Unidirectional cross-feeding enhances type IV pili–mediated transformation of antibiotic resistance gene

• T4P/T6SS associate more strongly with ARGs than T4SS, and uncovers widespread metabolic dependencies. • Unidirectional cross-feeding strength is positively correlated with ARG transformation frequency. • Strong unidirectional cross-feeding promotes ARG transfer via T6SS-T4P, coordinated by quorum sensing and two-component systems. • Quorum sensing and two-component systems are functionally coupled with T4P/T6SS activity. • Microbial interactions shape ecological dissemination of antibiotic resistance genes. The horizontal spread of antibiotic resistance genes (ARGs) poses a serious global-health threat. Microbial interactions are increasingly recognized as influential factors in the spread of ARGs, yet the role of metabolic dependencies remains poorly understood. Through functional association analysis of genomic features, this study indicates that type IV pili (T4P) and type VI secretion systems (T6SS) are strongly associated with the presence of ARGs. Moreover, non-antibiotic-resistant microbes (Non-ARMs) are predicted to potentially rely metabolically on antibiotic-resistant microbes (ARMs). Among the metabolites supplied exclusively by ARMs, organic compounds dominated (76.3%), followed by inorganic compounds (18.4%) and complex biomolecules (5.3%). To experimentally investigate the effects of such dependencies on T4P-mediated ARGs transformation, we established coculture systems with varying strengths of unidirectional cross-feeding by modulating the carbon source composition. The frequency of ARG transformation increased significantly with the strength of cross-feeding (Spearman’s ρ > 0.8, p < 0.05). Transcriptomic analysis revealed the activation of two-component systems and quorum sensing pathways, which are known global regulators of bacterial stress responses and cell–cell communication. This activation was associated with increased expression of T4P and T6SS genes, suggesting a potential regulatory link with enhanced ARG acquisition. This study suggests that unidirectional metabolic dependency promotes ARG transformation, and fills a specific research gap by linking the strength of metabolic dependence with the frequency of ARG transformation, and raises the possibility that metabolic interactions could inform future efforts to model resistance spread.