Novel antibiotic resistance genes from the hospital effluent are disseminated into the marine environment in Norway

Hospital effluent comprises feces of many individuals, including patients undergoing antibiotic treatment. Although, hospital effluent is an important source for contamination of the environment with antibiotic resistance genes (ARGs) and pathogens, how hospital effluent in a low resistance setting contributes to antimicrobial resistance (AMR) in the environment is largely understudied. The aim of our study was to understand the microbiota and resistome of hospital effluent, and its role in the spread of AMR in the marine environment in Norway. We further aimed at describing/characterizing novel resistance factors from hospital effluent and the receiving sewage treatment plant (STP). 24-hour composite samples of the hospital effluent and the influent and effluent of the receiving STP were collected at two sampling time-points (February and April 2023) in Bergen city, Norway. Isolation of Escherichia coli and Klebsiella spp. was performed, using ECC and SCAI plates with cefotaxime, tigecycline or meropenem, followed by antibiotic susceptibility testing, using EUVSEC3 plates. Whole-genome sequencing of selected strains (n=36) and shotgun metagenomics of sewage samples (n=6) were performed, using Illumina NovaSeq. ARGs were identified with USEARCH, and known and novel ARGs were assembled with fARGene. All E. coli strains (n=66) were multidrug-resistant (MDR), while 92.3% of the Klebsiella spp. strains (n=55) showed MDR phenotype. The sequenced strains carried multiple clinically important ARGs, including carbapenemases such as NDM-5 (n=3) and KPC-3 (n=3). We obtained 238 Gigabases of sequence data from which we identified 676 unique ARGs with >200 ARGs shared across samples. We assembled 1,205 ARGs using fARGene, 365 gene sequences represented novel ARGs (< 90% amino acid (aa) identity). Both known and novel ARGs (n=54) were shared between the hospital effluent and the treated effluent of the receiving STP. We show that hospital effluent in Norway has a high diversity of both known and novel ARGs. Our study demonstrates that hospital effluent is a source of clinically relevant pathogens, as well as known and novel ARGs, reaching the marine environment in Norway through treated sewage.