Structured Ethical Review for Wastewater-Based Testing in Support of Public Health.

Wastewater-based testing (WBT) for SARS-CoV-2 has rapidly expanded over the past three years due to its ability to provide a comprehensive measurement of disease prevalence independent of clinical testing. The development and simultaneous application of WBT measured biomarkers for research activities and for the pursuit of public health goals, both areas with well-established ethical frameworks. Currently, WBT practitioners do not employ a standardized ethical review process, introducing the potential for adverse outcomes for WBT professionals and community members. To address this deficiency, an interdisciplinary workshop developed a framework for a structured ethical review of WBT. The workshop employed a consensus approach to create this framework as a set of 11 questions derived from primarily public health guidance. This study retrospectively applied these questions to SARS-CoV-2 monitoring programs covering the emergent phase of the pandemic (3/2020-2/2022 (n = 53)). Of note, 43% of answers highlight a lack of reported information to assess. Therefore, a systematic framework would at a minimum structure the communication of ethical considerations for applications of WBT. Consistent application of an ethical review will also assist in developing a practice of updating approaches and techniques to reflect the concerns held by both those practicing and those being monitored by WBT supported programs.

Composition of soil bacterial communities associated with urban stormwater detention basins and their predicted functional roles in N cycle.

AIMS: Stormwater detention basins serve as vital components in mitigating the adverse effects of urban runoff, and investigating the microbial dynamics within these systems is crucial for enhancing their performance and pollutant removal capabilities. The aim of this study was to examine and compare the soil bacterial communities in two stormwater detention basins located on the Edwards Aquifer in Bexar County, Texas, USA, and evaluate how soil physiochemical properties may affect them. METHODS AND RESULTS: Each basin soil was sampled in two different seasons at varying depths and the structure of microbial communities was examined using paired end Illumina sequencing using V3 and V4 region of 16S rRNA gene. PICRUSt2 was used to predict functional genes in the nitrogen cycle. In addition, soil physicochemical properties such as pH, carbon, nitrogen, and phosphorus and particle size were examined. A beta diversity analysis revealed that basins had distinctive microbial communities. Additionally, soil particle size, phosphorus and ammonia significantly correlated with some of the dominant phyla in the basins. Proteobacteria and Acidobacteria showed a positive correlation with the relative abundances of nitrogen-cycling genes, while Actinobacteria showed a negative correlation. CONCLUSIONS: This study evaluated the associations between soil physicochemical properties and microbial community dynamics in stormwater basins. The study also predicts the relative abundance of nitrogen cycling genes, suggesting shared functional traits within microbial communities. The findings have implications for understanding the potential role of microbial communities in nitrogen cycling processes and contribute to developing sustainable stormwater management strategies and protecting water quality in urban areas.

Evaluation of nitrogen removal, functional gene abundance and microbial community structure in a stormwater detention basin.

Stormwater control measures such as detention basins are used to mitigate the negative effects of urban stormwater resulting from watershed development. In this study, the performance of a detention basin in mitigating nitrogen pollution was examined and the abundance of N-cycling genes (amoA, nirK, nosZ, hzsB and Ntsp-amoA) present in the soil media of the basin was measured using quantitative PCR. Results showed a net export of nitrogen from the basin, however, differences between in- and outflow concentrations were not significant. Furthermore, the quantitative PCR showed that nirK (denitrification gene) was more abundant in the winter season, whereas amoA (nitrification gene) was more abundant in the summer season. The abundance of nirK, Ntsp-amoA and hzsB genes also varied with the sampling depth of soil and based on 16S rRNA gene sequencing of soil samples, Actinobacteria and Proteobacteria were the most dominant phyla. Species diversity appeared higher in summer, while the top and bottom layer of soil clustered separately based on the bacterial community structure. These results underline the importance of understanding nitrogen dynamics and microbial processes within stormwater control measures to enhance their design and performance.

Detection of human fecal pollution in environmental waters using human mitochondrial DNA and correlation with general and human-associated fecal genetic markers.

Human mitochondrial DNA (mtDNA) genetic markers are abundant in sewage and highly human-specific, suggesting a great potential for the environmental application as human fecal pollution indicators. Limited data are available on the occurrence and co-occurrence of human mtDNA with fecal bacterial markers in surface waters, and how the abundance of these markers is influenced by rain events. A 1-year sampling study was conducted in a suburban watershed impacted by human sewage contamination to evaluate the performance of a human mtDNA-based marker along with the bacterial genetic markers for human-associated Bacteroidales (BacHum and HF183) and Escherichia coli. Additionally, the human mtDNA-based assay was correlated with rain events and other markers. The mtDNA marker was detected in 92% of samples (n = 140) with a mean concentration of 2.96 log10 copies/100 ml throughout the study period. Human mtDNA was detected with greater abundance than human-associated Bacteroidales that could be attributed to differences in the decay of these markers in the environment. The abundance of all markers was positively correlated with rain events, and human mtDNA abundance was significantly correlated with various bacterial markers. In general, these results should support future risk assessment for impacted watersheds, particularly those affected by human fecal pollution, by evaluating the performance of these markers during rain events.

Effects of metal oxide nanoparticles on nitrification in wastewater treatment systems: A systematic review.

While the variety of engineered nanoparticles used in consumer products continues to grow, the use of metal oxide nanoparticles in electronics, textiles, cosmetics and food packaging industry has grown exponentially in recent years, which will inevitably result in their release into wastewater streams in turn impacting the important biological processes in wastewater treatment plants. Among these processes, nitrification play a critical role in nitrogen removal during wastewater treatment, however, it is sensitive to a wide range of inhibitory substances including metal oxide nanoparticles. Therefore, it is essential to systematically asses the effects of metal oxide nanoparticles on nitrification in biological wastewater treatment systems. In this review we discuss the present scenario of metal oxide nanoparticles and their impact on biological wastewater treatment processes, specifically nitrogen removal through nitrification. We also summarize the various methods used to measure nitrification inhibition by metal oxide nanoparticles and highlight corresponding results obtained using those methods. Finally, the key research gaps that need to be addressed in future are discussed.

To Assess and Evaluate the Variation of Mandibular Anatomy Using Cone Beam Computed Tomography Before Planning an Implant Surgery: A Population-based Retrospective Study.

AIM: The accurate placement of implants in mandible requires consideration for angulations of the bone along with the vertical dimensions. The aim of this present study was to assess the variation of mandibular anatomy using computed tomography (CT) radiography and to evaluate the effect of presence and absence of teeth on the mandibular anatomy before planning an implant surgery. MATERIALS AND METHODS: The present population-based retrospective study was conducted using 167 digital CT scan images those selected from departmental archives. The samples were sub-divided two groups: group 1included digital CT of edentulous mandible while group 2 included digital CT of edentulous mandible. The axial height, vertical height, and angulations were recorded separately for each group. RESULTS: The results of the present study showed a gradual increase in mandibular angle in both the groups with a statistically significant difference only in the canine-premolar area. The axial height showed a significant difference at canine and first premolar area and a second premolar and the first molar area. However, available height showed a significant difference in canine and first premolar area and distal to the second molar area. When both the sides were compared, no statistically significant difference was observed between right and left sides. CONCLUSION: It was concluded that due to the variability of the mandibular anatomy and because of the effects of various imperative factors, 3D imaging like CT scans should be recommended for safer and secure presurgical planning. CLINICAL SIGNIFICANCE: In surgical osteotomies and implant placement especially in post-extraction sockets, two-dimensional (2D) image of panoramic radiographs should not be considered that reliable as these three-dimensional (3D) imaging radiographs. Therefore CT scans of various angulations and sections must be considered by the clinicians to rationally study the mandibular anatomy and their risk associated areas.

Whole-Community Metagenomics in Two Different Anammox Configurations: Process Performance and Community Structure.

Anaerobic ammonia oxidation (anammox) combined with partial nitritation (PN) is an innovative treatment process for energy-efficient nitrogen removal from wastewater. In this study, we used genome-based metagenomics to investigate the overall community structure and anammox species enriched in suspended growth (SGR) and attached growth packed-bed (AGR) anammox reactors after 220 days of operation. Both reactors removed more than 85% of the total inorganic nitrogen. Metagenomic binning and phylogenetic analysis revealed that two anammox population genomes, affiliated with the genus Candidatus Brocadia, were differentially abundant between the SGR and AGR. Both of the genomes shared an average nucleotide identify of 83%, suggesting the presence of two different species enriched in both of the reactors. Metabolic reconstruction of both population genomes revealed key aspects of their metabolism in comparison to known anammox species. The community composition of both the reactors was also investigated to identify the presence of flanking community members. Metagenomics and 16S rRNA gene amplicon sequencing revealed the dominant flanking community members in both reactors were affiliated with the phyla Anaerolinea, Ignavibacteria, and Proteobacteria. Findings from this research adds two new species, Ca. Brocadia sp. 1 and Ca. Brocadia sp. 2, to the genus Ca. Brocadia and sheds light on their metabolism in engineered ecosystems.

Distribution of human-specific bacteroidales and fecal indicator bacteria in an urban watershed impacted by sewage pollution, determined using RNA- and DNA-based quantitative PCR assays.

The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as "naked DNA" in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.

Impact of Heavy Metals on Transcriptional and Physiological Activity of Nitrifying Bacteria.

Heavy metals can inhibit nitrification, a key process for nitrogen removal in wastewater treatment. The transcriptional responses of amoA, hao, nirK, and norB were measured in conjunction with specific oxygen uptake rate (sOUR) for nitrifying enrichment cultures exposed to different metals (Ni(II), Zn(II), Cd(II), and Pb(II)). There was significant decrease in sOUR with increasing concentrations for Ni(II) (0.03-3 mg/L), Zn(II) (0.1-10 mg/L), and Cd(II) (0.03-1 mg/L) (p < 0.05). However, no considerable changes in sOUR were observed with Pb(II) (1-100 mg/L), except at a dosage of 1000 mg/L causing 84% inhibition. Based on RT-qPCR data, the transcript levels of amoA and hao decreased when exposed to Ni(II) dosages. Slight up-regulation of amoA, hao, and nirK (0.5-1.5-fold) occurred after exposure to 0.3-3 mg/L Zn(II), although their expression decreased for 10 mg/L Zn(II). With the exception of 1000 mg/L Pb(II), stimulation of all genes occurred on Cd(II) and Pb(II) exposure. While overall the results show that RNA-based function-specific assays can be used as potential surrogates for measuring nitrification activity, the degree of inhibition inferred from sOUR and gene transcription is different. We suggest that variations in transcription of functional genes may supplement sOUR based assays as early warning indicators of upsets in nitrification.

Sequencing human mitochondrial hypervariable region II as a molecular fingerprint for environmental waters.

To protect environmental water from human fecal contamination, authorities must be able to unambiguously identify the source of the contamination. Current identification methods focus on tracking fecal bacteria associated with the human gut, but many of these bacterial indicators also thrive in the environment and in other mammalian hosts. Mitochondrial DNA could solve this problem by serving as a human-specific marker for fecal contamination. Here we show that the human mitochondrial hypervariable region II can function as a molecular fingerprint for human contamination in an urban watershed impacted by combined sewer overflows. We present high-throughput sequencing analysis of hypervariable region II for spatial resolution of the contaminated sites and assessment of the population diversity of the impacting regions. We propose that human mitochondrial DNA from public waste streams may serve as a tool for identifying waste sources definitively, analyzing population diversity, and conducting other anthropological investigations.

Engineering bacterial efflux pumps for solar-powered bioremediation of surface waters.

Antibiotics are difficult to selectively remove from surface waters by present treatment methods. Bacterial efflux pumps have evolved the ability to discriminately expel antibiotics and other noxious agents via proton and ATP driven pathways. Here, we describe light-dependent removal of antibiotics by engineering the bacterial efflux pump AcrB into a proteovesicle system. We have created a chimeric protein with the requisite proton motive force by coupling AcrB to the light-driven proton pump Delta-rhodopsin (dR) via a glycophorin A transmembrane domain. This creates a solar powered protein material capable of selectively capturing antibiotics from bulk solutions. Using environmental water and direct sunlight, our AcrB-dR vesicles removed almost twice as much antibiotic as the treatment standard, activated carbon. Altogether, the AcrB-dR system provides an effective means of extracting antibiotics from surface waters as well as potential antibiotic recovery through vesicle solubilization.

A comparative evaluation of marginal fit and microleakage of computer-aided design/computer-aided manufacturing-milled zirconia and prefabricated posterior occlusal veneers: An in vitrostudy.

AIMS: The aim of this study was to compare the marginal fit of prefabricated occlusal veneers with computer-aided design/computer-aided manufacturing (CAD-CAM)-milled zirconia occlusal veneers in the posterior teeth. SETTINGS AND DESIGN: Forty extracted human maxillary premolars were divided into two groups of 20 each. Group 1 was prepared to receive prefabricated occlusal veneers, and Group 2 was prepared to receive CAD-CAM-milled zirconia occlusal veneers. MATERIALS AND METHODS: Prefabricated samples (Edelweiss) were selected for Group 1, whereas for Group 2, the tooth preparations were scanned, and occlusal veneers were fabricated using Exocad designing software and milling machine. After luting, both the groups were submerged in dye, sectioned, and evaluated for marginal fit and microleakage under a stereomicroscope using the microscope imaging software and its measurement tool. STATISTICAL ANALYSIS USED: Data collected were subjected to statistical analysis using SPSS 27.0. Intragroup and intergroup comparison was done using the Mann-Whitney U test. The Chi-square test was applied to check the depth of penetration of dye based on percentages. RESULTS: The marginal gap of zirconia occlusal veneers fabricated with CAD-CAM is higher compared to that of prefabricated occlusal veneers. Similarly, the depth of penetration of dye is higher in CAD-CAM-milled zirconia occlusal veneers than prefabricated occlusal veneers. CONCLUSION: The marginal fit of prefabricated occlusal veneer is better than the marginal fit of zirconia occlusal veneers fabricated with CAD-CAM. Similarly, it can also be concluded that the microleakage of prefabricated occlusal veneer is less compared to the CAD-CAM-milled zirconia occlusal veneers.

Correlative assessment of fecal indicators using human mitochondrial DNA as a direct marker.

Identifying the source of surface water fecal contamination is paramount to mitigating pollution and risk to human health. Fecal bacteria such as E. coli have been staple indicator organisms for over a century, however there remains uncertainty with E. coli-based metrics since these bacteria are abundant in the environment. The relationships between the presence of direct indicator of human waste (human mitochondrial DNA), human-specific Bacteroidales, and E. coli were studied for water samples taken from an urban creek system (Duck Creek Watershed, Cincinnati, OH) impacted by combined sewer overflows. Logistic regression analysis shows that human-specific Bacteroidales correlates much more closely to human mitochondrial DNA (R = 0.62) relative to E. coli (R = 0.33). We also examine the speciation of Bacteroidales within the Duck Creek Watershed using next-generation sequencing technology (Ion Torrent) and show the most numerous populations to be associated with sewage. Here we demonstrate that human-specific Bacteroidales closely follow the dynamics of human mitochondrial DNA concentration changes, indicating that these obligate anaerobes are more accurate than E. coli for fecal source tracking, lending further support to risk overestimation using coliforms, especially fecal coliforms and E. coli.

Water quality performance assessment of two stormwater detention basins located in the recharge zone of a karst aquifer.

Stormwater detention basins are used to minimize peak discharges and improve water quality mainly through sedimentation; however, limited studies have evaluated the water quality performance of detention basins located over karst aquifers. Karst aquifers are vital sources of drinking water for many regions of the world and their recharge areas are susceptible to contamination from surface water resources. In this study, an analysis of two stormwater detention basins (namely, Kyle and TPC) located in the recharge zone of one of the most prolific karst aquifers in the world (Edwards Aquifer, San Antonio, Texas), were conducted over a period of one year to quantify the water quality and hydrologic performance of the basins. Automated samples were collected during the storm events and analyzed for nitrate (NO3--N), nitrite (NO2--N), ammonia (NH3-N), total dissolved nitrogen (TDN), phosphorus (PO43-), total suspended solids (TSS), total dissolved solids (TDS), total carbon (TC), total organic carbon (TOC), and chemical oxygen demand (COD). Both basins reduced NH3-N, TSS and COD concentrations significantly while NO3--N and PO43- concentrations exhibited a net export. Furthermore, TPC showed greater reductions in NO2--N, TOC and TC concentrations compared to Kyle. Higher TSS removal was observed at TPC due to differences in retention time. A volume reduction of 44% and 64% was observed in TPC and Kyle, respectively. The results of this study demonstrate that stormwater detention basins located over the Edwards Aquifer effectively remove particulate pollutants while also being a potential source of dissolved pollutants such as nitrate. Overall, the results presented here have important implications for operation and maintenance of stormwater basins constructed over recharge zones of Edwards Aquifer.

Occurrence and removal of fecal bacteria and microbial source tracking markers in a stormwater detention basin overlying the Edwards Aquifer recharge zone in Texas.

The Edwards Aquifer is the primary water resource for over 2 million people in Texas and faces challenges including fecal contamination of water recharging the aquifer, while effectiveness of best management practices (BMPs) such as detention basins in mitigating fecal pollution remains poorly understood. For this study, the inlet and outlet of a detention basin overlying the aquifer's recharge zone were sampled following storm events using automated samplers. Microbial source tracking and culture-based methods were used to determine the occurrence and removal of fecal genetic markers and fecal coliform bacteria in collected water samples. Markers included E. coli (EC23S857), Enterococcus (Entero1), human (HF183), canine (BacCan), and bird (GFD). Fecal coliforms, EC23S857, and Entero1 were detected following each storm event. GFD was the most frequent host-associated marker detected (91% of samples), followed by BacCan (46%), and HF183 (17%). Wilcoxon signed rank tests indicated significantly lower outlet concentrations for fecal coliforms, EC23S857, and Entero1, but not for HF183, GFD, and BacCan. Higher GFD and BacCan outlet concentrations may be due to factors independent of basin design, such as the non-point source nature of bird fecal contamination and domestic dog care practices in neighborhoods contributing to the basin. Mann-Whitney tests showed marker concentrations were not significantly higher during instances of fecal coliform water quality criterion exceedance, except for E. coli, and that fecal coliform concentrations were not significantly different based on marker detection. Overall, results suggest that the detention basin is effective in attenuating fecal contamination associated with fecal coliforms and the general markers, but not for host-associated markers. Consequently, management efforts should focus on mitigating dog and bird-associated fecal pollution in the study region.

Structured Ethical Review for Wastewater-Based Testing.

Wastewater-based testing (WBT) for SARS-CoV-2 has rapidly expanded over the past three years due to its ability to provide a comprehensive measurement of disease prevalence independent of clinical testing. The development and simultaneous application of the field blurred the boundary between measuring biomarkers for research activities and for pursuit of public health goals, both areas with well-established ethical frameworks. Currently, WBT practitioners do not employ a standardized ethical review process (or associated data management safeguards), introducing the potential for adverse outcomes for WBT professionals and community members. To address this deficiency, an interdisciplinary group developed a framework for a structured ethical review of WBT. The workshop employed a consensus approach to create this framework as a set of 11-questions derived from primarily public health guidance because of the common exemption of wastewater samples to human subject research considerations. This study retrospectively applied the set of questions to peer- reviewed published reports on SARS-CoV-2 monitoring campaigns covering the emergent phase of the pandemic from March 2020 to February 2022 (n=53). Overall, 43% of the responses to the questions were unable to be assessed because of lack of reported information. It is therefore hypothesized that a systematic framework would at a minimum improve the communication of key ethical considerations for the application of WBT. Consistent application of a standardized ethical review will also assist in developing an engaged practice of critically applying and updating approaches and techniques to reflect the concerns held by both those practicing and being monitored by WBT supported campaigns. Synopsis: Development of a structured ethical review facilitates retrospective analysis of published studies and drafted scenarios in the context of wastewater-based testing.

Ritual Revision During a Crisis: The Case of Indian Religious Rituals During the COVID-19 Pandemic.

Rituals, particularly religious rituals, may play a significant role in times of crises. Often, these rituals undergo revision to adapt to the changing needs of the time. This article investigates recent unofficially revised Hindu religious rituals as performed during the COVID-19 pandemic. The multifarious creative interplay between Hindu tradition and change is illustrated through four cases: the religious festival of Durga Puja, the devotional songs or bhajans, the ritual of lighting lamps or diyas, and the fire rituals or havans. The authors offer a systematic discourse analysis of online news articles and YouTube posts that illuminate several aspects of ritual revision during unsettled times. They focus on the changes that were made to ritual elements: who controlled these alterations, how these modifications were made, and what potential benefits these revisions offered to the community of ritual participants. The authors highlight public policy implications regarding the involvement of diverse social actors, the creation of faith in science, the creation of feelings of unity and agency, and the amplification of local ritual modifications on a national scale.

Wastewater surveillance for SARS-CoV-2 to support return to campus: Methodological considerations and data interpretation.

The COVID-19 pandemic has been challenging for various institutions such as school systems due to widespread closures. As schools re-open their campuses to in-person education, there is a need for frequent screening and monitoring of the virus to ensure the safety of students and staff and to limit risk to the surrounding community. Wastewater surveillance (WWS) of SARS-CoV-2 is a rapid and economical approach to determine the extent of COVID-19 in the community. The focus of this review is on the emergence of WWS as a tool for safe return to school campuses, taking into account methodological considerations such as site selection, sample collection and processing, SARS-CoV-2 quantification, and data interpretation. Recently published studies on the implementation of COVID-19 WWS on school and college campuses were reviewed. While there are several logistical and technical challenges, WWS can be used to inform decision-making at the school campus and/or building level.

Assessment of Concentration, Recovery, and Normalization of SARS-CoV-2 RNA from Two Wastewater Treatment Plants in Texas and Correlation with COVID-19 Cases in the Community.

The purpose of this study was to conduct a correlative assessment of SARS-CoV-2 RNA concentrations in wastewater with COVID-19 cases and a systematic evaluation of the effect of using different virus concentration methods and recovery and normalization approaches. We measured SARS-CoV-2 RNA concentrations at two different wastewater treatment plants (WWTPs) in the Bexar County of Texas from October 2020 to May 2021 (32 weeks) using reverse transcription droplet digital PCR (RT-ddPCR). We evaluated three different adsorption-extraction (AE) based virus concentration methods (acidification, addition of MgCl2, or without any pretreatment) using bovine coronavirus (BCoV) as surrogate virus and observed that the direct AE method showed the highest mean recovery. COVID-19 cases were correlated significantly with SARS-CoV-2 N1 concentrations in Salitrillo (ρ = 0.75, p < 0.001) and Martinez II (ρ = 0.68, p < 0.001) WWTPs, but normalizing to a spiked recovery control (BCoV) or a fecal marker (HF183) reduced correlations for both treatment plants. The results generated in this 32-week monitoring study will enable researchers to prioritize the virus recovery method and subsequent correlation studies for wastewater surveillance.

Fecal pollution source characterization in the surface waters of recharge and contributing zones of a karst aquifer using general and host-associated fecal genetic markers.

Fecal pollution of surface waters in the karst-dominated Edwards aquifer is a serious concern as contaminated waters can rapidly transmit to groundwaters, which are used for domestic purposes. Although microbial source tracking (MST) detects sources of fecal pollution, integrating data related to environmental processes (precipitation) and land management practices (septic tanks) with MST can provide better understanding of fecal contamination fluxes to implement effective mitigation strategies. Here, we investigated fecal sources and their spatial origins at recharge and contributing zones of the Edwards aquifer and identified their relationship with nutrients in different environmental/land-use conditions. During March 2019 to March 2020, water samples (n = 295) were collected biweekly from 11 sampling sites across four creeks and analyzed for six physico-chemical parameters and ten fecal indicator bacteria (FIB) and MST-based qPCR assays targeting general (E. coli, Enterococcus, and universal Bacteroidales), human (BacHum and HF183), ruminant (Rum2Bac), cattle (BacCow), canine (BacCan), and avian (Chicken/Duck-Bac and GFD) fecal markers. Among physico-chemical parameters, nitrate-N (NO3-N) concentrations at several sites were higher than estimated national background concentrations for streams. General fecal markers were detected in the majority of water samples, and among host-associated MST markers, GFD, BacCow, and Rum2Bac were more frequently detected than BacCan, BacHum, and HF183, indicating avian and ruminant fecal contamination is a major concern. Cluster analysis results indicated that sampling sites clustered based on precipitation and septic tank density showed significant correlation (p < 0.05) between nutrients and FIB/MST markers, indicating these factors are influencing the spatial and temporal variations of fecal sources. Overall, results emphasize that integration of environmental/land-use data with MST is crucial for a better understanding of nutrient loading and fecal contamination.