Similar Prophage Induction but Divergent Antibiotic Resistance Gene Occurrence: Leachates and Free Radicals Drive Differential Activation Mechanisms in Aged Tire Crumb Rubber

Indigenous phages play a key role in the spread of extracellular antibiotic resistance genes (eARGs), and micro/nanocontaminants can exacerbate this process. However, the specific roles and mechanisms of phage communities in this process are still not fully understood. In this study, machine learning models were used to explore how micrometer-sized tire crumb rubber (mTCR) affects the transmission of ARGs via phage communities. The results showed that mTCR at environmentally relevant concentrations significantly activated prophages, promoting the spread of the ARGs. The activation mechanisms of shade- and photoaged mTCR differed, with eARGs carried by phage being more abundant in photoaged mTCR-treated sludge despite similar activation efficiencies. The random forests algorithm revealed that prophage activation induced by shade-aged mTCR was primarily driven by the metals and organic components leached from the mTCR. Antibiotic-resistant lysogens tolerated shade-aged mTCR due to their resistance to metals and organic matter, reducing internal prophage activation. In contrast, photoaged mTCR particles harbored more persistent free radicals, leading to the generation of extracellular O₂^•- and ^•OH, which damaged cell membranes and triggered prophage activation. These findings highlight the ecological risk associated with eARGs spread via prophage induction and deepen our understanding of the need to control discarded TCR.