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

Abstract

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 with 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 support the hypothesis that Vibrio 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 V. cholerae 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 Vibrio 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.

Keywords: ETEC; Salmonella; Shigella; cholera; phages; wastewater.

Fig 1

Temporal dynamics of V. cholerae O1 (VCO1) and corresponding phages in clinical and environmental specimens. (A) Weekly isolation rate of VCO1 in diarrheal specimens (n = 3,123) collected between January and December 2024. (B) Weekly isolation rate of VCO1 phages from diarrheal specimens (n = 1,068) from January to December 2024. (C) Correlation between weekly percentage of cholera cases and phages isolated from diarrheal stools. (D) Correlation between weekly percentage of cholera cases and phages isolated from environmental wastewater specimens (E) Weekly isolation rate of VCO1 phages in environmental wastewater samples collected between July and December 2024. Correlation analyses were performed using the Spearman test.

Fig 2

Time-lagged cross-correlation analyses of V. cholerae O1-specific phages and cholera cases. Correlation between the percentage of (A) V. cholerae O1 phages in diarrheal stool specimens and cholera cases and (B) V. cholerae O1 phages in environmental wastewater samples and cholera cases across a weekly time lag (phage leads). The dashed red lines represent the 95% confidence limits for statistical significance. Correlation values above or below these thresholds indicate significant associations at corresponding lag weeks.

Fig 3

Association of rainfall and temperature with Vibrio cholerae O1 (VCO1) and phages from clinical and environmental sources. (A) Weekly rainfall (mm/day), temperature (°C), and isolation rates of VCO1 and phages from clinical specimens (January–December 2024) and environmental wastewater specimens (July–December 2024) are shown as different lines. (B) Cross-correlation analysis (B) between clinical VCO1 (blue bars) or phages (red bars) and rainfall or temperature across weekly time lags and (C) between environmental VCO1-specific phages (green bars) and rainfall or temperature across weekly time lags. The dashed red lines represent the 95% confidence limits for statistical significance. Correlation values above or below these thresholds indicate significant associations at corresponding lag weeks.

Fig 4

Temporal dynamics of ETEC and corresponding phages in clinical and environmental specimens. (A) Weekly isolation rate of ETEC and ETEC phages from diarrheal specimens collected between January and December 2024. (B) Correlation between the weekly percentage of ETEC and its phages isolated from diarrheal stools. (C) Weekly isolation rate of ETEC and ETEC phages from environmental wastewater specimens collected between July and December 2024. (D) Correlation between the weekly percentage of ETEC and its phages isolated from environmental wastewater specimens. Correlation analyses were performed using the Spearman test.

Fig 5

Age-specific burden of Vibrio cholerae O1 bacteria (A) and phages (B) in diarrheal specimens. For statistical analysis, χ² tests were performed between the <5 year age group versus 5–17 year or ≥18 year groups. ***P < 0.0001.

Fig 6

Host ranges of phages isolated against Vibrio cholerae, ETEC, Shigella spp., and Salmonella spp.