Earthworm gut digestion drives the transfer behavior of antibiotic resistance genes in layers of extracellular polymeric substances during vermicomposting of dewatered sludge.

Gut digestion by earthworms (GDE) is a crucial step in vermicomposting, affecting the fate of antibiotic resistance genes (ARGs) in vermicompost sludge. The extracellular polymeric substance (EPS) matrix of sludge is an important space for ARG transfer. However, the effect of GDE on EPS-associated ARGs remains unclear. Therefore, this study explored the role of GDE in driving the transfer of ARGs within different EPS layers in sludge. For this, the changes in intracellular ARGs and EPS-associated ARGs in sludge were analyzed after 5 days of the GDE process. The results showed that after the GDE process, both nitrate and dissolved organic carbon significantly increased in all EPS layers of sludge, while the proteins and polysaccharides only enhanced in soluble and loosely bound EPS of sludge. In addition, a 7.0% decrease in bacterial diversity was recorded after the GDE process, with a functional bacterial community structure emerging. Moreover, the absolute abundance of total ARGs and mobile genetic elements decreased by 90.71% and 61.83%, respectively, after the GDE process. Intracellular ARGs decreased by 92.1%, while EPS-associated ARGs increased by 4.9%, indicative of intracellular ARG translocation into the EPS during the GDE process. Notably, the ARGs exhibited significant enrichment in both the soluble and loosely bound EPS, whereas they were reduced in the tightly bound EPS. The structural equation modeling revealed that the GDE process effectively mitigated the ARG dissemination risk by modulating both the EPS structure and microenvironment, with the organic structure representing a primary factor influencing ARGs in the EPS.