Antioxidant-producing extremophile regulates gut microbiota of black soldier fly larvae to inhibit conjugative transfer mediated by plasmids in municipal sludge.

Oxidative stress induced by heavy metals and antibiotics in municipal sludge inhibits the gut development of black soldier fly larvae (BSFL), compromising pollutant-processing capacity of their gut microbiota. Herein, Deinococcus radiodurans was colonized into the BSFL gut and established an antioxidant cascade regulatory system in concert with the gut microbiota, synergistically reducing the enrichment of antibiotic resistance genes (ARGs) in municipal sludge. Results showed that bioaugmentation with Deinococcus radiodurans scavenges reactive oxygen species and upregulates the expression of G-protein and s14e genes in BSFL gut, maintaining normal gut function. On this basis, we propose two potential mechanisms responsible for inhibiting the horizontal transfer of ARGs (HGT). At the molecular level, bioaugmentation reduces environmental co-selection pressure through heavy metal immobilization mediated by extracellular polymeric substances and SlpA protein, as well as antibiotic degradation driven by gut microbiota, reducing the abundances of the class 1 integron integrase gene (intI1), conjugative transfer gene (traG), and ARGs (aphA and tetA) in municipal sludge by more than one order of magnitude. At the ecological level, bioaugmentation reshapes the microbial community structure of sludge, reducing the abundance of Pseudomonadota carrying mobile genetic elements and ARGs by approximately 26%. Moreover, bioaugmentation disrupted the co-occurrence of the mer operon and ARGs (sul1). Conjugative experiments revealed that co-cultivation with Deinococcus radiodurans reduced the conjugative transfer frequency of the ARG-carrying RP4 plasmid by 91%. Therefore, this study elucidates how antioxidant-producing extremophiles can strengthen BSFL system to inhibit HGT and reduce ARG enrichment.