Environmental Surveillance for Salmonella Typhi and its Association With Typhoid Fever Incidence in India and Malawi.

BACKGROUND: Environmental surveillance (ES) for Salmonella Typhi potentially offers a low-cost tool to identify communities with a high burden of typhoid fever. METHODS: We developed standardized protocols for typhoid ES, including sampling site selection, validation, characterization; grab or trap sample collection, concentration; and quantitative PCR targeting Salmonella genes (ttr, staG, and tviB) and a marker of human fecal contamination (HF183). ES was implemented over 12 months in a historically high typhoid fever incidence setting (Vellore, India) and a lower incidence setting (Blantyre, Malawi) during 2021-2022. RESULTS: S. Typhi prevalence in ES samples was higher in Vellore compared with Blantyre; 39/520 (7.5%; 95% confidence interval [CI], 4.4%-12.4%) vs 11/533 (2.1%; 95% CI, 1.1%-4.0%) in grab and 79/517 (15.3%; 95% CI, 9.8%-23.0%) vs 23/594 (3.9%; 95% CI, 1.9%-7.9%) in trap samples. Detection was clustered by ES site and correlated with site catchment population in Vellore but not Blantyre. Incidence of culture-confirmed typhoid in local hospitals was low during the study and zero some months in Vellore despite S. Typhi detection in ES. CONCLUSIONS: ES describes the prevalence and distribution of S. Typhi even in the absence of typhoid cases and could inform vaccine introduction. Expanded implementation and comparison with clinical and serological surveillance will further establish its public health utility.

Wastewater surveillance for bacterial targets: current challenges and future goals.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs.

Enhanced Biological Trace Organic Contaminant Removal: A Lab-Scale Demonstration with Bisphenol A-Degrading Bacteria Sphingobium sp. BiD32.

Discharge of trace organic contaminants (TOrCs) from wastewater treatment plants (WWTPs) may contribute to deleterious effects on aquatic life. Release to the environment occurs both through WWTP effluent discharge and runoff following land applications of biosolids. This study introduces Enhanced Biological TOrC Removal (EBTCR), which involves continuous bioaugmentation of TOrC-degrading bacteria for improved removal in WWTPs. Influence of bioaugmentation on enhanced degradation was investigated in two lab-scale sequencing batch reactors (SBRs), using bisphenol A (BPA) as the TOrC. The reactors were operated with 8 cycles per day and at two solids retention times (SRTs). Once each day, the test reactor was bioaugmented with Sphingobium sp. BiD32, a documented BPA-degrading culture. After bioaugmentation, BPA degradation (including both the dissolved and sorbed fractions) was 2-4 times higher in the test reactor than in a control reactor. Improved removal persisted for >5 cycles following bioaugmentation. By the last cycle of the day, enhanced BPA removal was lost, although it returned with the next bioaugmentation. A net loss of Sphingobium sp. BiD32 was observed in the reactors, supporting the original hypothesis that continuous bioaugmentation (rather than single-dose bioaugmentation) would be required to improve TOrCs removal during wastewater treatment. This study represents a first demonstration of a biologically based approach for enhanced TOrCs removal that both reduces concentrations in wastewater effluent and prevents transfer to biosolids.

Genomic, Proteomic, and Metabolite Characterization of Gemfibrozil-Degrading Organism Bacillus sp. GeD10.

Gemfibrozil is a widely used hypolipidemic and triglyceride lowering drug. Excess of the drug is excreted and discharged into the environment primarily via wastewater treatment plant effluents. Bacillus sp. GeD10, a gemfibrozil-degrader, was previously isolated from activated sludge. It is the first identified bacterium capable of degrading gemfibrozil. Gemfibrozil degradation by Bacillus sp. GeD10 was here studied through genome sequencing, quantitative proteomics and metabolite analysis. From the bacterial proteome of Bacillus sp. GeD10 1974 proteins were quantified, of which 284 proteins were found to be overabundant by more than 2-fold (FDR corrected p-value ≤0.032, fold change (log2) ≥ 1) in response to gemfibrozil exposure. Metabolomic analysis identified two hydroxylated intermediates as well as a glucuronidated hydroxyl-metabolite of gemfibrozil. Overall, gemfibrozil exposure in Bacillus sp. GeD10 increased the abundance of several enzymes potentially involved in gemfibrozil degradation as well as resulted in the production of several gemfibrozil metabolites. The potential catabolic pathway/modification included ring-hydroxylation preparing the substrate for subsequent ring cleavage by a meta-cleaving enzyme. The identified genes may allow for monitoring of potential gemfibrozil-degrading organisms in situ and increase the understanding of microbial processing of trace level contaminants. This study represents the first omics study on a gemfibrozil-degrading bacterium.

Identification of Putative Genes Involved in Bisphenol A Degradation Using Differential Protein Abundance Analysis of Sphingobium sp. BiD32.

Discharge of the endocrine disrupting compound bisphenol A (BPA) with wastewater treatment plant (WWTP) effluents into surface waters results in deleterious effects on aquatic life. Sphingobium sp. BiD32 was previously isolated from activated sludge based on its ability to degrade BPA. This study investigated BPA metabolism by Sphingobium sp. BiD32 using label-free quantitative proteomics. The genome of Sphingobium sp. BiD32 was sequenced to provide a species-specific platform for optimal protein identification. The bacterial proteomes of Sphingobium sp. BiD32 in the presence and absence of BPA were identified and quantified. A total of 2155 proteins were identified; 1174 of these proteins were quantified, and 184 of these proteins had a statistically significant change in abundance in response to the presence/absence of BPA (p ≤ 0.05). Proteins encoded by genes previously identified to be responsible for protocatechuate degradation were upregulated in the presence of BPA. The analysis of the metabolites from BPA degradation by Sphingobium sp. BiD32 detected a hydroxylated metabolite. A novel p-hydroxybenzoate hydroxylase enzyme detected by proteomics was implicated in the metabolic pathway associated with the detected metabolite. This enzyme is hypothesized to be involved in BPA degradation by Sphingobium sp. BiD32, and may serve as a future genetic marker for BPA degradation.

Development of a computational model to inform environmental surveillance sampling plans for Salmonella enterica serovar Typhi in wastewater.

Typhoid fever-an acute febrile disease caused by infection with the bacterium Salmonella enterica serotype Typhi (S. Typhi)-continues to be a leading cause of global morbidity and mortality, particularly in developing countries with limited access to safe drinking water and adequate sanitation. Environmental surveillance, the process of detecting and enumerating disease-causing agents in wastewater, is a useful tool to monitor the circulation of typhoid fever in endemic regions. The design of environmental surveillance sampling plans and the interpretation of sampling results is complicated by a high degree of uncertainty and variability in factors that affect the final measured pathogens in wastewater samples, such as pathogen travel time through a wastewater network, pathogen dilution, decay and degradation, and laboratory processing methods. Computational models can, to an extent, assist in the design of sampling plans and aid in the evaluation of how different contributing factors affect sampling results. This study presents a computational model combining dynamic and probabilistic modeling techniques to estimate-on a spatial and temporal scale-the approximate probability of detecting S. Typhi within a wastewater system. This model may be utilized to inform environmental surveillance sampling plans and may provide useful insight into selecting appropriate sampling locations and times and interpreting results. A simulated applied modeling scenario is presented to demonstrate the model's functionality for aiding an environmental surveillance study in a typhoid-endemic community.

Development, confirmation, and application of a seeded Escherichia coli process control organism to validate Salmonella enterica serovar Typhi environmental surveillance methods.

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of Typhoid fever. Blood culture is the gold standard for clinical diagnosis, but this is often difficult to employ in resource limited settings. Environmental surveillance of waste-impacted waters is a promising supplement to clinical surveillance, however validating methods is challenging in regions where S. Typhi concentrations are low. To evaluate existing S. Typhi environmental surveillance methods, a novel process control organism (PCO) was created as a biosafe surrogate. Using a previous described qPCR assay, a modified PCR amplicon for the staG gene was cloned into E. coli. We developed a target region that was recognized by the Typhoid primers in addition to a non-coding internal probe sequence. A multiplex qPCR reaction was developed that differentiates between the typhoid and control targets, with no cross-reactivity or inhibition of the two probes. The PCO was shown to mimic S. Typhi in lab-based experiments with concentration methods using primary wastewater: filter cartridge, recirculating Moore swabs, membrane filtration, and differential centrifugation. Across all methods, the PCO seeded at 10 CFU/mL and 100 CFU/mL was detected in 100% of replicates. The PCO is detected at similar quantification cycle (Cq) values across all methods at 10 CFU/mL (Average = 32.4, STDEV = 1.62). The PCO was also seeded into wastewater at collection sites in Vellore (India) and Blantyre (Malawi) where S. Typhi is endemic. All methods tested in both countries were positive for the seeded PCO. The PCO is an effective way to validate performance of environmental surveillance methods targeting S. Typhi in surface water.

Cultivation and characterization of bacterial isolates capable of degrading pharmaceutical and personal care products for improved removal in activated sludge wastewater treatment.

Pharmaceutical and personal care products (PPCPs) discharged with wastewater treatment plant (WWTP) effluents are an emerging surface water quality concern. Biological transformation has been identified as an important removal mechanism during wastewater treatment. The aim of this research was the identification of bacteria with characteristics for potential bioaugmentation to enhance PPCP removal. We report here the cultivation and characterization of bacteria capable of degrading PPCPs to ng/L concentrations. An isolation approach was developed using serial enrichment in mineral medium containing 1 mg/L of an individual PPCP as the sole organic carbon source available to heterotrophs until the original activated sludge inocula was diluted to ~10(-8) of its initial concentration, followed by colony growth on solid R2A agar. Eleven bacteria were isolated, eight that could remove triclosan, bisphenol A, ibuprofen, or 17ß-estradiol to below 10 ng/L, one that could remove gemfibrozil to below 60 ng/L, and two that could remove triclosan or E2, but not to ng/L concentrations. Most bacterial isolates degraded contaminants during early growth when grown utilizing rich carbon sources and were only able to degrade the PPCPs on which they were isolated. Seven of the bacterial isolates were sphingomonads, including all the triclosan and bisphenol A degraders and the ibuprofen degrader. The study results indicate that the isolated bacteria may have a positive influence on removal in WWTPs if present at sufficient concentrations and may be useful for bioaugmentation.

Estimates of the cost to build a stand-alone environmental surveillance system for typhoid in low- and middle-income countries.

The typhoid conjugate vaccine is a safe and effective method for preventing Salmonella enterica serovar Typhi (typhoid) and the WHO's guidance supports its use in locations with ongoing transmission. However, many countries lack a robust clinical surveillance system, making it challenging to determine where to use the vaccine. Environmental surveillance is one alternative approach to identify ongoing transmission, but the cost to implement such a strategy is previously unknown. This paper estimated the cost of setting up and operating an environmental surveillance program for thirteen protocols that are in development, including thirteen cost components and twenty-seven pieces of equipment. Unit costs were obtained from research labs involved in protocol development and equipment information was obtained from manufacturers and the expert opinion of individuals in participating labs. We used Monte Carlo simulations to estimate the costs and the input parameters were modeled as distributions to incorporate the uncertainty. Total costs per sample including setup, overhead, and operational costs, range from $357-794 at a scale of 25 sites to $116-532 at 125 sites. Operational costs (ongoing expenditures) range from $218-584 per sample at a scale of 25 sites to $74-421 at 125 sites. Eleven of the thirteen protocols have operational costs below $200, at this higher scale. Protocols with higher up-front equipment costs benefit more from scale efficiencies and sensitivity analyses show that laboratory labor, processes, and consumables are the primary drivers of uncertainty. At scale, environmental surveillance for typhoid may be affordable (depending on the protocol, scale, and geographic context), though cost will need to be considered alongside future evaluations of test sensitivity. Opportunities to leverage existing infrastructure and multi-disease platforms may be necessary to further reduce costs.

Development and Validation of the Skimmed Milk Pellet Extraction Protocol for SARS-CoV-2 Wastewater Surveillance.

Wastewater surveillance for SARS-CoV-2 may serve as a useful source of data for public health departments as the virus is shed in the stool of infected individuals. However, for wastewater data to be actionable, wastewater must be collected, concentrated, and analyzed in a timely manner. This manuscript presents modifications on a skimmed milk concentration protocol to reduce processing time, increase the number of samples that can be processed at once, and enable use in resource-limited settings. Wastewater seeded with Human coronavirus OC43 (OC43) was concentrated using a skimmed milk flocculation protocol, and then pellets were directly extracted with the QIAamp Viral RNA Mini kit. This protocol has a higher average effective volume assayed (6.35 mL) than skimmed milk concentration methods, with and without Vertrel XF™, which involve resuspension of the pellets in PBS extraction prior to nucleic acid extraction (1.28 mL, 1.44 mL, respectively). OC43 was selected as a recovery control organism because both it and SARS-CoV-2 are enveloped respiratory viruses that primarily infect humans resulting in respiratory symptoms. The OC43 percent recovery for the direct extraction protocol (3.4%) is comparable to that of skimmed milk concentration with and without Vertrel XF™ extraction (4.0%, 2.6%, respectively). When comparing SARS-CoV-2 detection using McNemar's chi-square test, the pellet extraction method is not statistically different from skimmed milk concentration, with and without Vertrel XF™ extraction. This suggests that the method performs equally as well as existing methods. Added benefits include reduced time spent per sample and the ability to process more samples at a single time. Direct extraction of skimmed milk pellets is a viable method for quick turnaround of wastewater data for public health interventions.

Persistence of poliovirus types 2 and 3 in waste-impacted water and sediment.

Eradication of poliovirus (PV) is a global public health priority, and as clinical cases decrease, the role of environmental surveillance becomes more important. Persistence of PV and the environmental factors that influence it (such as temperature and sample type) are an important part of understanding and interpreting positive environmental surveillance samples. The objective of this study was to evaluate the persistence of poliovirus type 2 (PV2) and type 3 (PV3) in wastewater and sediment. Microcosms containing either 1) influent wastewater or 2) influent wastewater with a sediment matrix were seeded with either PV2 or PV3, and stored for up to 126 days at three temperatures (4°C, room temperature [RT], and 30°C). Active PV in the liquid of (1), and the sediment and liquid portions of (2) were sampled and quantified at up to 10 time points via plaque assay and RT-qPCR. A suite of 17 models were tested for best fit to characterize decay of PV2 and PV3 over time and determine the time points at which >90% (T90) and >99% (T99) reduction was reached. Linear models assessed the influence of experimental factors (matrix, temperature, virus type and method of detection) on the predicted T90 and T99 values. Results showed that when T90 was the dependent variable, virus type, matrix, and temperature significantly affected decay, and there was a clear interaction between the sediment matrix and temperature. When T99 was the dependent variable, only temperature and matrix type significantly influenced the decay metric. This study characterizes the persistence of both active and molecular PV2 and PV3 in relevant environmental conditions, and demonstrates that temperature and sediment both play important roles in PV viability. As eradication nears and clinical cases decrease, environmental surveillance and knowledge of PV persistence will play a key role in understanding the silent circulation in endemic countries.

Survey of rapid development of environmental surveillance methods for SARS-CoV-2 detection in wastewater.

Environmental surveillance as a part of wastewater-based epidemiology (WBE) of SARS-CoV-2 can provide an early, cost-effective, unbiased community-level indicator of circulating COVID-19 in a population. The objective of this study was to determine how widely SARS-CoV-2 detection in wastewater is being investigated and what methods are used. A survey was developed and distributed, with results showing that methods were rapidly applied to conduct SARS-CoV-2 WBE, primarily to test wastewater influent from large urban wastewater treatment plants. Additionally, most methods utilized small wastewater volumes and the primary concentration methods used were polyethylene glycol precipitation, membrane filtration and centrifugal ultrafiltration followed by nucleic acid extraction and assay for primarily nucleocapsid gene targets (N1, N2, and/or N3). Since this survey was performed, many laboratories have continued to optimize and implement a variety of methods for SARS-CoV-2 WBE. Method comparison studies completed since this survey was conducted will assist in developing WBE as a supplemental tool to support public health and policy decision making responses.

A comparison of SARS-CoV-2 wastewater concentration methods for environmental surveillance.

Wastewater1 surveillance of SARS-CoV-2 may be a useful supplement to clinical surveillance as it is shed in feces, there are many asymptomatic cases, and diagnostic testing can have capacity limitations and extended time to results. Although numerous studies have utilized wastewater surveillance for SARS-CoV-2, the methods used were developed and/or standardized for other pathogens. This study evaluates multiple methods for concentration and recovery of SARS-CoV-2 and seeded human coronavirus OC43 from municipal primary wastewater and/or sludge from the Greater Seattle Area (March-July 2020). Methods evaluated include the bag-mediated filtration system (BMFS), with and without Vertrel™ extraction, skimmed milk flocculation, with and without Vertrel™ extraction, polyethylene glycol (PEG) precipitation, ultrafiltration, and sludge extraction. Total RNA was extracted from wastewater concentrates and analyzed for SARS-CoV-2 and OC43 with RT-qPCR. Skimmed milk flocculation without Vertrel™ extraction performed consistently over time and between treatment plants in Seattle-area wastewater with the lowest average OC43 Cq value and smallest variability (24.3; 95% CI: 23.8-24.9), most frequent SARS-CoV-2 detection (48.8% of sampling events), and highest average OC43 percent recovery (9.1%; 95% CI: 6.2-11.9%). Skimmed milk flocculation is also beneficial because it is feasible in low-resource settings. While the BMFS had the highest average volume assayed of 11.9 mL (95% CI: 10.7-13.1 mL), the average OC43 percent recovery was low (0.7%; 95% CI: 0.4-1.0%). Ultrafiltration and PEG precipitation had low average OC43 percent recoveries of 1.0% (95% CI: 0.5-1.6%) and 3.2% (95% CI: 1.3-5.1%), respectively. The slopes and efficiency for the SARS-CoV-2 standard curves were not consistent over time, confirming the need to include a standard curve each run rather than using a single curve for multiple plates. Results suggest that the concentration and detection methods used must be validated for the specific water matrix using a recovery control to assess performance over time.

Feasibility of the Bag-Mediated Filtration System for Environmental Surveillance of Poliovirus in Kenya.

The bag-mediated filtration system (BMFS) was developed to facilitate poliovirus (PV) environmental surveillance, a supplement to acute flaccid paralysis surveillance in PV eradication efforts. From April to September 2015, environmental samples were collected from four sites in Nairobi, Kenya, and processed using two collection/concentration methodologies: BMFS (> 3 L filtered) and grab sample (1 L collected; 0.5 L concentrated) with two-phase separation. BMFS and two-phase samples were analyzed for PV by the standard World Health Organization poliovirus isolation algorithm followed by intratypic differentiation. BMFS samples were also analyzed by a cell culture independent real-time reverse transcription polymerase chain reaction (rRT-PCR) and an alternative cell culture method (integrated cell culture-rRT-PCR with PLC/PRF/5, L20B, and BGM cell lines). Sabin polioviruses were detected in a majority of samples using BMFS (37/42) and two-phase separation (32/42). There was statistically more frequent detection of Sabin-like PV type 3 in samples concentrated with BMFS (22/42) than by two-phase separation (14/42, p = 0.035), possibly due to greater effective volume assayed (870 mL vs. 150 mL). Despite this effective volume assayed, there was no statistical difference in Sabin-like PV type 1 and Sabin-like PV type 2 detection between these methods (9/42 vs. 8/42, p = 0.80 and 27/42 vs. 32/42, p = 0.18, respectively). This study demonstrated that BMFS can be used for PV environmental surveillance and established a feasible study design for future research.

Improvement of the Bag-Mediated Filtration System for Sampling Wastewater and Wastewater-Impacted Waters.

Environmental surveillance of poliovirus (PV) plays an important role in the global program for eradication of wild PV. The bag-mediated filtration system (BMFS) was first developed in 2014 and enhances PV surveillance when compared to the two-phase grab method currently recommended by the World Health Organization (WHO). In this study, the BMFS design was improved and tested for its usability in wastewater and wastewater-impacted surface waters in Nairobi, Kenya. Modifications made to the BMFS included the size, color, and shape of the collection bags, the filter housing used, and the device used to elute the samples from the filters. The modified BMFS concentrated 3-10 L down to 10 mL, which resulted in an effective volume assayed (900-3000 mL) that was 6-20 times greater than the effective volume assayed for samples processed by the WHO algorithm (150 mL). The system developed allows for sampling and in-field virus concentration, followed by transportation of the filter for further analysis with simpler logistics than the current methods. This may ultimately reduce the likelihood of false-negative samples by increasing the effective volume assayed compared to samples processed by the WHO algorithm, making the BMFS a valuable sampling system for wastewater and wastewater-impacted surface waters.

Kinetics modeling predicts bioaugmentation with Sphingomonad cultures as a viable technology for enhanced pharmaceutical and personal care products removal during wastewater treatment.

Pharmaceutical and personal care products (PPCPs) discharged with wastewater treatment effluents are a surface water quality concern. PPCPs are partially removed during wastewater treatment and biological transformation is an important removal mechanism. To investigate the potential for enhanced PPCP removal using bioaugmentation, bacteria were previously isolated from activated sludge capable of degrading PPCPs to ng/L concentrations. This study examined the degradation kinetics of triclosan and bisphenol A by five of these bacteria, both in pure culture and when augmented to activated sludge. Sorption coefficients were determined to account for the influence of partitioning during bioremoval. When the bacteria were added to activated sludge, degradation increased. Experimentally determined kinetic parameters were used to model a full-scale continuous treatment process, showing that low biomass could achieve reduced effluent PPCP concentrations. These results demonstrated that bioaugmentation may improve PPCP removal using established wastewater infrastructure under conditions of high solids partitioning.