Antibiotic resistance (AR), a natural phenomenon, has evolved into a global public health challenge due to the misuse of antibiotics. Human activities have accelerated the spread and impact of AR to the natural environments, which serve as reservoirs for antibiotic resistance genes (ARGs). Pristine caves offer an unaltered environment that reflect a pre-antibiotic overese era, which may give a unique perspective on the natural formation of microbiomes and resistomes, i.e., collection of ARGs within a community. Comparing cave resistomes and agricultural soils may offer valuable insights into the impacts of natural versus human-modified ecosystems on resistome dynamics. Our comparative study compared cave sediments with 55 metagenome sequences derived from two distinct agricultural soils; with organic fertiliser or inorganic fertiliser soils. The results showed that multidrug resistance (MDR) genes, particularly multidrug efflux pumps (rpoB2), dominated both the cave sediments and the agricultural soils. In addition, specific samples had distinct resistome profiles, with cave sediments exhibiting the lowest total ARGs abundance and ARGs subtypes number, whereas agricultural soils with organic fertilizer had the highest ARGs abundance and ARGs subtypes. Some types of ARGs showed a preference for enrichment in particular sample types. Bacitracin and fluoroquinolone antibiotic families were consistently enriched in cave sediments, whereas sulfonamide, phenicol, and tetracycline families were consistently enrichment in agricultural soils with organic fertilizer. Limited ARGs in cave sediments and differences in MDR and sul-, tet-ARG subtypes in agricultural soils with organic fertilizer contributed to the variations in cave and agricultural soil resistomes. These studies have implications for mitigating AR in agriculture and understanding resistance reservoirs in cave ecosystems. The results highlight the importance of MDR genes, particularly multidrug efflux pumps, in resistome formation and development.

Keywords: Resistome, Multidrug resistance (MDR) genes, Metagenomics, Environmental resistome