Proximity-Ligation Metagenomic Sequence Analysis Reveals That the Antibiotic Resistome Makes Significant Transitions During Municipal Wastewater Treatment

Shotgun and proximity-ligation metagenomic sequencing were used to generate thousands of metagenome assembled genomes (MAGs) from the untreated wastewater, activated sludge bioreactors, and anaerobic digesters from two full-scale municipal wastewater treatment facilities. Analysis of the antibiotic resistance genes (ARGs) in the pool of contigs from the shotgun metagenomic sequences revealed significantly different relative abundances and types of ARGs in the untreated wastewaster compared to the activated sludge bioreactors or the anaerobic digesters (p < 0.05). In contrast, these results were statistically similar when comparing the ARGs in the pool of MAGs, suggesting that proximity-ligation metagenomic sequencing is particularly useful for pairing ARGs with their hosts but less adept at discerning quantitative differences in ARG types and relative abundances. For example, numerous MAGs of the genera Acinetobacter, Enterococcus, Klebsiella and Pseudomonas were identified in the untreated wastewater, many of which harboured plasmid-borne and/or chromosomal-borne ARGs; none of these MAGs, however, were detected in the activated sludge bioreactors or anaerobic digesters. In conclusion, this research demonstrates that the antibiotic resistome undergoes significant transitions in both the relative abundance and the host organisms during the municipal wastewater treatment process.