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.

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.

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.

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.

Next-generation sequencing reveals fecal contamination and potentially pathogenic bacteria in a major inflow river of Taihu Lake.

Taihu Lake is one of the largest freshwater lakes in China and serves as an important source for drinking water. This lake is suffering from eutrophication, cyanobacterial blooms and fecal pollution, and the inflow Tiaoxi River is one of the main contributors. The goal here was to characterize the bacterial community structure of Tiaoxi River water by next-generation sequencing (NGS), paying attention to bacteria that are either fecal-associated or pathogenic, and to examine the relationship between environmental parameters and bacterial community structure. Water samples collected from 15 locations in three seasons, and fecal samples collected from different hosts and wastewater samples were used for bacterial community analysis. The phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Cyanobacteria were predominant in most of the water samples tested. In fecal samples, Bacteroidetes, Firmicutes, and Proteobacteria were abundant, while wastewater samples were dominated by Proteobacteria, Bacteroidetes, Acidobacteria, and Chloroflexi. The cluster analysis and principal coordinate analysis indicated that bacterial community structure was significantly different between water, fecal and sewage samples. Shared OTUs between water samples and chicken, pig, and human fecal samples ranged from 4.5 to 9.8% indicating the presence of avian, pig and human fecal contamination in Tiaoxi River. At genus level, five bacterial genera of fecal origin and sequences of seven potential pathogens were detected in many locations and their presence was correlated well with the land use pattern. The sequencing data revealed that Faecalibacterium could be a potential target for human-associated microbial source-tracking qPCR assays. Our results suggest that pH, conductivity, and temperature were the main environmental factors in shaping the bacterial community based on redundancy analysis. Overall, NGS is a valuable tool for preliminary investigation of environmental samples to identify the potential human health risk, providing specific information about fecal and potentially pathogenic bacteria that can be followed up by specific methods.

Urbanization Impacts the Physicochemical Characteristics and Abundance of Fecal Markers and Bacterial Pathogens in Surface Water.

Urbanization is increasing worldwide and is happening at a rapid rate in China in line with economic development. Urbanization can lead to major changes in freshwater environments through multiple chemical and microbial contaminants. We assessed the impact of urbanization on physicochemical characteristics and microbial loading in canals in Suzhou, a city that has experienced rapid urbanization in recent decades. Nine sampling locations covering three urban intensity classes (high, medium and low) in Suzhou were selected for field studies and three locations in Huangshan (natural reserve) were included as pristine control locations. Water samples were collected for physicochemical, microbiological and molecular analyses. Compared to medium and low urbanization sites, there were statistically significant higher levels of nutrients and total and thermotolerant coliforms (or fecal coliforms) in highly urbanized locations. The effect of urbanization was also apparent in the abundances of human-associated fecal markers and bacterial pathogens in water samples from highly urbanized locations. These results correlated well with land use types and anthropogenic activities at the sampling sites. The overall results indicate that urbanization negatively impacts water quality, providing high levels of nutrients and a microbial load that includes fecal markers and pathogens.

Quantification of Microbial Source Tracking and Pathogenic Bacterial Markers in Water and Sediments of Tiaoxi River (Taihu Watershed).

Taihu Lake is one of the largest freshwater lakes in China, serving as an important source of drinking water; >60% of source water to this lake is provided by the Tiaoxi River. This river faces serious fecal contamination issues, and therefore, a comprehensive investigation to identify the sources of fecal contamination was carried out and is presented here. The performance of existing universal (BacUni and GenBac), human (HF183-Taqman, HF183-SYBR, BacHum, and Hum2), swine (Pig-2-Bac), ruminant (BacCow), and avian (AV4143 and GFD) associated microbial source tracking (MST) markers was evaluated prior to their application in this region. The specificity and sensitivity results indicated that BacUni, HF183-TaqMan, Pig-2-Bac, and GFD assays are the most suitable in identifying human and animal fecal contamination. Therefore, these markers along with marker genes specific to selected bacterial pathogens were quantified in water and sediment samples of the Tiaoxi River, collected from 15 locations over three seasons during 2014 and 2015. Total/universal Bacteroidales markers were detected in all water and sediment samples (mean concentration 6.22 log10 gene copies/100 ml and 6.11 log10 gene copies/gram, respectively), however, the detection of host-associated MST markers varied. Human and avian markers were the most frequently detected in water samples (97 and 89%, respectively), whereas in sediment samples, only human-associated markers were detected more often (86%) than swine (64%) and avian (8.8%) markers. The results indicate that several locations in the Tiaoxi River are heavily polluted by fecal contamination and this correlated well with land use patterns. Among the five bacterial pathogens tested, Shigella spp. and Campylobacter jejuni were the most frequently detected pathogens in water (60% and 62%, respectively) and sediment samples (91% and 53%, respectively). Shiga toxin-producing Escherichia coli (STEC) and pathogenic Leptospira spp. were less frequently detected in water samples (55% and 33%, respectively) and sediment samples (51% and 13%, respectively), whereas E. coli O157:H7 was only detected in sediment samples (11%). Overall, the higher prevalence and concentrations of Campylobacter jejuni, Shigella spp., and STEC, along with the MST marker detection at a number of locations in the Tiaoxi River, indicates poor water quality and a significant human health risk associated with this watercourse. GRAPHICAL ABSTRACTTracking fecal contamination and pathogens in watersheds using molecular methods.