Analysis of extracellular and intracellular antibiotic resistance genes in commercial organic fertilizers reveals a non-negligible risk posed by extracellular genes

Livestock manure is known to be a significant reservoir of antibiotic resistance genes (ARGs), posing a major threat to human health and animal safety. ARGs are found in both intracellular and extracellular DNA fractions. However, there has been no comprehensive analysis of these fractions in commercial organic fertilizers (COFs). The present study conducted a systematic survey of the profiles of intracellular ARGs (iARGs) and extracellular ARGs (eARGs) and their contributing factor in COFs in Northern China. Results showed that the ARG diversity in COFs (i.e., 57 iARGs and 53 eARGs) was significantly lower than that in cow dung (i.e., 68 iARGs and 69 eARGs). The total abundance of iARGs and eARGs decreased by 85.7% and 75.8%, respectively, after compost processing, and there were no significant differences between iARGs and eARGs in COFs (P > 0.05). Notably, the relative abundance of Campilobacterota decreased significantly (99.1-100.0%) after composting, while that of Actinobacteriota and Firmicutes increased by 21.1% and 29.7%, respectively, becoming the dominant bacteria in COFs. Co -occurrence analysis showed that microorganisms and mobile genetic elements (MGEs) were more closely related to eARGs than iARGs in COFs. And structural equation models (SEMs) further verified that microbial community was an essential factor regulating iARGs and eARGs variation in COFs, with a direct influence (lambda = 0.74 and 0.62, P < 0.01), following by similar effects of MGEs (lambda = 0.59 and 0.43, P < 0.05). These findings indicate the need to separate eARGs and iARGs when assessing the risk of dissemination and during removal management in the environment.