Divalent Cations and Chlorine Synergistically Reshape T6SS-Mediated Microbial Competition in Water Distribution System Biofilms

Abstract Microbial interactions shape the community structure and function in drinking water distribution system (DWDS) biofilms, yet how the dynamic competition is regulated in biofilms remains poorly understood. Here, we show that divalent cations (Ca2+, Mg2+) and chlorine in drinking water synergistically activate the type VI secretion system (T6SS) in opportunistic pathogens within biofilms. Field-collected biofilms serve as ecological hotspots that selectively enrich specific T6SS-harboring pathogens. Using simulated pipe systems and mutant validation, we further establish that divalent cations and chlorine enhance c-di-GMP signaling to promote T6SS-mediated colonization. This activation stimulates EPS production, whose polysaccharides mitigate T6SS attack, dampening the competitive advantage during mid-stage maturation but allowing gradual resurgence over extended succession. This divalent cation-chlorine-driven interplay shapes ecological succession, selectively retaining T6SS-proficient cells and establishing a “biofilm reservoir effect.” Under chlorine decay, T6SS-active pathogens can be released into bulk water, elevating exposure risks. Conversely, reducing divalent cations via nanofiltration suppresses T6SS across multiple pathogens, limiting reservoir accumulation and release. Our findings identify divalent cation availability as a key environmental signal associated with T6SS-mediated competition, linking water chemistry to pathogen persistence. This work suggests integrating membrane-based divalent cation removal with chlorine management as a chemistry-based strategy to mitigate microbial risks, advancing an integrated water quality paradigm for drinking water safety.