Application of single-cell Raman-deuterium isotope probing to reveal the resistance of marine ammonia-oxidizing archaea SCM1 against common antibiotics

Antimicrobial resistance (AMR) in oceans poses a significant threat to human health through the seafood supply chain. Ammonia-oxidizing archaea (AOA) are important marine microorganisms and play a key role in the biogeochemical nitrogen cycle around the world. However, the AMR of marine AOA to aquicultural antibiotics is poorly explored. Here, Raman-deuterium isotope probing (Raman-DIP), a single-cell tool, was developed to reveal the AMR of a typical marine species of AOA, Nitrosopumilus maritimus (designated SCM1), against six antibiotics, including erythromycin, tetracycline, novobiocin, neomycin, bacitracin, and vancomycin. The D2O concentration (30% v/v) and culture period (9 days) were optimized for the precise detection of metabolic activity in SCM1 cells through Raman-DIP. The relative metabolic activity of SCM1 upon exposure to antibiotics was semi-quantitatively calculated based on single-cell Raman spectra. SCM1 exhibited high resistance to erythromycin, tetracycline, novobiocin, neomycin, and vancomycin, with minimum inhibitory concentration (MIC) values between 100 and 400 mg/L, while SCM1 is very sensitive to bacitracin (MIC: 0.8 mg/L). Notably, SCM1 cells were completely inactive under the metabolic activity minimum inhibitory concentration conditions (MA-MIC: 1.6–800 mg/L) for the six antibiotics. Further genomic analysis revealed the antibiotic resistance genes (ARGs) of SCM1, including 14 types categorized into 33 subtypes. This work increases our knowledge of the AMR of marine AOA by linking the resistant phenome to the genome, contributing to the risk assessment of AMR in the underexplored ocean environment. As antibiotic resistance in marine microorganisms is significantly affected by the concentration of antibiotics in coastal environments, we encourage more studies concentrating on both the phenotypic and genotypic antibiotic resistance of marine archaea. This may facilitate a comprehensive evaluation of the capacity of marine microorganisms to spread AMR and the implementation of suitable control measures to protect environmental safety and human health.