Isolation of a single Serratia fonticola strain from interstage samples in a drinking water treatment plant: genomic insights and floc-shielding effects during chlorination

Abstract

Ensuring microbial safety in drinking water remains a challenge, particularly for ageing infrastructure and complex treatment processes. Because water treatment works sit at the interface between source-water ecosystem integrity and public-health protection, failures in particle control also have broader environmental-safety implications. Here, we investigated the repeated detection of Serratia fonticola (a coliform) across multiple stages of a water treatment works (WTW). Sixty-seven isolates, obtained from raw, interstage, and final waters, were analysed via whole-genome sequencing. Phylogenomic and average nucleotide identity analyses (>99.94%) confirmed a single, near-clonal strain that is adapted to the WTW and is genetically distinct from raw-water isolates. Variant calling revealed minimal differences, suggesting the organism's repeated detection was not caused by multiple sources but rather a single persistence variant. The variant carried antimicrobial resistance genes, including a FONA-family β-lactamase and blaSFDC, together with plasmid-encoded toxin-antitoxin systems; however, none of the isolates possessed known high-level chlorine-resistance genes. Flow cytometry-based disinfection assays demonstrated that free-floating S. fonticola was rapidly inactivated by modest chlorine doses, achieving a > 3-log reduction with a Ct of around 10 mg·min L⁻¹ . However, floc-associated cells showed markedly higher resilience, requiring a Ct of approximately 100 mg·min L⁻¹ to achieve a ∼2.5-log reduction, indicating strong physicochemical protection of the organism within flocs. These findings suggest that physical shielding, rather than intrinsic chlorine tolerance, facilitates S. fonticola survival through the treatment process. The invariable presence of a specific plasmid in nearly all of the WTW isolates suggests that it may confer traits enhancing resilience to chlorine and promote survival within biofilms. Optimising filtration and minimising floc carry-over, alongside strategies to remove biofilm, may therefore be key to preventing final-water detections of this opportunistic organism.