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[Preprint]. 2025 Jul 30:2025.07.30.25332375.
doi: 10.1101/2025.07.30.25332375.

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

Marjahan Akhtar  1 , Md Ariful Amin  1 , Subah Nuzhat Hussain  1 , Nazia Nazrul Nafsi  1 , Nasrin Parvin  1 , Farhana Khanam  1 , Md Taufiqul Islam  1 , Md Amirul Islam Bhuiyan  1 , Rahima Afroz  1 , Md Golam Firoj  1 , Fahima Chowdhury  1 , Ashraful Islam Khan  1 , Mohammad Jubair  1 , Edward T Ryan  2   3   4 , B Jesse Shapiro  5 , Nicholas R Thomson  6   7 , Eric J Nelson  8 , Md Mustafizur Rahman  1 , Yasmin Ara Begum  1 , Taufiqur Rahman Bhuiyan  1 , Firdausi Qadri  1
Affiliations

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

Marjahan Akhtar et al. medRxiv. .

Update in

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 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.

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Conflict of interest statement

Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1.
Figure 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=3123) collected between January and December 2024. (B) Weekly isolation rate of VCO1 phages from diarrheal specimens (n=1068) 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.
Figure 2.
Figure 2.. Time-lagged correlation analyses of V. cholerae O1-specific phages and cholera cases.
(A) Correlation between the percentage of weekly isolation of V. cholerae O1 phages in diarrheal stool specimens and cholera cases in preceding and subsequent weeks. (B) Correlation between the percentage of weekly detection of V. cholerae O1 phages in environmental wastewater samples and cholera cases in preceding and subsequent weeks. Positive lag weeks represent phage detection preceding cholera cases, while negative lags represent phage detection following cholera cases. Spearman correlation coefficients were calculated for each lag week. The blue horizontal line indicates the 95% significance threshold (p < 0.05), suggesting statistically significant correlations above this line.
Figure 3.
Figure 3.. 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 to December 2024. (C) Correlation between weekly percentage of ETEC and its phages isolated from environmental wastewater specimens. Correlation analyses were performed using the Spearman test.
Figure 4.
Figure 4.. Age-specific burden of Vibrio cholerae O1 bacteria (A) and phages (B) in diarrheal specimens.
For statistical analysis, chi square tests were performed between <5 years age group versus 5–17 years or ≥18 years. ***P<0.0001.
Figure 5.
Figure 5.
Host ranges of phages isolated against Vibrio cholerae, ETEC, Shigella spp., and Salmonella spp.
See this image and copyright information in PMC

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