Do Free-Living Amoebae Serve as a Shelter for Helicobacter pylori in Istanbul’s Water Treatment Plants?

Understanding microbial survival mechanisms in aquatic environments is essential for public health. This study investigates the potential role of free-living amoebae (FLA) as environmental shelters for Helicobacter pylori in Istanbul’s potable and wastewater treatment plants. Sampling was conducted during wet (February–March 2024) and dry (May–June 2024) seasons from influent and effluent points across various facilities. Standardized protocols for water collection, filtration, DNA extraction, and sequencing were employed. Metagenomic analysis revealed the presence of Dictyostelium discoideum as the only detected FLA and identified Helicobacter DNA fragments, with an average of 31.8% assigned to H. pylori within this genus. Given that D. discoideum is primarily a soil-dwelling social amoebae rather than a typical aquatic FLA of public health concern, its detection in the water treatment systems likely reflects soil runoff following precipitation events or environmental contamination during sample collection and filtration. However, qPCR and propidium monoazide–droplet digital PCR (PMA-ddPCR)—used to assess bacterial viability—yielded negative results for all water samples. Similarly, axenic culturing and microscopic analysis failed to isolate viable amoebae. Although metagenomic signals suggested prior microbial presence, the absence of viable organisms indicates possible inactivation due to environmental or treatment-related factors. This study is the first to apply PMA-ddPCR for H. pylori detection, offering high specificity for viable cells. The findings underscore a key limitation in using DNA-based assays alone to assess pathogen viability and interspecies interactions. Despite negative results for viable H. pylori and FLA, the study raises concerns about waterborne transmission and highlights the importance of integrating viability-focused techniques. Broader sampling and longitudinal monitoring are recommended to fully assess the risk of amoebae-mediated survival and environmental persistence of pathogenic bacteria like H. pylori. These findings support a One Health approach, linking environmental microbiology to human and animal health risk management in urban water systems.