Metagenomic analysis after selective culture enrichment of wastewater demonstrates increased burden of antibiotic resistant genes in hospitals relative to the community

Antimicrobial resistance (AMR) is an ever-increasing threat to global health. Wastewater-based surveillance is an emerging methodology that objectively enables an inclusive and comprehensive assessment of population AMR in an observed sewershed. Here we compared the resistome of two tertiary-care hospitals with two separate neighborhoods, using complimentary targeted qPCR and metagenomics of wastewater before and after selective culture enrichment for clinically important Gram negatives. In total 26 ARG-type (1225 ARG-subtypes) were found across all samples, in which β-lactam ARG was the richest (the number of different ARG-subtypes found) followed by multidrug, fluoroquinolone, macrolide-lincosamide-streptogramin (MLS) and aminoglycoside. The composition of ARGs in wastewater differed between raw wastewater pellets and culture-enriched wastewater samples and the resistomes clustered based on the type of location (Hospitals vs neighborhoods). Hospital wastewater was found to have higher diversity and greater abundance of ARGs compared to neighborhood wastewater when the composition profiles of ARGs in both raw and culture-enriched wastewater pellets. Clinically relevant ARG (i.e., VIM, NDM metallo-ß-lactamases) were detected in culture enrichment samples that were not identified in raw samples, despite a lower targeted sequencing depth. Wastewater-based surveillance is an effective, and potentially extremely important and powerful tool that could be developed to augment hospital-based infection control and antimicrobial stewardship programs, creating a safer space for those receiving care.