Hospital wastewater-based surveillance as an early-warning system for toxigenic Vibrio cholerae: first detection, isolation, characterization, and traceback investigation in Beijing, China

ABSTRACT

Toxigenic Vibrio cholerae (V. cholerae) was first sensitively and rapidly detected in hospital wastewater in Beijing, China, on 12 June 2025. Viable V. cholerae was successfully isolated from the wastewater sample and identified as the O1 Ogawa serotype. Whole-genome sequencing and comparative genomic analysis revealed that the isolate was closely related to locally circulating strains but clearly distinct from the seventh pandemic lineage, suggesting its link to indigenous transmission chains. In addition, we immediately initiated a comprehensive and multi-source traceback investigation upon detection of this positive signal, integrating clinical, environmental, and epidemiological data. This case highlights the early-warning and situational-awareness value of wastewater surveillance in detecting emerging pathogens and emphasizes its indispensable role in infectious disease prevention and control systems, providing a scientific basis for timely public health decision-making.

Wastewater surveillance has rapidly emerged as an innovative, population-level monitoring tool that does not rely on healthcare-seeking behaviours and can systematically capture cryptic infections that escape clinical detection, thereby providing a powerful and timely complement to traditional, case-based surveillance systems [1–3]. Vibrio cholerae (V. cholerae), a Gram-negative, comma-shaped bacterium primarily transmitted through contaminated water and food, including seafood and raw or undercooked products, comprises more than 200 serogroups based on surface O antigens, among which O1 and O139 are responsible for epidemic and pandemic cholera [4–6]. Although infection can result in acute watery diarrhoea and severe dehydration, the majority of V. cholerae infections remain undetected due to asymptomatic or mild clinical presentations or failure to seek medical care [7,8]. These characteristics limit the sensitivity of case-based surveillance and highlight the potential of wastewater-based approaches to reveal otherwise unrecognized community-level circulation of V. cholerae and to strengthen early risk assessment.

With the support of the Chinese Urban Wastewater Surveillance System (CWSS) [9], we have established a city-wide, multi-tiered, and continuous wastewater surveillance in Beijing, covering both centralized wastewater treatment plants and high-risk venues (e.g. large general hospitals), since January 2023. The sampling information is presented in Figure 1A. On 12 June 2025, we for the first time sensitively detected toxigenic V. cholerae nucleic acid positive in wastewater at a hospital surveillance site, with a ctxA gene Ct value of 32.99. We immediately performed urgent recheck testing, targeting the rfb and ctxAB genes of V. cholerae. The results showed that the universal primer of V. cholerae was positive (Ct = 23.27), the O1 rfb gene was positive (Ct = 34.28), and the ctxAB gene was also positive (Ct = 33.96), confirming the presence of toxigenic V. cholerae O1 in the hospital wastewater system.

Figure 1.

The workflow for the first detection, isolation, characterization, and traceback investigation of toxigenic V. cholerae from a hospital wastewater surveillance site. (A) The wastewater surveillance network system of Beijing. (B) wgSNP phylogenetic tree of V. cholerae strains. The tree on the left showed the global phylogeny, while the tree on the right compared it with local strains from recent years. The red fivepointed star marked the first detection of V. cholerae viral strain in wastewater in Beijing. (C) Timeline of actions and outcomes following positive detection of V. cholerae in wastewater: testing, isolation and culture, genetic sequencing, and traceback investigation. (D) Location map of positive detection site and the associated hospital.

Subsequently, we conducted bacterial isolation from the wastewater sample and successfully isolated V. cholerae, and the isolate was designated BJ25VC004. Serological agglutination identified the isolate as the O1 Ogawa serotype. Whole-genome sequencing was performed using both the Illumina NovaSeq PE150 platform (Illumina, USA) and the Oxford Nanopore platform (Oxford, UK). A hybrid genome assembly of the two datasets was generated using Unicycler v0.5.1. We compared the BJ25VC004 genome with V. cholerae strains collected through intestinal pathogen surveillance in Beijing, and further compared it with strains from other provinces in China (including Guangdong, Tianjin, Jiangsu, Zhejiang, and Shanghai) as well as Norway, France, USA and other countries, encompassing representative strains from the seventh cholera pandemic (Figure 1B). A maximum likelihood phylogeny was reconstructed based on the core-genome SNPs (159908) using Snippy (v4.0.2). To clearly illustrate the relationship between this strain and those detected in the local circulation, another tree was constructed (Figure 1B). This tree consisted of V. cholerae strains recently circulating in Beijing, with the root being the representative seventh pandemic strain N16961. This analysis identified a total of 4752 SNPs within the core genome of these strains, with BJ25VC004 differing from other local genomes by 11–24 SNPs. Genome sequencing also revealed that BJ25VC004 genome harboured two tandem copies of the CTX prophage, each carrying the ctxB10 allele, classical rstR_and a variant of the tcpA_ET. In contrast to the seventh pandemic reference strain, neither Vibrio seventh pandemic island I (VSPI) nor VSPII was identified in this isolate, which resembled the genomic sequences obtained from clinical isolates identified in 2021 [10]. In contrast, it was more distantly related to strains from other regions of China and from overseas. All the information suggested that the wastewater-derived isolate was closely linked to indigenous transmission chains rather than representing a novel introduction, and highlighted the value of wastewater genomics for resolving the geographic source and transmission context of detected pathogens.

As cholera is classified as a category A infectious disease in China, we promptly initiated a comprehensive, multi-sectoral traceback investigation following the detection of V. cholerae positive in wastewater (Figure 1C). The surveillance site received wastewater from a tertiary Grade A general hospital with an annual outpatient volume exceeding 2.3 million visits. The wastewater sample was a composited sample of 24 h period before 10:00 AM on 11 June 2025, representing aggregated excreta from a large catchment population over the preceding day. Based on the temporal window, we investigated 28 cases recorded in the hospital’s gastrointestinal clinic from June 9 to 11. Four of these cases had stool samples collected, and all of which tested negative for V. cholerae. For the remaining 24 cases without stool samples, phone inquiries revealed that all patients reported that their symptoms had largely resolved, and their housemates did not exhibit gastrointestinal symptoms such as diarrhoea or vomiting.

During the investigation, an epidemiological survey was conducted for a patient who consulted on June 10 with relatively severe clinical symptoms (self-reported 10 episodes of diarrhoea). The patient stated that she had no travel history out of Beijing in 5 days prior to onset, no suspicious dietary history, and the symptoms were mostly resolved by the time of the investigation, and she refused to provide a sample. We advised her to monitor her health, to seek prompt medical attention if symptoms such as diarrhoea or vomiting recurred, and to remain at home as much as possible in the near term. Concurrently, an expanded investigation of other related departments, such as the department of gastroenterology, also did not identify any suspicious cases. Although no definitive cholera case could be linked to this wastewater positive signal, the investigation demonstrated the feasibility of rapidly moving from wastewater detection to targeted clinical and epidemiological follow-up, thereby narrowing the potential source population within a short time frame.

To further verify the contamination, an additional instantaneous water sample was collected from the site on June 13, which tested negative. Subsequently, routine weekly monitoring showed that nucleic acid testing for toxicogenic V. cholerae remained negative at all surveillance sites, indicating that the measures were timely and effective in preventing widespread transmission of the pathogen. This event exemplifies how hospital wastewater-based surveillance can function as a sensitive early-warning and situational-awareness tool for toxigenic V. cholerae, triggering rapid public health response in the absence of confirmed clinical cases, and underscores its value in strengthening cholera prevention and control systems.

Discussion

V. cholerae is commonly found in wastewater and environmental waters, but its distribution is highly clustered in cholera-endemic regions like South Asia and Africa, where pathogenic serogroups O1/O139 and virulence genes such as ctxA/B are frequently detected [11–13]. Detection in hospital wastewater is rare. To the best of our knowledge, toxigenic V. cholerae has only been reported in hospital wastewater in South Africa [14]. In our study, within a non-endemic area, toxigenic V. cholerae O1 was identified in a single sample from systematic monitoring of 1785 wastewater samples (Figure 1A). This extremely low detection rate, obtained under intensive and systematic surveillance, underscores that capturing such events in non-endemic regions is highly incidental with current approaches and would be very likely missed in routine practice. All samples from other high-risk venues and wastewater treatment plants were negative, further highlighting the crucial importance of targeted monitoring of hospital wastewater for timely detection of such sporadic and atypical pathogens. These findings collectively suggest that large tertiary hospitals, which serve as hubs for patients with acute gastrointestinal symptoms and international travel histories, constitute strategic sentinels within wastewater-based surveillance networks for rare, high-consequence enteric pathogens.

Wastewater sampling in high-risk venues is generally performed biweekly, whereas weekly sampling was adopted in our study. However, during the early phase of pathogen emergence, infections are typically sporadic, and pathogen shedding into the sewer system is limited, resulting in weak and highly transient signals in wastewater. Even with increased sampling frequency, detections under such conditions remain largely stochastic. Our results indicate that conventional grab or composite sampling at discrete time points is intrinsically constrained in its ability to reliably capture low-abundance, short-lived contamination events. These constraints highlight the need for in-sewer real-time monitoring systems equipped with automated sampling and on-site testing capabilities. Continuous 24-h surveillance could substantially enhance detection sensitivity, reduce dependence on chance timing of sampling, and improve capture of transient signals at the very onset of pathogen introduction.

The surveillance point for this study was situated approximately 800 m downstream of the hospital, where it received a mixture of hospital effluent and surrounding community sewage (Figure 1D). The dilution and mixing not only weakened pathogen signals, but also introduced additional sources of contamination that complicated traceback investigations. This configuration reflects a common trade-off in wastewater surveillance between broad population coverage and the ability to localize infection sources. To improve the sensitivity of early detection and enable reliable source tracing of emerging infectious diseases, it is advisable to establish sampling points inside hospitals to monitor untreated wastewater. By shortening transit time and reducing dilution, in-hospital sampling, ideally stratified by ward or functional unit, would allow finer spatiotemporal resolution, facilitate linkage of wastewater signals to specific patient cohorts, and markedly strengthen the interpretability of positive detections.

In addition, real-time data-sharing and feedback mechanisms between wastewater surveillance institutions and hospitals have not yet been established. As a result, positive signals cannot be communicated to medical institutions in a timely manner, preventing clinical systems from conducting coordinated investigations and responses and delaying outbreak management. This fragmentation between environmental and clinical surveillance substantially undermines the translational value of wastewater data, which currently functions more as a retrospective indicator than as an operational trigger for intervention. The source-tracing process in this study further revealed the absence of formal regulations or operational guidelines specifying mandatory follow-up actions after the detection of a positive wastewater signal. This institutional gap leaves subsequent public health responses without an enforceable procedural basis, rendering them effectively non-compulsory. For example, in this case, the patient had the right to refuse sampling, leading to the termination of our traceback investigation. These challenges highlight the urgent need to develop clear governance frameworks, including predefined alert thresholds, standardized risk-communication pathways, and legally supported response protocols that delineate the responsibilities of public health agencies and healthcare facilities when wastewater signals for high-priority pathogens are detected.

In summary, this study demonstrates the sensitivity of hospital wastewater surveillance in detecting emerging or reemerging pathogens, while also revealing some incompleteness in the current wastewater surveillance system. Our findings provide evidence that environmental signals can precede or occur in the absence of confirmed clinical cases, underscoring the potential of hospital wastewater surveillance to serve as an early-warning layer within integrated infectious disease surveillance architectures. With continued advances in analytical technologies and surveillance frameworks, wastewater surveillance is expected to play an increasingly important role in the detection and source-tracing of emerging infectious diseases. Future priorities should include the deployment of real-time monitoring infrastructure, refinement of sampling strategies at critical nodes within hospitals, and the formal integration of wastewater data into routine public health decision-making to realize the promise of this approach for cholera and other enteric pathogens.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Funding Statement

This study was supported by the Beijing Science and Technology Project (241100009024047), Beijing Research Center for Respiratory Infectious Diseases Project (BJRID2025-003), the National Natural Science Foundation of China (22306017), Capital’s Funds for Health Improvement and Research (2024-1G-4361) and the Talent Support Program of Guangdong Provincial Center for Disease Control and Prevention (2024D374).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The sequencing data are available upon reasonable request.

Supplemental Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2026.2653278.