Metagenomic analysis after selective culture enrichment of hospital and community wastewater enhances antimicrobial resistance gene detection

Antimicrobial resistance is an accelerating threat to global health. Wastewater-based surveillance (WBS) enables objective, inclusive, and comprehensive assessments of population-level antimicrobial resistance; however, it is limited in its ability to detect rare antibiotic resistance genes (ARG). We compared traditional high-depth metagenomic sequencing of raw wastewater with lower-depth sequencing following semi-selective culture enrichment for gram negatives for rare ARGs in wastewater from two tertiary-care hospitals and two nearby urban neighborhoods. In total, 26 antibiotic resistance gene types (1,225 subtypes) were identified, with beta-lactamase genes being the most prevalent. Resistomes differed between raw and culture-enriched wastewater metagenomes and clustered based on sample type (hospitals versus neighborhoods). Hospital wastewater had higher diversity and a greater abundance of ARGs relative to both raw and culture-enriched neighborhood wastewater metagenomes. Lower coverage sequencing following culture enrichment proved superior to deeper sequencing for identifying rare, clinically relevant targets, including carbapenemase genes. In particular, enrichment with meropenem proved the most sensitive to identifying clinically relevant genes and enabled significant cost savings. ARG WBS has enormous potential for augmenting hospital-based infection prevention and control and antimicrobial stewardship programs.