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Objective: Evaluate the effects of five disinfection methods on bacterial concentrations in hospital sink drains, focusing on three opportunistic pathogens (OPs): Serratia marcescens, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. Design: Over two years, three sampling campaigns were conducted in a neonatal intensive care unit (NICU). Samples from 19 sink drains were taken at three time points: before, during, and after disinfection. Bacterial concentration was measured using culture-based and flow cytometry methods. High-throughput short sequence typing was performed to identify the three OPs and assess S. marcescens persistence after disinfection at the genotypic level. Setting: This study was conducted in a pediatric hospitals NICU in Montréal, Canada, which is divided in an intensive and intermediate care side, with individual rooms equipped with a sink. Interventions: Five treatments were compared: self-disinfecting drains, chlorine disinfection, boiling water disinfection, hot tap water flushing, and steam disinfection. Results: This study highlights significant differences in the effectiveness of disinfection methods. Chlorine treatment proved ineffective in reducing bacterial concentration, including the three OPs. In contrast, all other drain interventions resulted in an immediate reduction in culturable bacteria (4–8 log) and intact cells (2–3 log). Thermal methods, particularly boiling water and steam treatments, exhibited superior effectiveness in reducing bacterial loads, including OPs. However, in drains with well-established bacterial biofilms, clonal strains of S. marcescens recolonized the drains after heat treatments. Conclusions: Our study supports thermal disinfection (>80°C) for pathogen reduction in drains but highlights the need for additional trials and the implementation of specific measures to limit biofilm formation. [ABSTRACT FROM AUTHOR]