The polyspecificity and adaptability of multidrug resistance shaped the antibiotic resistome in anaerobic digestion amended by nZVI: Evidence from efflux pumps and rRNA methyltransferase genes

Nano zero-valent iron (nZVI) assisted anaerobic digestion (AD) is considered an effective approach for mediating antibiotic resistance genes (ARGs) dissemination in activated sludge. However, little is known about the mechanisms that drive antibiotic resistomes evolution in relation with their physiological roles distinct from protection of bacteria against antimicrobials. Metagenomic sequencing showed that efflux pumps and rRNA methyltransferase genes were the most prevalent ARGs in anaerobes, being responsible for the variations of antibiotic resistome upon nZVI addition. Particularly, to withstand the oxidative stimuli derived from initially 9 days nZVI exposure, genes involved in the two component systems ( PhoRB , BaeSR , EvgSA ) and global regulator networks ( MarA , SoxS ) were increased, activating the chromosomally encoded intrinsic efflux pumps expression. Furthermore, to benefit for survival, the polyspecific efflux pumps were involved in the microbial physiological adaptations of enhanced siderophores, lipid moieties, and quorum sensing molecules exportation, which induced the adaptative efflux pumps expression. On the other hand, to maintain the protein synthesis under oxidative condition, genes encoding Rsm, Erm, and Rlu family methyltransferases were notably up-regulated via horizontal transfer, inducing the acquired rRNA methylation resistance. Importantly, after prolonged exposure to nZVI for 18 and 27 days, the antibiotic resistomes were measurably decreased, due to the recovered oxidative state and increased membrane rigidity. Overall, these findings provide a deep understanding on the necessity of antibiotic resistome evolution for anaerobes to adapt to nZVI exposure for survival, which can be introduced to improve the ARGs risks analysis.