Metagenomics-Based Characterization of Antibiotic Resistome in Hospital Wastewater from Tertiary Health Facilities in Cross River State

Hospital wastewater serves as a complex mixture of antibiotic compounds, resistant bacteria, and mobile genetic elements, constituting a major environmental reservoir for antibiotic resistance gene (ARG) dissemination. Metagenomics enables culture-independent comprehensive resistome profiling of such complex microbial communities. This study characterized the antibiotic resistome in hospital wastewater from three tertiary health facilities in Calabar and Ogoja, Cross River State, South-South Nigeria, using shotgun metagenomics. Wastewater samples were collected from influent and effluent streams at each facility over a two-month period. Total community DNA was extracted by bead-beating homogenization. Sequencing was performed on the Illumina NovaSeq platform, and reads were taxonomically classified by Kraken2. ARG annotation was performed against the Comprehensive Antibiotic Resistance Database (CARD) using DeepARG. Mobile genetic elements (integrons, transposons) were characterized using MobileElementFinder. The hospital wastewater resistome was dominated by ARGs conferring resistance to beta-lactams (blaTEM, blaCTX-M), aminoglycosides (aac(3), aph(3)), and sulfonamides (sul1, sul2). Effluent ARG abundance was not significantly reduced compared to influent, suggesting inadequate wastewater treatment capacity. Over 80% of detected ARGs were co-localized with Class 1 integrons, indicating high horizontal gene transfer potential. These findings demonstrate that hospital wastewater in Cross River State constitutes an active ARG environmental reservoir requiring policy-level intervention. Keywords: metagenomics, resistome, hospital wastewater, antibiotic resistance genes, Cross River State.