From grasslands to genes: exploring the major microbial drivers of antibiotic-resistance in microhabitats under persistent overgrazing

The extensive use of antibiotics in the global livestock industry in recent decades has accelerated the accumulation and dissemination of antibiotic-resistance genes (ARGs) within terrestrial ecosystems. This occurs due to the limited absorption of most antibiotics, leading to their release into the environment through feces and urine. This poses a significant threat to both the environment and human health. However, the response of antibiotic-resistant microorganisms and their ARGs in grasslands to prolonged grazing, as well as the primary microbial taxa driving the ARG distribution, remain poorly understood, especially within various microhabitats. In this study, we characterized ARGs in the phyllosphere, litter, and soil after decades of livestock grazing in a meadow steppe. We particularly focused on identifying the major members of the microbial community influencing ARGs and the distinction between microbial generalists and specialists. Our findings indicate that a core set of ARGs accounted for 90% of the abundance in this plant-soil ecosystem. While the soil exhibited the highest ARG abundance, the phyllosphere, and litter displayed higher ARG diversity and diverse distribution patterns after overgrazing. Grazing increased ARG abundance by elevating the proportion of core ARGs and suppressing stochastic ARGs in the phyllosphere and litter, while it had little effect on the ARGs in the soil. Additionally, microbial generalist abundance increased, but specialist abundance decreased in the phyllosphere and litter, with no effect in the soil, under grazed conditions. Ultimately, microbial microhabitats and grazing influenced ARG community characteristics through direct (i.e., feces and other exogenous ARG input) and indirect (i.e., trampling and selective feeding) effects on nutrient availability, microbial community composition, and mobile genetic elements. The generalist community, with its broad ecological niches and phylogenetic composition, made the most significant contribution to the ARG characteristics. This study underscores the impact of environmental disturbances on the distributional patterns of ARGs in ecosystems, mediated by the regulation of microbial generalized species. These insights enhance our understanding of microbial control over ARGs and facilitate predictions regarding the dynamics and risk of ARGs in diverse ecological niches subjected to anthropogenic disturbances.