Cyanobacteria-mediated carbon-nitrogen coupling promotes the enrichment of antibiotic resistance genes in the Yangtze estuarine biofilms

The metabolism of biogenic carbon-nitrogen elements mediated by Cyanobacteria is a fundamental process for microbial physiological and metabolic activities, playing vital roles in the proliferation and dissemination of antibiotic resistance genes (ARGs). However, the relationships between carbon-nitrogen metabolism and ARG patterns among Cyanobacteria remain understudied. Here, the study investigated the relationships between ARGs and carbon-nitrogen metabolism in naturally occurring biofilms, sediments, and water bodies in the Yangtze Estuary, via metagenomic analysis combined with DNA-based stable isotope probing (DNA-SIP). The results showed that Cyanobacteria were the dominant ARG hosts in biofilms, contributing 39% of ARGs, compared with only 0.2% and 2.5% in water and sediment samples, respectively. The Calvin cycle and nitrogen fixation genes were significantly positively correlated with ARGs, explaining 13.3% and 54.1% of the variation in the ARGs, respectively. DNA-SIP further confirmed that organisms involved in the Calvin cycle and nitrogen fixation, such as Cyanobacteria, were dominant hosts of ARGs. Several metagenome-assembled genomes, belonging to Cyanobacteria, were labelled with 13C and identified as Microcoleaceae sp. and Geitlerinemaceae sp. Certain ARGs were annotated in nitrogen transformation genes (nifH, NAR, NIR, and nirB) and carbon-related metabolic pathways, further confirming the genetic co-occurrence of ARGs and microbial carbon-nitrogen genes. This study provides new insights into the dual roles of ARGs in antibiotic resistance and metabolic regulation of Cyanobacteria in estuarine biofilms.