibiotic use and the emergence of antimicrobial resistance (AMR). Traditionally, analysis of the resistome (collection of AMR) has been based on microbial culture techniques or molecular assays of a limited number of antibiotic resistant genes (ARG). Recently, metagenomic approaches have been used to study the whole community. However, these methods are not able to elucidate the bacteria conferring AMR. For this purpose, we examined the resistome of raw sludge, digestive sludge, and dewatered cake biosolids (Class B) samples collected from a WWTP in Ohio, United States, and utilized an in vivo proximity-ligation molecular method (Hi-C) to link the AMR to their microbial hosts. In addition, DNA libraries were deep sequenced using an Illumina HiSeq platform (short-reads) and ONT sequencer MinION (long-reads). Metagenomic results highlighted marked microbial community differences, revealing a highly dynamic and diverse microbial community. Metagenomic binning yields 258 genomes (250 bacterial and 8 archaeal) with 76 bins representing metagenome-assembled genomes (MAGs) that had a >50% completeness and <10% contamination representing 4.4% to 24% of the assemblies and consisting of several phyla representatives. Metabolic complexity and functional potential of the microbiome is evident in wastewater ecosystems. ARGs were characterized among all samples with the highest diversity in samples collected from raw sludge and decreased in biosolids. The recovered ARGs were associated with resistance against tetracyclines, sulfonamides, lipopeptides, fluoroquinolones, phenicol, bacitracin, beta-lactam, streptothricins, aminoglycoside and MLS (macrolides-lincosamides-streptogramines). A comprehensive characterization of microbial communities (including their resistome) is essential for risk management. Such information can be incorporated into ecosystem models and greatly enhance our ability to estimate the risk of dissemination of resistance genes from environmental these reservoirs.