Fecal Indicator Research Articles

Escherichia coli Phylogenetic and Antimicrobial Pattern as an Indicator of Anthropogenic Impact on Threatened Freshwater Mussels.

Freshwater bivalves are widely used as accumulation indicators and monitoring tools for assessing contaminant effects on different levels of biological integration. This pilot study aimed to explore the phylogenetic diversity of Escherichia coli isolated from freshwater mussels (Margaritifera margaritifera and Potomida littoralis) and characterize their phenotypes and antibiotic resistance profiles. Samples were collected in the Rabaçal and Tua Rivers, in the Douro basin, Portugal-two sites representing different levels of anthropogenic contamination. Antimicrobial susceptibility testing was performed via the disk diffusion method with 21 antibiotics. Results showed that 31% of isolates were multidrug-resistant (MDR). Thus, freshwater mussels provide an effective and time-integrated approach for identifying/quantifying fecal indicators, including MDR bacteria. PCR-based assays were designed for assessing phylogenetic E. coli groups. Among the E. coli isolates, the highest prevalence (44%) was observed in group D or E, followed by group E or Clade I (25%), group A (19%), and group B1 (13%). E. coli isolated from M. margaritifera predominantly exhibited a higher prevalence of phylogroups D or E, whereas E. coli from P. littoralis showed associations with phylogroups E or clade I, B1, A, and D or E. Our results provide new insights into the phylogenetic diversity of E. coli in freshwater bivalves. Additionally, the findings highlight the possible linkage of phylogroups with the host species, the geographical location in the water stream, and human activity. Using E. coli as a bioindicator isolated from freshwater mussels helps us grasp how human activities affect the environment. This study has important implications for those interested in safeguarding water resources, especially in tackling antibiotic resistance in aquatic ecosystems.

Fecal Impairment Framework, A New Conceptual Framework for Assessing Fecal Contamination in Recreational Waters.

Fecal pollution of surface water is a pervasive problem that negatively affects waterbodies concerning both public health and ecological functions. Current assessment methods monitor fecal indicator bacteria (FIB) to identify pollution sources using culture-based quantification and microbial source tracking (MST). These types of information assist stakeholders in identifying likely sources of fecal pollution, prioritizing them for remediation, and choosing appropriate best management practices. While both culture-based quantification and MST are useful, they yield different kinds of information, potentially increasing uncertainty in prioritizing sources for management. This study presents a conceptual framework that takes separate human health risk estimates based on measured MST and E. coli concentrations as inputs and produces an estimate of the overall fecal impairment risk as its output. The proposed framework is intended to serve as a supplemental screening tool for existing monitoring programs to aid in identifying and prioritizing sites for remediation. In this study, we evaluated the framework by applying it to two primarily agricultural watersheds and several freshwater recreational beaches using existing routine monitoring data. Based on a combination of E. coli and MST results, the proposed fecal impairment framework identified four sites in the watersheds as candidates for remediation and identified temporal trends in the beach application. As these case studies demonstrate, the proposed fecal impairment framework is an easy-to-use and cost-effective supplemental screening tool that provides actionable information to managers using existing routine monitoring data, without requiring specialized expertize.

Analysis of Trace Mineral Elements and Microorganisms in Two Hot Springs in Jazan Region of Saudi Arabia

Thermal springs have hydrothermal or volcanic activity, which causes the water to boil up and dissolve any metal that may be present there. It helps in recovery to reduce stress, promote sleep, boost blood circulation, relieve pain and heal skin problems. However, they consist of some heavy and trace metals and some non-metals, such as Mg, Mo, Na, As, Hg, Cd, Ni, Fe, Cu, Mn, Zn, Co, Ti, Cr and Pb are present. These hot springs are also known to exhibit natural microbiota, which is helpful in maintaining the public health and preventing consumer outbreaks with the advancement of research, microbiological monitoring of hot spring water can be done by utilizing high throughput sequencing. Taking these facts in concern, the present work, hot spring samples were collected from different locations of Al-Harth and Alarda hot springs, Jazan region, Saudi Arabia. The samples collected were analyzed for trace minerals and microbiological activity. These samples were found to exhibit the temperature range from 60 to 70 ºC and an average pH of 6.5 to 8.5. Almost all the samples had shown the presence of trace elements with electrical conductivity and total dissolved solids (TDS) ranged from 0.02 to 0.045 dms and 27 to 43 mg/L, respectively. Scattergun metagenomic analysis was performed to determine the microbiological content. The preliminary study reports the microbiome (bacteria, archea and viruses) of alarath hot spring water employing whole-genome high-throughput sequencing and bioinformatics. Bacterial species detected were members of the phyla 64% Proteobacteria, 20% Actinobacteria, 6.3% unclassified, 2.2% Deinococcus thermus, 1.4 Firmcutes and 1.6% others. Fecal indicator bacteria, i.e. Escherichia coli or enterococci, were not detected in any samples examined in this study. Both archaeal DNA (0.4%) and viral DNA (0.009%) were found.

Assessment of seasonality and normalization techniques for wastewater-based surveillance in Ontario, Canada.

Wastewater-based surveillance is at the forefront of monitoring for community prevalence of COVID-19, however, continued uncertainty exists regarding the use of fecal indicators for normalization of the SARS-CoV-2 virus in wastewater. Using three communities in Ontario, sampled from 2021-2023, the seasonality of a viral fecal indicator (pepper mild mottle virus, PMMoV) and the utility of normalization of data to improve correlations with clinical cases was examined. Wastewater samples from Warden, the Humber Air Management Facility (AMF), and Kitchener were analyzed for SARS-CoV-2, PMMoV, and crAssphage. The seasonality of PMMoV and flow rates were examined and compared by Season-Trend-Loess decomposition analysis. The effects of normalization using PMMoV, crAssphage, and flow rates were analyzed by comparing the correlations to clinical cases by episode date (CBED) during 2021. Seasonal analysis demonstrated that PMMoV had similar trends at Humber AMF and Kitchener with peaks in January and April 2022 and low concentrations (troughs) in the summer months. Warden had similar trends but was more sporadic between the peaks and troughs for PMMoV concentrations. Flow demonstrated similar trends but was not correlated to PMMoV concentrations at Humber AMF and was very weak at Kitchener (r = 0.12). Despite the differences among the sewersheds, unnormalized SARS-CoV-2 (raw N1-N2) concentration in wastewater (n = 99-191) was strongly correlated to the CBED in the communities (r = 0.620-0.854) during 2021. Additionally, normalization with PMMoV did not improve the correlations at Warden and significantly reduced the correlations at Humber AMF and Kitchener. Flow normalization (n = 99-191) at Humber AMF and Kitchener and crAssphage normalization (n = 29-57) correlations at all three sites were not significantly different from raw N1-N2 correlations with CBED. Differences in seasonal trends in viral biomarkers caused by differences in sewershed characteristics (flow, input, etc.) may play a role in determining how effective normalization may be for improving correlations (or not). This study highlights the importance of assessing the influence of viral fecal indicators on normalized SARS-CoV-2 or other viruses of concern. Fecal indicators used to normalize the target of interest may help or hinder establishing trends with clinical outcomes of interest in wastewater-based surveillance and needs to be considered carefully across seasons and sites.

Novel photoelectrochemical 3D-system for water disinfection by deposition of modified carbon nitride on vitreous carbon foam

Graphitic carbon nitride (GCN) is an optical semiconductor with excellent photoactivity under visible light irradiation. It has been widely applied for organic micropollutant removal from contaminated water, and less investigated for microorganisms’ inactivation. The photocatalytic degradation mechanism using GCN is attributed to a series of reactions with reactive oxygen species and photogenerated holes that can be boosted by modifying its physical-chemical structure. This work reports a successful improvement of the overall photocatalytic and electrocatalytic activities of the pristine material by thermal and chemical modification by a copolymerisation synthesis method. The copolymerisation of dicyandiamide as a precursor with barbituric acid strongly reduced photoluminescence due to the enhanced charge separation thus improving the catalyst efficiency under visible light irradiation. The material with 1.6 wt% of barbituric acid showed the best photocatalytic performance and electrochemical properties. This photocatalyst was selected for immobilisation on a conductive carbon foam, which promotes a higher electrochemical active surface area and enhanced mass transfer. This three-dimensional metal-free electrode was employed for the photoelectrochemical inactivation of two different microorganisms, Escherichia coli, and Enterococcus faecalis, obtaining removals below the detection limit after 30 min in simulated faecal-contaminated waters. This photoelectrochemical reactor was also applied to treat polluted river and urban waste waters, and the faecal contamination indicators were vastly reduced to values below the detection limit in 60 min in both cases, showing the wide applicability of this innovative photoelectrode for different types of polluted aqueous matrices.

Global microbial water quality data and predictive analytics: Key to health and meeting SDG 6

Microbial water quality is an integral to water security and is directly linked to human health, food safety, and ecosystem services. However, specifically pathogen data and even faecal indicator data (e.g., E. coli), are sparse and scattered, and their availability in different water bodies (e.g., groundwater) and in different socio-economic contexts (e.g., low- and middle-income countries) are inequitable. There is an urgent need to assess and collate microbial data across the world to evaluate the global state of ambient water quality, water treatment, and health risk, as time is running out to meet Sustainable Development Goal (SDG) 6 by 2030. The overall goal of this paper is to illustrate the need and advocate for building a robust and useful microbial water quality database and consortium worldwide that will help achieve SDG 6. We summarize available data and existing databases on microbial water quality, discuss methods for producing new data on microbial water quality, and identify models and analytical tools that utilize microbial data to support decision making. This review identified global datasets (7 databases), and regional datasets for Africa (3 databases), Australia/New Zealand (6 databases), Asia (3 databases), Europe (7 databases), North America (12 databases) and South America (1 database). Data are missing for low- and middle-income countries. Increased laboratory capacity (due to COVID-19 pandemic) and molecular tools can identify potential pollution sources and monitor directly for pathogens. Models and analytical tools can support microbial water quality assessment by making geospatial and temporal inferences where data are lacking. A genomics, information technology (IT), and data revolution is upon us and presents unprecedented opportunities to develop software and devices for real-time logging, automated analysis, standardization, and modelling of microbial data to strengthen knowledge of global water quality. These opportunities should be leveraged for achieving SDG 6 around the world.

How clean is your ice machine? Revealing microbial amplification and presence of opportunistic pathogens in hospital ice-water machines

Ice machines in healthcare facilities have been suspected and even linked to outbreaks and pseudo-outbreaks. Guidelines exist for maintenance of these devices but there is no clear independent infection control standard, and little is known on their microbial contamination. To evaluate the microbial contamination, amplification, and presence of opportunistic pathogens in ice-water machines in a healthcare facility. Concentrations of general microbial indicators (heterotrophic plate counts (HPC), total and intact cells), faecal indicators (enterococci) and opportunistic pathogens (Pseudomonas aeruginosa, nontuberculous mycobacteria (NTM), Candida spp.) were measured in 36 ice-water machines on patient wards of a 772-bed hospital. Profile sampling was performed on 5 ice-water machines and adjacent faucets to identify sites of microbial proliferation. Candida spp. were found in half of ice-water samples while enterococci and P. aeruginosa were present in 6 and 11 drain inlets respectively. NTM were measured in all ice-water samples and 35/36 biofilms. Pre-filters and ice machines are sites for additional amplification: NTM densities were on average 1.3 log higher in water of ice machine flushed 5 min compared to flushed adjacent tap water. Ice machines design needs to be adapted to reduce microbial proliferation. The absence of correlation between HPC densities (current microbial indicators) and NTM concentrations suggests a need for cleaning efficiency indicators better correlated with opportunistic pathogens. Cleaning and disinfection guidelines of ice machines in healthcare facilities need to be improved, especially when ice is given to the most vulnerable patients, and NTM may be an efficiency indicator.

Persistence of pathogens and bacterial community dynamics in tropical soil after application of raw sewage

The objective of this work was to evaluate the persistence of faecal indicators and pathogenic organisms (Salmonella spp., Escherichia coli and viable helminth eggs) and the structure/diversity of bacterial communities in soil receiving raw sewage (RS) for an extended period of application (3 uninterrupted years). In the experimental design, three treatments were defined: (1) Control soil, characterized by the analysis of a composite sample collected in an area of similar soil, but not a recipient of RS (TSC); (2) Soil receiving conventional mineral fertilization, and furrow irrigation with supply water (TW); and (3) Fertirrigated soil with RS applied by furrows (TF). The results of persistence of pathogenic organisms and indicators in TF indicated a sanitary quality similar to the control soil (TSC), thus potentially bringing low risks of contamination with pathogens present in the soil. The presence of viable helminth eggs was not identified in any treatment studied, because of its low concentration in the raw sewage of the studied system. The TW, TF and TSC treatments had 34.8% of bacterial diversity in common. The bacterial composition of the soil showed a predominance of the Proteobacteria phylum in all treatments studied; however, TF was the one with the highest relative abundance of this phylum (44.8%).

Gastropods as a source for fecal indicator bacteria in drinking water

Microbial water quality is routinely examined using the fecal indicator bacteria Escherichia coli, coliform bacteria and enterococci. Several practical cases in German drinking water distribution systems indicated invertebrates such as insects or gastropods as a source for the microbiological deterioration. Therefore, we examined three genera of Gastropoda (Arion, Helix and Cepaea) for the presence of fecal indictor bacteria in excreta using standard methods. Enterococci and coliform bacteria were detected in high concentrations (mean values of 1.5 × 106 and 6.3 × 106 per gram feces, respectively). E. coli was also detected, still specification revealed that what was assigned by standard ISO-methods to be E. coli was indeed a novel species of Buttiauxella, exhibiting β-D-glucuronidase activity, thus, explaining the false-positive results. Microbiome analyses confirmed the cultural results. Enterobacteriaceae were dominant in the samples, yet only very few sequences could be assigned to Escherichia. Our study suggests, that enterococci and coliform bacteria are an integral component in the gastropod microbiome, whereas E. coli might be derived from other sources with gastropods being a vector. The results further indicate, that the current concept of fecal indicator bacteria needs to be extended, as not only humans and homeothermic animals could be a source for fecal indictor bacteria, but also gastropods need to be taken into consideration.

Monitoring Intertidal Habitats for Effects from Biosolids Applications onto an Adjacent Forestry Plantation

Stabilised organic solids derived from sewage sludge (“biosolids”) are applied to land as an alternative to disposal as landfill. This study evaluated the long-term effects of biosolids applied to forestry plantations on the adjacent intertidal habitats of Rabbit Island (New Zealand). On this island, biosolids are applied to enhance the growth of trees (Pinus radiata). Shoreline topography, macroalgal cover, sediment grain size, the concentrations of nutrients, trace metals, and faecal indicator bacteria, and benthic infaunal communities were studied in 2008, 2014, and 2019 at twelve intertidal transect sites (four “reference” and eight “application”) adjacent to forestry blocks where biosolids have been applied over a period of 24 years. The sediment composition did not differ significantly between the survey years or between the reference and application sites. Total nitrogen concentrations in the sediments increased over time at some transects, but such increases were not consistent among the application transects. No symptoms of excessive algal growth, sediment anoxia, and hydrogen sulphide odours were observed at most sites. Key infaunal taxa were similar between the reference and application transects. Overall, no long-term adverse changes to intertidal habitats attributed to biosolids application were detected between the reference and application sites. This study shows that biosolids application can co-occur without detectable adverse effects on nearby intertidal environments. In a global context of rising concern over climate change, environmental pollution, and resource scarcity, forest fertilisation with biosolids can facilitate biomass production and soil development while protecting valued coastal ecosystems.

Role of suspended solids on the co-precipitation of pathogenic indicators and antibiotic resistance genes with struvite from digested swine wastewater

Struvite recovered from wastewater contains high concentration of fecal indicator bacteria (FIB), porcine adenoviruses (PAdV) and antibiotic resistance genes (ARGs), becoming potential resources of these microbial hazards. Understanding the precipitation behavior of pathogenic indicators and ARGs with suspended solids (SS) will provide the possible strategy for the control of co-precipitation. In this study, SS was divided into high-density SS (separated by centrifugation) and low-density SS (further separated by filtration), and the role of SS on the co-precipitation of FIB, PAdV and ARGs was investigated. The distribution analysis showed that 35.5–73.0% FIB, 79.6% PAdV and 64.5–94.8% ARGs existed in high-density SS, while the corresponding values were 26.9–64.4%, 11.7% and 3.5–24.3% in low-density SS. During struvite generation, 82.7−96.9% FIB, 75.5% PAdV and 56.3–86.5% ARGs were co-precipitated into struvite. High-density SS contributed 20.7−68.5% FIB, 63.9% PAdV and 38.7–87.2% ARGs co-precipitation, and the corresponding contribution of low-density SS was 31.4−79.2%, 3.9% and 6.2–54.7%. Moreover, the precipitated SS in struvite obviously decreased inactivation efficiency of FIB and ARGs in drying process. These results provide a potential way to control the co-precipitation and inactivation of FIB, PAdV and ARGs in struvite through removing high-density SS prior to struvite recovery.

Pathogens and Fecal Indicators in Stormwater

Pathogens and Fecal Indicators in Stormwater

Performance of bacterial and mitochondrial qPCR source tracking methods: A European multi-center study

With the advent of molecular biology diagnostics, different quantitative PCR assays have been developed for use in Source Tracking (ST), with none of them showing 100% specificity and sensitivity. Most studies have been conducted at a regional level and mainly in fecal slurry rather than in animal wastewater. The use of a single molecular assay has most often proven to fall short in discriminating with precision the sources of fecal contamination. This work is a multicenter European ST study to compare bacterial and mitochondrial molecular assays and was set to evaluate the efficiency of nine previously described qPCR assays targeting human-, cow/ruminant-, pig-, and poultry-associated fecal contamination. The study was conducted in five European countries with seven fecal indicators and nine ST assays being evaluated in a total of 77 samples. Animal fecal slurry samples and human and non-human wastewater samples were analyzed. Fecal indicators measured by culture and qPCR were generally ubiquitous in the samples. The ST qPCR markers performed at high levels in terms of quantitative sensitivity and specificity demonstrating large geographical application. Sensitivity varied between 73% (PLBif) and 100% for the majority of the tested markers. On the other hand, specificity ranged from 53% (CWMit) and 97% (BacR). Animal-associated ST qPCR markers were generally detected in concentrations greater than those found for the respective human-associated qPCR markers, with mean concentration for the Bacteroides qPCR markers varying between 8.74 and 7.22 log10 GC/10 mL for the pig and human markers, respectively. Bacteroides spp. and mitochondrial DNA qPCR markers generally presented higher Spearman's rank coefficient in the pooled fecal samples tested, particularly the human fecal markers with a coefficient of 0.79. The evaluation of the performance of Bacteroides spp., mitochondrial DNA and Bifidobacterium spp. ST qPCR markers support advanced pollution monitoring of impaired aquatic environments, aiming to elaborate strategies for target-oriented water quality management.

Https://www.onlinescientificresearch.com/articles/new-discoveries-in-number-theory-and-its-practical-application.pdf

Assessment of faecal contamination of water is a key factor in assessing the quality and safety of food. It also reveals the level of hygiene adopted by food handlers in the course of preparation and serving of such foods. This study assessed the risk of microbial contamination of water used by food vendors in Owerri munipality, Imo State Nigeria between January-December 2019 with an emphasis on total coliforms and the faecal coliforms/ faecal indicator bacteria (FIB) – Escherichia coli (E. coli) and thermotolerant coliforms (TTC). One hundred and thirty water samples collected across three vending types (street vendors, high level and low level restaurants) were evaluated for microbial contamination using standard methods. Data obtained was statistically analyzed with p-value set at 0.05 significance level. The result identified Enterobacter spp. and Klebsiella spp. The result showed that 48.46% (63) of the 130 water samples examined were contaminated with coliform bacteria, of which 20 (22.22%) samples had faecal coliform bacteria, representing 10.77% of the entire water samples examined. Of the 64 samples from water used for food preparation, 12.50% (8) had faecal coliform bacteria as against 9.09% (6) of 66 samples collected from water offered to customers for drinking. The result showed that 116 (89.23%) of the 130 water samples recorded no faecal coliform contamination, hence they were considered excellent based on the guidelines for determination of faecal contamination of water. Two samples had faecal coliform counts of 1-3 per 100 ml of water accounting for 1.54% of water samples, which is considered Satisfactory. The result revealed suspicious (4-9 per 100 ml) coliform count in 9 (6.92%) water samples, while three water samples (2.31%) had ≥10 count and are classified as unsatisfactory. The result revealed that 89.23% (116) of the examined water samples were of ‘no risk’ to the public, which was significantly higher than 8.46% (11) and 2.31% (3) recognized as being of low and intermediate risk respectively (p <0.05). High and very high risk water samples were not encountered in the study. This trend in qualification of risk of water samples reflected in the two types of water usages, and the observed differences was however not statistically significant (p>0.05).

Comparison of Cefotaxime-Resistant Escherichia coli and sul1 and intI1 by qPCR for Monitoring of Antibiotic Resistance of Wastewater, Surface Water, and Recycled Water.

Awareness of the need for surveillance of antimicrobial resistance (AMR) in water environments is growing, but there is uncertainty regarding appropriate monitoring targets. Adapting culture-based fecal indicator monitoring to include antibiotics in the media provides a potentially low-tech and accessible option, while quantitative polymerase chain reaction (qPCR) targeting key genes of interest provides a broad, quantitative measure across the microbial community. The purpose of this study was to compare findings obtained from the culture of cefotaxime-resistant (cefR) Escherichia coli with two qPCR methods for quantification of antibiotic resistance genes across wastewater, recycled water, and surface waters. The culture method was a modification of US EPA Method 1603 for E. coli, in which cefotaxime is included in the medium to capture cefR strains, while qPCR methods quantified sul1 and intI1. A common standard operating procedure for each target was applied to samples collected by six water utilities across the United States and processed by two laboratories. The methods performed consistently, and all three measures reflected the same overarching trends across water types. The qPCR detection of sul1 yielded the widest dynamic range of measurement as an AMR indicator (7-log versus 3.5-log for cefR E. coli), while intI1 was the most frequently detected target (99% versus 96.5% and 50.8% for sul1 and cefR E. coli, respectively). All methods produced comparable measurements between labs (p < 0.05, Kruskal-Wallis). Further study is needed to consider how relevant each measure is to capturing hot spots for the evolution and dissemination of AMR in the environment and as indicators of AMR-associated human health risk.

Quantitative microbial risk assessment of the gastrointestinal risks to swimmers at Southeast Asian urban beaches using site-specific and combined autochthonous and fecal bacteria exposure data

Recreational exposure to microbial pollution at urban beaches poses a health risk to beachgoers. The accurate quantification of such risks is crucial in managing beaches effectively and establishing warning guidelines. In this study, we employed a quantitative microbial risk assessment (QMRA) framework to assess marine water quality and estimate the risks associated with Vibrio parahaemolyticus, an autochthonous pathogen that causes gastrointestinal illnesses, and enterococci, a traditional fecal bacteria indicator. The microbial contamination levels of V. parahaemolyticus and enterococci were determined from 48 water samples collected at two beaches in Thailand during dry and wet seasons. The accidentally ingested water volumes were obtained through a survey involving 438 respondents. The probability of illness (Pill) was estimated using dose–response models and Monte Carlo simulation. The results revealed that enterococci posed a higher risk of illness than V. parahaemolyticus at all seven study sites. The median combined gastrointestinal (GI) risk from both bacteria at all sites met the US EPA risk benchmark of 0.036 and the 0.05 benchmark set by the WHO, but the 95th percentile risk data at all sites exceeded the benchmarks. This emphasizes the need for the continuous monitoring and management of microbial pollution at these sites. The site-specific exposure data showed higher estimated risks with increased variations compared to the WHO-referenced values, which highlights the significance of locally measured microbial concentrations and survey exposure data to avoid underestimation. Estimating the risks from recreational exposure to waterborne bacteria can inform beach management policies aimed at reducing public health risks to swimmers. The study findings improve the understanding of the risks associated with water recreation activities at Southeast Asian beaches and offer valuable insights for the development of water quality guidelines, which are crucial for the sustainable development of the blue economy.

High-Throughput Microfluidic Quantitative PCR Platform for the Simultaneous Quantification of Pathogens, Fecal Indicator Bacteria, and Microbial Source Tracking Markers.

Contamination of water with bacterial, viral, and protozoan pathogens can cause human diseases. Both humans and nonhumans can release these pathogens through their feces. To identify the sources of fecal contamination in the water environment, microbial source tracking (MST) approaches have been developed; however, the relationship between MST markers and pathogens is still not well understood most likely due to the lack of comprehensive datasets of pathogens and MST marker concentrations. In this study, we developed a novel microfluidic quantitative PCR (MFQPCR) platform for the simultaneous quantification of 37 previously validated MST markers, two fecal indicator bacteria (FIB), 22 bacterial, 11 viral, and five protozoan pathogens, and three internal amplification/process controls in many samples. The MFQPCR chip was applied to analyze pathogen removal rates during the wastewater treatment processes. In addition, multiple host-specific MST markers, FIB, and pathogens were successfully quantified in human and avian-impacted surface waters. While the genes for pathogens were relatively infrequently detected, positive correlations were observed between some potential pathogens such as Clostridium perfringens and Mycobacterium spp., and human MST markers. The MFQPCR chips developed in this study, therefore, can provide useful information to monitor and improve water quality.

Assessment and Mitigation of Fecal Bacteria Exports from a Coastal North Carolina Watershed

Urban runoff from the Boat House Creek watershed was suspected as a main delivery mechanism for fecal indicator bacteria (FIB) to the lower White Oak River Estuary in coastal North Carolina, but the dominant source of waste (animal or human) was unknown. Water samples from eight locations within the watershed were collected approximately monthly for two years for enumeration of Escherichia coli (E. coli), enterococci, physicochemical characterization, and microbial source tracking analyses. Concentrations and loadings of E. coli and enterococci were typically elevated during stormflow relative to baseflow conditions, and most samples (66% of enterococci and 75% of E. coli) exceeded the US EPA statistical threshold values. Concentrations of FIB were significantly higher during warm relative to colder months. Human sources of FIB were not observed in the samples, and FIB concentrations increased in locations with wider buffers, thus wildlife was the suspected main FIB source. Stormwater control measures including a rain garden, water control structures, swale modifications, and check dams were implemented to reduce runoff and FIB loadings to the estuary. Stormflow reductions of &gt;5700 m3 year−1 are estimated from the installation of the practices. More work will be needed to improve/maintain water quality as watershed development continues.

Characterization of crAss-like phage isolates highlights Crassvirales genetic heterogeneity and worldwide distribution

Crassvirales (crAss-like phages) are an abundant group of human gut-specific bacteriophages discovered in silico. The use of crAss-like phages as human fecal indicators is proposed but the isolation of only seven cultured strains of crAss-like phages to date has greatly hindered their study. Here, we report the isolation and genetic characterization of 25 new crAss-like phages (termed crAssBcn) infecting Bacteroides intestinalis, belonging to the order Crassvirales, genus Kehishuvirus and, based on their genomic variability, classified into six species. CrAssBcn phage genomes are similar to ΦCrAss001 but show genomic and aminoacidic differences when compared to other crAss-like phages of the same family. CrAssBcn phages are detected in fecal metagenomes around the world at a higher frequency than ΦCrAss001. This study increases the known crAss-like phage isolates and their abundance and heterogeneity open the question of what member of the Crassvirales group should be selected as human fecal marker.

Transport and Retention of Fecal Indicator Bacteria in Unsaturated Porous Media: Effect of Transient Water Flow.

For production of clean drinking water, the processes governing bacterial remobilization in the unsaturated zone at transient water flow are critical. Although managed aquifer recharge is an effective way to dispose of pathogens, there are concerns about recontamination after heavy precipitation. To better understand how bacteria that were initially retained in porous media can be released to groundwater due to transient water content, transport experiments and modeling for Escherichia coli and Enterococcus moraviensis were conducted at the soil column scale. After inoculating dune sand columns with a bacteria suspension for 4 h, three rainfall events were performed at 24-h intervals. The effluent from sand columns was collected to analyze bacteria breakthrough curves (BTCs). After the rainfall experiments, the bacteria distribution in the sand column was determined. The collected BTCs and profile retentions were modeled with HYDRUS-1D, using different model concepts, including one-site kinetic attachment/detachment (M1), Langmuirian (M2), Langmuirian and blocking (M3), and two-site attachment/detachment (M4). After inoculation, almost 99% of the bacteria remained in the soil. The M1 and M2 bacteria models had a high agreement between observed and modeled concentrations, and attachment and detachment were two significant mechanisms for regulating bacteria movement in a porous medium with fluctuations in water flow. At the end of the experiment, the majority of bacteria were still found within the depth range of 5 cm to 15 cm. Our experiments show that E. coli is more mobile in sandy soils than E. moraviensis. The results of this study also suggest that the unsaturated zone is an important barrier between microbial contamination at the soil surface and groundwater. Follow-up studies are needed to completely understand the variables that regulate bacteria remobilization in the unsaturated zone of dune sands. IMPORTANCE At managed artificial recharge sites in the Netherlands, recontamination of infiltrated water with fecal indicator bacteria has been observed. The results of this study suggest that the unsaturated zone is an important barrier between microbial contamination at the soil surface and groundwater. Bacteria that accumulate in the unsaturated zone, on the other hand, can multiply to such an extent that they can be released into the saturated zone when saturation increases due to major rain events or a rise in groundwater level.