Integrating socio-economic vulnerability factors improves neighborhood-scale wastewater-based epidemiology for public health applications.

Wastewater Based Epidemiology (WBE) of COVID-19 is a low-cost, non-invasive, and inclusive early warning tool for disease spread. Previously studied WBE focused on sampling at wastewater treatment plant scale, limiting the level at which demographic and geographic variations in disease dynamics can be incorporated into the analysis of certain neighborhoods. This study demonstrates the integration of demographic mapping to improve the WBE of COVID-19 and associated post-COVID disease prediction (here kidney disease) at the neighborhood level using machine learning. WBE was conducted at six neighborhoods in Seattle during October 2020 - February 2022. Wastewater processing and RT-qPCR were performed to obtain SARS-CoV-2 RNA concentration. Census data, clinical data of COVID-19, as well as patient data of acute kidney injury (AKI) cases reported during the study period were collected and the distribution across the city was studied using Geographic Information System (GIS) mapping. Further, we analyzed the data set to better understand socioeconomic impacts on disease prevalence of COVID-19 and AKI per neighborhood. The heterogeneity of eleven demographic factors (such as education and age among others) was observed within neighborhoods across the city of Seattle. Dynamics of COVID-19 clinical cases and wastewater SARS-CoV-2 varied across neighborhood with different levels of demographics. Machine learning models trained with data from the earlier stages of the pandemic were able to predict both COVID-19 and AKI incidence in the later stages of the pandemic (Spearman correlation coefficient of 0·546 - 0·904), with the most predictive model trained on the combination of wastewater data and demographics. The integration of demographics strengthened machine learning models' capabilities to predict prevalence of COVID-19, and of AKI as a marker for post-COVID sequelae. Demographic-based WBE presents an effective tool to monitor and manage public health beyond COVID-19 at the neighborhood level.

Wastewater surveillance of SARS-CoV-2 at intra-city level demonstrated high resolution in tracking COVID-19 and calibration using chemical indicators.

Wastewater-based epidemiology has proven to be a supportive tool to better comprehend the dynamics of the COVID-19 pandemic. As the disease moves into endemic stage, the surveillance at wastewater sub-catchments such as pump station and manholes is providing a novel mechanism to examine the reemergence and to take measures that can prevent the spread. However, there is still a lack of understanding when it comes to wastewater-based epidemiology implementation at the smaller intra-city level for better granularity in data, and dilution effect of rain precipitation at pump stations. For this study, grab samples were collected from six areas of Seattle between March-October 2021. These sampling sites comprised five manholes and one pump station with population ranging from 2580 to 39,502 per manhole/pump station. The wastewater samples were analyzed for SARS-CoV-2 RNA concentrations, and we also obtained the daily COVID-19 cases (from individual clinical testing) for each corresponding sewershed, which ranged from 1 to 12 and the daily incidence varied between 3 and 64 per 100,000 of population. Rain precipitation lowered viral RNA levels and sensitivity of viral detection but wastewater total ammonia (NH4+-N) and phosphate (PO43--P) were shown as potential chemical indicators to calibrate/level out the dilution effect. These chemicals showed the potential in improving the wastewater surveillance capacity of COVID-19.

Case Study: Impact of Diurnal Variations and Stormwater Dilution on SARS-CoV-2 RNA Signal Intensity at Neighborhood Scale Wastewater Pumping Stations.

Wastewater based epidemiology (WBE) has emerged as a tool to track the spread of SARS-CoV-2. However, sampling at wastewater treatment plants (WWTPs) cannot identify transmission hotspots within a city. Here, we sought to understand the diurnal variations (24 h) in SARS-CoV-2 RNA titers at the neighborhood level, using pump stations that serve vulnerable communities (e.g., essential workers, more diverse communities). Hourly composite samples were collected from wastewater pump stations located in (i) a residential area and (ii) a shopping district. In the residential area, SARS-CoV-2 RNA concentration (N1, N2, and E assays) varied by up to 42-fold within a 24 h period. The highest viral load was observed between 5 and 7 am, when viral RNA was not diluted by stormwater. Normalizing peak concentrations during this time window with nutrient concentrations (N and P) enabled correcting for rainfall to connect sewage to clinical cases reported in the sewershed. Data from the shopping district pump station were inconsistent, probably due to the fluctuation of customers shopping at the mall. This work indicates pump stations serving the residential area offer a narrow time period of high signal intensity that could improve the sensitivity of WBE, and tracer compounds (N, P concentration) can be used to normalize SARS-CoV-2 signals during rainfall.

Diversity and health risk potentials of the Enterococcus population in tropical coastal water impacted by Hurricane Lane.

Hurricane-caused stormwater runoffs transport diverse terrestrial pollutants, adversely impact microbiological water quality, and introduce fecal and other pathogens to coastal water environments. This study investigated the genotypic diversity, phylogenetic composition, antibiotic resistance patterns, and virulence gene repertoire of the Enterococcus population in the Hilo Bay coastal water after the immediate impact of Hurricane Lane. DNA fingerprinting of Enterococcus isolates exhibited large genotypic diversity, while 16S rRNA gene sequencing identified four major species, including E. faecalis (34.7%), E. faecium (22.4%), E. hirae (22.4%), and E. durans (18.4%). Four common enterococcal virulence genes (cylA, esp, asa1, and gelE) were detected in the Enterococcus population, with significant portions of E. durans (33.3%), E. faecalis (41.2%), E. faecium (36.4%), and E. hirae (27.3%) isolates possessing two or more virulence genes. Considerable antibiotic resistance to rifampin, erythromycin, tetracycline, and nitrofurantoin was detected in the Enterococcus population, with one E. durans isolate showing vancomycin resistance. The results indicate considerable health implications associated with Enterococcus spp. in the hurricane-impacted tropical coastal water, illustrating the needs for more comprehensive understanding of the microbiological risks associated with storm-impacted coastal water.

Fine-Scale Temporal Dynamics of SARS-CoV-2 RNA Abundance in Wastewater during A COVID-19 Lockdown.

Wastewater is a pooled sampling instrument that may provide rapid and even early disease signals in the surveillance of COVID-19 disease at the community level, yet the fine-scale temporal dynamics of SARS-CoV-2 RNA in wastewater remains poorly understood. This study tracked the daily dynamics of SARS-CoV-2 RNA in the wastewater from two wastewater treatment plants (WWTPs) in Honolulu during a rapidly expanding COVID-19 outbreak and a responding four-week lockdown that resulted in a rapid decrease of daily clinical COVID-19 new cases. The wastewater SARS-CoV-2 RNA concentration from both WWTPs, as measured by three quantification assays (N1, N2, and E), exhibited both significant inter-day fluctuations (101.2-105.1 gene copies or GC/L in wastewater liquid fractions, or 101.4-106.2 GC/g in solid fractions) and an overall downward trend over the lockdown period. Strong and significant correlation was observed in measured SARS-CoV-2 RNA concentrations between the solid and liquid wastewater fractions, with the solid fraction containing majority (82.5%-92.5%) of the SARS-CoV-2 RNA mass and the solid-liquid SARS-CoV-2 RNA concentration ratios ranging from 103.6 to 104.3 mL/g. The measured wastewater SARS-CoV-2 RNA concentration was normalized by three endogenous fecal RNA viruses (F+ RNA coliphages Group II and III, and pepper mild mottle virus) to account for variations that may occur during the multi-step wastewater processing and molecular quantification, and the normalized abundance also exhibited similar daily fluctuations and overall downward trend over the sampling period.

Immediate Impact of Hurricane Lane on Microbiological Quality of Coastal Water in Hilo Bay, Hawaii.

Hurricanes and associated stormwater runoff events are expected to greatly impact coastal marine water quality, yet little is known about their immediate effects on microbiological quality of near-shore water. This study sampled Hilo Bay immediately after the impact of Hurricane Lane to understand the spatial and temporal variations of the abundance and diversity of fecal indicator enterococci, common fecal pathogens, and antibiotic resistance genes (ARGs). Water samples from seven sampling sites over 7 days were collected and analyzed, which showed that the overall microbiological water quality parameters [enterococci geometric mean (GM): 6-22 cfu/100 mL] fell within water quality standards and that the temporal dynamics indicated continuing water quality recovery. However, considerable spatial variation was observed, with the most contaminated site exhibiting impaired water quality (GM = 144 cfu/100 mL). The Enterococcus population also showed distinct genotypic composition at the most contaminated site. Although marker genes for typical fecal pathogens (invA for Salmonella, hipO for Campylobacter, mip for Legionella pneumophila, and eaeA for enteropathogenic Escherichia coli) were not detected, various ARGs (ermB, qurS, tetM, blaTEM, and sul1) and integron-associated integrase intI1 were detected at high levels. Understanding the temporal and spatial variation of microbiological water quality at fine granularity is important for balancing economic and recreational uses of coastal water and the protection of public health post the impact of major hurricane events.

Low-dosage ozonation in gas-phase biofilter promotes community diversity and robustness.

BACKGROUND: The ozonation of biofilters is known to alleviate clogging and pressure drop issues while maintaining removal performances in biofiltration systems treating gaseous volatile organic compounds (VOCs). The effects of ozone on the biofilter microbiome in terms of biodiversity, community structure, metabolic abilities, and dominant taxa correlated with performance remain largely unknown. METHODS: This study investigated two biofilters treating high-concentration toluene operating in parallel, with one acting as control and the other exposed to low-dosage (200 mg/m3) ozonation. The microbial community diversity, metabolic rates of different carbon sources, functional predictions, and microbial co-occurrence networks of both communities were examined. RESULTS: Consistently higher biodiversity of over 30% was observed in the microbiome after ozonation, with increased overall metabolic abilities for amino acids and carboxylic acids. The relative abundance of species with reported stress-tolerant and biofilm-forming abilities significantly increased, with a consortium of changes in predicted biological pathways, including shifts in degradation pathways of intermediate compounds, while the correlation of top ASVs and genus with performance indicators showed diversifications in microbiota responsible for toluene degradation. A co-occurrence network of the community showed a decrease in average path distance and average betweenness with ozonation. CONCLUSION: Major degrading species highly correlated with performance shifted after ozonation. Increases in microbial biodiversity, coupled with improvements in metabolizing performances of multiple carbon sources including organic acids could explain the consistent performance commonly seen in the ozonation of biofilters despite the decrease in biomass, while avoiding acid buildup in long-term operation. The increased presence of stress-tolerant microbes in the microbiome coupled with the decentralization of the co-occurrence network suggest that ozonation could not only ameliorate clogging issues but also provide a microbiome more robust to loading shock seen in full-scale biofilters. Video abstract.

A Trace Amount of Surfactants Enables Diffusiophoretic Swimming of Bacteria.

From birth to health, surfactants play an essential role in our lives. Due to the importance, their environmental impacts are well understood. One of the aspects that has been extensively studied is their impact on bacteria, particularly on their motility. Here, we uncover an alternate chemotactic strategy triggered by surfactants-diffusiophoresis. We show that even a trace amount of ionic surfactants, down to a single ppm level, can promote the bacterial diffusiophoresis by boosting the surface charge of the cells. Because diffusiophoresis is driven by the surface-solute interactions, surfactant-enhanced diffusiophoresis is observed regardless of the types of bacteria. Whether Gram-positive or -negative, flagellated or nonflagellated, the surfactants enable fast migration of freely suspended bacteria, suggesting a ubiquitous locomotion mechanism that has been largely overlooked. We also demonstrate the implication of surfactant-enhanced bacterial diffusiophoresis on the rapid formation of biofilms in flow networks, suggesting environmental and biomedical implications.

Fecal indicator bacteria, direct pathogen detection, and microbial community analysis provide different microbiological water quality assessment of a tropical urban marine estuary.

Urban marine estuaries are often impacted by microbiological contamination that impairs use and affects human health acutely, while limited is known about microbiological water quality in urban marine estuaries in the absence of reported sewage spills. This study used a tropical urban marine estuary, the Ala Wai Canal in Honolulu, Hawaii, as the model system to compare fecal indicator bacteria (FIB) concentrations, bacterial pathogen profiles, and microbial community structures. The FIB Escherichia coli exhibited higher geometric mean 132 CFU/100mL (n=28) than those of enterococci (18 CFU/100mL) and Clostridium perfringens (21 CFU/100mL). Amongst the four pathogens targeted by cultivation methods (Salmonella, Campylobacter, Listeria monocytogenes and Vibrio parahaemolyticus), only was V. parahaemolyticus detected and was detected at high frequency. Microbial community analysis through 16S rRNA gene amplicon sequencing also indicated the high prevalence of Vibrio in the water. The pathogen detection patterns and microbial community structure showed no significant correlation with FIB concentration profiles. Together, the results highlight the limitation of using traditional FIB in assessing water microbiological quality in the tropical urban marine estuary environment, indicating the need for more comprehensive microbial risk assessment approaches such as direct detection of pathogens.

Multiplex PCR coupled with direct amplicon sequencing for simultaneous detection of numerous waterborne pathogens.

The current water quality monitoring and regulation approaches use fecal indicator bacteria (FIB) to indirectly assess health risks from fecal pathogens. Direct detection of waterborne pathogens is expected to provide more accurate and comprehensive risk assessment, which however has been hindered by the lack of methods for simultaneous detection of the numerous waterborne pathogens. This study aimed to develop a mPCR-NGS approach that uses the high sequencing depth of NGS and sequence-based detection to significantly increase the multiplex level of mPCR for direct pathogen detection in water. Individual PCR primers were designed for 16 target marker genes of nine different bacterial pathogens, and an optimal combination of primers with least primer complementarities was identified for the multiplex setting. Using an artificial tester sample, the mPCR system was optimized for annealing temperature and primer concentration, and bioinformatic procedures were developed to directly detect the target marker gene amplicons in NGS sequence reads, which showed simultaneous detection of 14 different target genes in one reaction. The effectiveness of the developed mPCR-NGS approach was subsequently demonstrated on DNA extracts from stream water samples and their counterparts that were spiked with various target pathogen DNA, and all target genes spiked into the environmental water samples were successfully detected. Several key issues for further improving the mPCR-NGS approach were also identified and discussed.

Effect of ozone injection on the long-term performance and microbial community structure of a VOCs biofilter.

For biofilters treating waste gases containing volatile organic compounds (VOCs), biomass accumulation is a common problem which will induce bed clogging and significant decrease in VOCs removal efficiency during long-term operation. In this study, ozone injection was developed as a biomass control strategy, and its effects on the biofilter performance and the microbial community structure were investigated in long-term operation. Two biofilters, identified as BF1 and BF2, were operated continuously for 160 days treating gaseous toluene under the same conditions, except that 200 mg/m3 ozone was continuously injected into BF1 during days 45-160. During the operation period, ozone injection did not change the toluene removal efficiency, while the pressure drop of BF1 with ozone injection was significantly lowered compared with BF2. The wet biomass accumulation rate of BF1 was 11 g/m3/hr, which was only 46% of that in BF2. According to the carbon balance result, ozone injection also increased the toluene mineralization rate from 83% to 91%, which could be an important reason for the low biomass accumulation. The PMA-qPCR result indicated that ozone injection increased the microbial viability of the biofilm. The high-throughput sequencing result also revealed that the dominant phyla and genera were not changed significantly by ozone injection, but some ozone-tolerant genera such as Rhodanobacter, Dokdonella and Rhodococcus were enhanced by ozone exposure. All the results verified that ozone injection is capable of sustaining the long-term performance of biofilters by lowering the biomass accumulation, increasing the microbial viability and changing the microbial community structure.

Use of amplicon sequencing to improve sensitivity in PCR-based detection of microbial pathogen in environmental samples.

DNA-based molecular detection of microbial pathogens in complex environments is still plagued by sensitivity, specificity and robustness issues. We propose to address these issues by viewing them as inadvertent consequences of requiring specific and adequate amplification (SAA) of target DNA molecules by current PCR methods. Using the invA gene of Salmonella as the model system, we investigated if next generation sequencing (NGS) can be used to directly detect target sequences in false-negative PCR reaction (PCR-NGS) in order to remove the SAA requirement from PCR. False-negative PCR and qPCR reactions were first created using serial dilutions of laboratory-prepared Salmonella genomic DNA and then analyzed directly by NGS. Target invA sequences were detected in all false-negative PCR and qPCR reactions, which lowered the method detection limits near the theoretical minimum of single gene copy detection. The capability of the PCR-NGS approach in correcting false negativity was further tested and confirmed under more environmentally relevant conditions using Salmonella-spiked stream water and sediment samples. Finally, the PCR-NGS approach was applied to ten urban stream water samples and detected invA sequences in eight samples that would be otherwise deemed Salmonella negative. Analysis of the non-target sequences in the false-negative reactions helped to identify primer dime-like short sequences as the main cause of the false negativity. Together, the results demonstrated that the PCR-NGS approach can significantly improve method sensitivity, correct false-negative detections, and enable sequence-based analysis for failure diagnostics in complex environmental samples.

Ozonation and Depolymerization of Extracellular Polymeric Substances (EPS) Extracted from a Biofilter Treating Gaseous Toluene.

Low-concentration ozonation was developed as a novel technique to control the excess biomass in volatile organic compound (VOC) biofilters. In order to understand the reaction mechanism between ozone and biomass, the changes in properties of ozone exposed extracellular polymeric substances (EPS) were investigated in this study. EPS was sequestered from the biofilm, obtained from a biofilter treating gaseous toluene, and then it was exposed to gaseous ozone at 272 ± 22 ppm continuously for 12 h. The total organic carbon (TOC) results indicated that low concentration ozone could not mineralize the EPS to carbon dioxide (CO2) completely. The excitation-emission matrix fluorescence spectroscopy (EEM) results demonstrated that ozone preferred to attack the benzene ring and specific amino acid residues (such as tryptophan) on the protein chain. High performance size-exclusion chromatography (HPSEC) results confirmed that the protein molecules were depolymerized after ozone attack, while the molecular weight of polysaccharides was not much affected by ozone. During ozonation, few volatile organic compounds (VOCs), such as carboxylic acids, aldehydes, ketones, benzaldehyde and by-products of toluene, were generated, which confirms a minor change in the TOC concentration of EPS. Results revealed that low concentration ozone can reduce the molecular weight of biofilter EPS which can be a key reason for controlling biomass accumulation. Additionally, this can be used to study the composition of biofilm EPS from biofilters.

Investigation of the characteristics of biofilms grown in gas-phase biofilters with and without ozone injection by CLSM technique.

Recently, ozone injection technique was developed as a novel biomass control method to reduce bed clogging in biofilters treating volatile organic compounds (VOCs). However, the effects of ozone on the characteristics of biofilms are still unknown. In this study, two identical lab-scale biofilters treating gaseous toluene were operated in parallel except that one was continuously injected with 200 mg/m(3) ozone. Four glass slides were placed inside each biofilter on day 57 and then were taken out sequentially after 1, 2, 4, and 6 weeks of cultivation. The biofilms grown on the glass slides were stained by the ViaGram™ Red + Bacterial Gram Stain and Viability Kit and observed through the confocal laser scanning microscopy (CLSM). According to the CLSM images of 1, 2, and 4 weeks, the ozonated biofilm was significantly thinner than the control biofilm, which demonstrated that ozone could effectively control the biomass in the biofilter. For the biofilter without ozone injection, the ratios of viable cells (0.51~0.89) and the ratios of Gram-positive bacteria (0.22~0.57) both decreased within 4 weeks of cultivation. The CLSM image analysis results also demonstrated that a continuous injection of 200 mg/m(3) ozone was able to significantly enhance the ratio of viable cells to 0.77~0.97 and allow the dominance of Gram-positive bacteria in the biofilms with the ratio 0.46~0.88 instead of Gram-negative bacteria. For the 6-week samples, the biofilm thickness of the control system was reduced significantly which indicated the detachment of accumulated biofilms might occur in the samples without ozone.

Effect of continuous ozone injection on performance and biomass accumulation of biofilters treating gaseous toluene.

Biofilters treating high-concentration gaseous volatile organic compounds (VOC) can be subject to bed clogging induced by excess biomass accumulation. In this study, O3 was continuously injected into biofilters to control biomass. Its effects on the performance of the biofilters and on biomass accumulation were investigated. Four identical biofilters designed to treat gaseous toluene were operated for 70 days, and three of them were continuously injected with O3 at different concentrations (from 80 to 320 mg/m(3)). The results showed that continuous O3 injection could effectively keep the bed pressure drop stable and had no adverse effect on toluene removal when O3 concentrations were 180-220 mg/m(3). The maximum toluene elimination capacity of the four biofilters was 140 g-toluene/m(3)/h, and the bed pressure drop of the biofilter fed with 180-220 mg/m(3) O3 remained below 3 mmH2O/m throughout the operation period. The biomass accumulation rates of the three biofilters with O3 at 80-320 mg/m(3) were lowered by 0.15-0.25 g/L/day compared with the biofilter without O3. The decreases in biomass accumulation resulted in higher void fractions of the filter beds with O3 injection. Carbon balance analysis indicated that CO2 production had increased while biomass accumulation and leachate waste production decreased in response to O3 injection. Based on the experimental results, it was concluded here that continuous O3 injection can reduce increases in bed pressure effectively, preserve VOC removal capacity, and prevent production of extra leachate waste.

Effect of continuous ozone injection on performance and biomass accumulation of biofilters treating gaseous toluene.

Biofilters treating high-concentration gaseous volatile organic compounds (VOC) can be subject to bed clogging induced by excess biomass accumulation. In this study, O3 was continuously injected into biofilters to control biomass. Its effects on the performance of the biofilters and on biomass accumulation were investigated. Four identical biofilters designed to treat gaseous toluene were operated for 70 days, and three of them were continuously injected with O3 at different concentrations (from 80 to 320 mg/m(3)). The results showed that continuous O3 injection could effectively keep the bed pressure drop stable and had no adverse effect on toluene removal when O3 concentrations were 180-220 mg/m(3). The maximum toluene elimination capacity of the four biofilters was 140 g-toluene/m(3)/h, and the bed pressure drop of the biofilter fed with 180-220 mg/m(3) O3 remained below 3 mmH2O/m throughout the operation period. The biomass accumulation rates of the three biofilters with O3 at 80-320 mg/m(3) were lowered by 0.15-0.25 g/L/day compared with the biofilter without O3. The decreases in biomass accumulation resulted in higher void fractions of the filter beds with O3 injection. Carbon balance analysis indicated that CO2 production had increased while biomass accumulation and leachate waste production decreased in response to O3 injection. Based on the experimental results, it was concluded here that continuous O3 injection can reduce increases in bed pressure effectively, preserve VOC removal capacity, and prevent production of extra leachate waste.