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, Vibrio cholerae O1 (VCO1), enterotoxigenic Escherichia coli (ETEC), Shigella spp., and Salmonella spp. in diarrheal patients and environmental wastewater specimens collected from six different sites in Dhaka, Bangladesh, in 2024. VCO1, ETEC, Shigella, and Salmonella 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 Shigella, and 9.2% for Salmonella-specific phages. Adults showed a significantly higher burden of VCO1 and corresponding phages compared to children <5 years (P<0.001). Seasonal analysis revealed significant correlations between VCO1 (37.3%) and corresponding phages (57.6%) peaking in late September in both clinical (R = 0.53, P< 0.0001) and environmental wastewater specimens (R = 0.65, P<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 (R = 0.65, P<0.001). Cross-specificity analysis demonstrated that VCO1 phages were highly specific to their targets, whereas ETEC and Shigella phages exhibited broader host ranges, with some Shigella phages capable of infecting ETEC and Salmonella spp. Overall, these findings underscore the potential of bacteriophages as an alternate or adjunctive tool for cholera surveillance.