Identification of diverse antibiotic resistant bacteria in agricultural soil with H₂¹⁸O stable isotope probing and metagenomics

Abstract Background In this study, we aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil, by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18 O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled “heavy” and the unlabelled “light” SIP fractions were sequenced. Results After incubations, an increase of the 16S rRNA copy numbers in the “heavy” fractions of the treatments with 18 O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) are highly abundant after two days of incubation with antibiotics. Several Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. On the other hand, only two ARGs from the unbinned-assembled light fractions were identified. Conclusions The results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil, and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.