Clinical and environmental wastewater-based bacteriophage surveillance for high-impact diarrheal diseases, including cholera, in Bangladesh

Bacteriophages (phages) likely play a critical role in modulating transmission dynamics of diarrheal pathogens. This study investigated the role of phages in modulating the prevalence and seasonal patterns of major diarrheal pathogens, <i>Vibrio cholerae</i> O1 (VCO1), enterotoxigenic <i>Escherichia coli</i> (ETEC), <i>Shigella</i> spp., and <i>Salmonella</i> spp. in diarrheal patients and environmental wastewater specimens collected from six different sites in Dhaka, Bangladesh, in 2024. VCO1, ETEC, <i>Shigella</i>, and <i>Salmonella</i> were detected in 10.1%, 7.8%, 1.7%, and 2.4% of diarrheal specimens, respectively. In contrast, phages targeting these pathogens were more frequently isolated, with detection rates of 20% for VCO1, 30% for ETEC, 57% for <i>Shigella</i>, and 9.2% for <i>Salmonella</i>-specific phages. Adults showed a significantly higher burden of VCO1 and corresponding phages compared with children <5 years (<i>P</i> < 0.001). Seasonal analysis revealed significant correlations between VCO1 (37.3%) and corresponding phages (57.6%) peaking in late September in both clinical (<i>r</i> = 0.53, <i>P</i> < 0.0001) and environmental wastewater specimens (<i>r</i> = 0.65, <i>P</i> < 0.001). The highest correlation (r = 0.68) was found between the increased rate of wastewater phages in the preceding week and a rise in cholera cases in the following week. ETEC and ETEC phages isolated from wastewater also showed strong correlations (<i>r</i> = 0.65, <i>P</i> < 0.001). Cross-specificity analysis demonstrated that VCO1 phages were highly specific to their targets, whereas ETEC and <i>Shigella</i> phages exhibited broader host ranges, with some <i>Shigella</i> phages capable of infecting ETEC and <i>Salmonella</i> spp. Overall, these findings support the hypothesis that <i>Vibrio</i> phages could serve as an alternative or complementary tool for cholera surveillance.IMPORTANCEUnderstanding the dynamics between phages and their bacterial hosts is critical for elucidating disease burden; however, their potential for surveillance remains underexplored. To our knowledge, this is the first study that longitudinally investigated major diarrheal pathogens and their phages in both clinical and environmental sources to assess the potential of bacteriophages as a tool to improve diarrheal surveillance. The high frequency of phages compared to the host bacterial counterparts suggests a valuable, yet underutilized, role for phages in surveillance systems. Strong seasonal alignment between <i>V. cholerae</i> O1 and its phages, both peaking in late September, suggests that phage dynamics may reflect pathogen transmission. These preliminary observations raise the possibility that wastewater-derived <i>Vibrio</i> phages could function as early indicators of cholera burden. Future research should aim to explore the complex and poorly understood interactions between phages and their bacterial hosts, particularly how these dynamics shape pathogen populations in endemic settings.