Metagenomic Comparison of Antibiotic Resistance Genes Associated with Liquid and Dewatered Biosolids

Municipal biosolids (MBs) that are land-applied in North America are known to possess an active microbial population that can include human pathogens. Activated sludge is a hotspot for the accumulation of antibiotics and has been shown to be a selective environment for microorganisms that contain antibiotic resistance genes (ARGs); however, the prevalence of ARGs in MBs is not well characterized. In this study, we enriched the plasmid metagenome from raw sewage sludge and two CP2 MBs, a mesophilic anaerobic digestate and a dewatered digestate, to evaluate the presence of ARGs in mobile genetic elements. The CP2-class biosolids are similar to Class B biosolids in the United States. The CP2 biosolids must meet a microbiological cut off of 2 x 10(6) colony-forming units (CFU) Escherichia coli per dry gram or 100 mL of biosolids. The enriched plasmid DNA was sequenced (Illumina MiSeq). Sequence matching against databases, including the Comprehensive Antibiotic Resistance Database (CARD), MG-RAST, and INTEGRALL, identified potential genes of interest related to ARGs and their ability to transfer. The presence and abundance of different ARGs varied between treatments with heterogeneity observed among the same sample types. The MBs plasmid-enriched metagenomes contained ARGs associated with resistance to a variety of antibiotics, including beta-lactams, rifampicin, quinolone, and tetracycline as well as the detection of extended spectrum beta-lactamase genes. Cultured bacteria from CP2 MBs possessed antibiotic resistances consistent with the MBs metagenome data including multiantibiotic-resistant isolates. The results from this study provide a better understanding of the ARG and MGE profile of the plasmid-enriched metagenome of CP2 MBs.