Microbial Diversity and Resistome of Hospital Wastewater from Tertiary Facilities in Ibadan, Oyo State: A Functional Metagenomics Study

Hospital wastewater harbors a complex microbiome enriched for antibiotic-resistant organisms and resistance genes that may be released into the broader environment through inadequate treatment. Functional metagenomics offers a culture-independent approach to comprehensively characterize both microbial diversity and resistome in complex water matrices. This study applied functional metagenomics to hospital wastewater collected from influent and effluent streams of two tertiary hospitals in Ibadan, Oyo State, South West Nigeria. DNA extracted from 0.22 micron membrane-filtered water was subjected to library construction and next-generation sequencing on the Illumina NovaSeq platform. Taxonomic classification was performed using Kraken2, and resistome profiling used the Comprehensive Antibiotic Resistance Database (CARD). Functional annotation of metagenomic contigs was performed via KEGG and COG databases. Results revealed high microbial diversity with 18 bacterial phyla detected, dominated by Proteobacteria (38%), Firmicutes (22%), and Bacteroidetes (19%). The resistome encompassed 341 ARG subtypes, with tetracycline, beta-lactam, and sulfonamide resistance genes as the most abundant. mcr-3 (colistin) and blaNDM were detected in both hospitals' effluent. Treatment significantly reduced microbial load but only marginally reduced resistome complexity (21% ARG reduction). Plasmid-associated ARG mobility elements were preserved through treatment. These findings demonstrate that conventional wastewater treatment is inadequate for resistome elimination and support the urgent development of tertiary treatment standards for Nigerian hospital wastewater. Keywords: hospital wastewater, functional metagenomics, resistome, ARGs, Ibadan.