Species-specific chlorine resistance and biofilm regulation by extracellular polymeric substances and quorum sensing in drinking water pipeline bacteria

ABSTRACT Biofilms in drinking water distribution systems pose significant risks by harboring chlorine-resistant bacteria. This study isolated five bacterial strains ( Sphingomonas ursincola , Sphingobium amiense , Gordonia amicalis , Microbacterium saccharophilum , Hydrogenophaga laconesensis ) from municipal pipelines to evaluate differences in biofilm formation and chlorine resistance. Biofilm formation ability varied notably, with S. ursincola showing the strongest capacity and H. laconesensis the weakest. Quorum sensing (QS) signal molecules (C 6 -HSL, 3-OXO-C 14 -HSL; 0.2240–0.2481 μg/L) and extracellular polymeric substances (EPSs: 19.940–32.407 mg/L) were critical in biofilm regulation, where QS molecules influenced EPS composition. Chlorine resistance assays revealed species-specific tolerance: at ≤1.0 mg/L, resistance ranked M. saccharophilum > S. amiense > G. amicalis > S. ursincola > H. laconesensis ; above 1.0 mg/L, S. ursincola and S. amiense exhibited robust resistance. Low chlorine levels (0.6 mg/L) damaged only 25.91%–34.80% of bacteria, insufficient to control the biofilm formation of the tested isolates. Optimal disinfection occurred at 1.0–1.5 mg/L, effectively controlling biofilm biomass. These findings highlight EPS-driven chlorine resistance mechanisms and QS-mediated biofilm regulation, providing actionable strategies for pipeline management. IMPORTANCE This study addressed a critical gap in understanding different bacterial biofilm dynamics and chlorine resistance mechanisms in drinking water systems. By linking quorum sensing to extracellular polymeric substance production, it reveals how bacteria modulate biofilm resilience, elucidating species-specific mechanisms underlying biofilm resilience to chlorine disinfection. The identification of chlorine-resistant species ( Sphingomonas ursincola , Sphingobium amiense ) and optimal disinfection thresholds (1.0–1.5 mg/L) directly informs municipal water treatment protocols, providing a practical chlorine concentration range (1.0–1.5 mg/L) that effectively controls biofilms while avoiding excessive disinfectant use. These results are pivotal for mitigating secondary contamination risks and safeguarding public health, particularly in aging infrastructure where biofilm-related outbreaks are prevalent.