Influence of Filter Pore Size on Composition and Relative Abundance of Bacterial Communities and Select Host-Specific MST Markers in Coastal Waters of Southern Lake Michigan.

Water clarity is often the primary guiding factor in determining whether a prefiltration step is needed to increase volumes processed for a range of microbial endpoints. In this study, we evaluate the effect of filter pore size on the bacterial communities detected by 16S rRNA gene sequencing and incidence of two host-specific microbial source tracking (MST) markers in a range of coastal waters from southern Lake Michigan, using two independent data sets collected in 2015 (bacterial communities) and 2016-2017 (MST markers). Water samples were collected from river, shoreline, and offshore areas. For bacterial communities, each sample was filtered through a 5.0-µm filter, followed by filtration through a 0.22-µm filter, resulting in 70 and 143 filter pairs for bacterial communities and MST markers, respectively. Following DNA extraction, the bacterial communities were compared using 16S rRNA gene amplicons of the V3-V4 region sequenced on a MiSeq Illumina platform. Presence of human (Bacteroides HF183) and gull (Gull2, Catellicoccus marimammalium) host-specific MST markers were detected by qPCR. Actinobacteriota, Bacteroidota, and Proteobacteria, collectively represented 96.9% and 93.9% of the relative proportion of all phyla in the 0.22- and 5.0-µm pore size filters, respectively. There were more families detected in the 5.0-µm pore size filter (368) than the 0.22-µm (228). There were significant differences in the number of taxa between the two filter sizes at all levels of taxonomic classification according to linear discriminant analysis (LDA) effect size (LEfSe) with as many as 986 taxa from both filter sizes at LDA effect sizes greater than 2.0. Overall, the Gull2 marker was found in higher abundance on the 5.0-µm filter than 0.22 µm with the reverse pattern for the HF183 marker. This discrepancy could lead to problems with identifying microbial sources of contamination. Collectively, these results highlight the importance of analyzing pre- and final filters for a wide range of microbial endpoints, including host-specific MST markers routinely used in water quality monitoring programs. Analysis of both filters may increase costs but provides more complete genomic data via increased sample volume for characterizing microbial communities in coastal waters.

Interaction of bacterial communities and indicators of water quality in shoreline sand, sediment, and water of Lake Michigan.

Shoreline sand harbors high concentrations of fecal indicator bacteria (FIB) that may be resuspended into the water column through washing and resuspension. Studies have explored coastal processes that influence this sand-water flux for FIB, but little is known about how microbial markers of contamination or the bacterial community interact in the sand-water interface. In this study, we take a three-tiered approach to explore the relationship between bacteria in sand, sediment, and overlying water at three shoreline sites and two associated rivers along an extended freshwater shoreline. Samples were collected over two years and analyzed for FIB, two microbial source tracking (MST) markers (Catellicoccus marimammalium, Gull2; Bacteroides HF183), and targeted metagenomic 16S rRNA gene analysis. FIB was much higher in sand than in water at all three sites. Gull2 marker was abundant in shoreline sand and water while HF183 marker was mostly present in rivers. Overall bacterial communities were dissimilar between sand/sediment and water, indicating little interaction. Sediment composition was generally unfavorable to bacterial resuspension. Results show that FIB and MST markers were effective estimates of short-term conditions at these locations, and bacterial communities in sand and sediment reflected longer-term conditions. Findings are useful for locating contamination sources and targeting restoration by evaluating scope of shoreline degradation.

Impacts of a changing earth on microbial dynamics and human health risks in the continuum between beach water and sand.

Although infectious disease risk from recreational exposure to waterborne pathogens has been an active area of research for decades, beach sand is a relatively unexplored habitat for the persistence of pathogens and fecal indicator bacteria (FIB). Beach sand, biofilms, and water all present unique advantages and challenges to pathogen introduction, growth, and persistence. These dynamics are further complicated by continuous exchange between sand and water habitats. Models of FIB and pathogen fate and transport at beaches can help predict the risk of infectious disease from beach use, but knowledge gaps with respect to decay and growth rates of pathogens in beach habitats impede robust modeling. Climatic variability adds further complexity to predictive modeling because extreme weather events, warming water, and sea level change may increase human exposure to waterborne pathogens and alter relationships between FIB and pathogens. In addition, population growth and urbanization will exacerbate contamination events and increase the potential for human exposure. The cumulative effects of anthropogenic changes will alter microbial population dynamics in beach habitats and the assumptions and relationships used in quantitative microbial risk assessment (QMRA) and process-based models. Here, we review our current understanding of microbial populations and transport dynamics across the sand-water continuum at beaches, how these dynamics can be modeled, and how global change factors (e.g., climate and land use) should be integrated into more accurate beachscape-based models.

Using Integrated Environmental Modeling to Assess Sources of Microbial Contamination in Mixed-Use Watersheds.

Microbial fate and transport in watersheds should include a microbial source apportionment analysis that estimates the importance of each source, relative to each other and in combination, by capturing their impacts spatially and temporally under various scenarios. A loosely configured software infrastructure was used in microbial source-to-receptor modeling by focusing on animal- and human-impacted mixed-use watersheds. Components include data collection software, a microbial source module that determines loading rates from different sources, a watershed model, an inverse model for calibrating flows and microbial densities, tabular and graphical viewers, software to convert output to different formats, and a model for calculating risk from pathogen exposure. The system automates, as much as possible, the manual process of accessing and retrieving data and completes input data files of the models. The workflow considers land-applied manure from domestic animals on undeveloped areas; direct shedding (excretion) on undeveloped lands by domestic animals and wildlife; pastureland, cropland, forest, and urban or engineered areas; sources that directly release to streams from leaking septic systems; and shedding by domestic animals directly to streams. The infrastructure also considers point sources from regulated discharges. An application is presented on a real-world watershed and helps answer questions such as: What are the major microbial sources? What practices contribute to contamination at the receptor location? What land-use types influence contamination at the receptor location? and Under what conditions do these sources manifest themselves? This research aims to improve our understanding of processes related to pathogen and indicator dynamics in mixed-use watershed systems.

Capturing Microbial Sources Distributed in a Mixed-use Watershed within an Integrated Environmental Modeling Workflow.

Many watershed models simulate overland and instream microbial fate and transport, but few provide loading rates on land surfaces and point sources to the waterbody network. This paper describes the underlying equations for microbial loading rates associated with 1) land-applied manure on undeveloped areas from domestic animals; 2) direct shedding (excretion) on undeveloped lands by domestic animals and wildlife; 3) urban or engineered areas; and 4) point sources that directly discharge to streams from septic systems and shedding by domestic animals. A microbial source module, which houses these formulations, is part of a workflow containing multiple models and databases that form a loosely configured modeling infrastructure which supports watershed-scale microbial source-to-receptor modeling by focusing on animal- and human-impacted catchments. A hypothetical application - accessing, retrieving, and using real-world data - demonstrates how the infrastructure can automate many of the manual steps associated with a standard watershed assessment, culminating in calibrated flow and microbial densities at the watershed's pour point.

Molecular tools for bathing water assessment in Europe: Balancing social science research with a rapidly developing environmental science evidence-base.

The use of molecular tools, principally qPCR, versus traditional culture-based methods for quantifying microbial parameters (e.g., Fecal Indicator Organisms) in bathing waters generates considerable ongoing debate at the science-policy interface. Advances in science have allowed the development and application of molecular biological methods for rapid (~2 h) quantification of microbial pollution in bathing and recreational waters. In contrast, culture-based methods can take between 18 and 96 h for sample processing. Thus, molecular tools offer an opportunity to provide a more meaningful statement of microbial risk to water-users by providing near-real-time information enabling potentially more informed decision-making with regard to water-based activities. However, complementary studies concerning the potential costs and benefits of adopting rapid methods as a regulatory tool are in short supply. We report on findings from an international Working Group that examined the breadth of social impacts, challenges, and research opportunities associated with the application of molecular tools to bathing water regulations.

Opportunities and limitations of molecular methods for quantifying microbial compliance parameters in EU bathing waters.

The debate over the suitability of molecular biological methods for the enumeration of regulatory microbial parameters (e.g. Faecal Indicator Organisms [FIOs]) in bathing waters versus the use of traditional culture-based methods is of current interest to regulators and the science community. Culture-based methods require a 24-48hour turn-around time from receipt at the laboratory to reporting, whilst quantitative molecular tools provide a more rapid assay (approximately 2-3h). Traditional culturing methods are therefore often viewed as slow and 'out-dated', although they still deliver an internationally 'accepted' evidence-base. In contrast, molecular tools have the potential for rapid analysis and their operational utility and associated limitations and uncertainties should be assessed in light of their use for regulatory monitoring. Here we report on the recommendations from a series of international workshops, chaired by a UK Working Group (WG) comprised of scientists, regulators, policy makers and other stakeholders, which explored and interrogated both molecular (principally quantitative polymerase chain reaction [qPCR]) and culture-based tools for FIO monitoring under the European Bathing Water Directive. Through detailed analysis of policy implications, regulatory barriers, stakeholder engagement, and the needs of the end-user, the WG identified a series of key concerns that require critical appraisal before a potential shift from culture-based approaches to the employment of molecular biological methods for bathing water regulation could be justified.

Dramatic improvements in beach water quality following gull removal.

Gulls are often cited as important contributors of fecal contamination to surface waters, and some recreational beaches have used gull control measures to improve microbial water quality. In this study, gulls were chased from a Lake Michigan beach using specially trained dogs, and water quality improvements were quantified. Fecal indicator bacteria and potentially pathogenic bacteria were measured before and during gull control using culture methods and quantitative polymerase chain reaction (qPCR). Harassment by dogs was an effective method of gull control: average daily gull populations fell from 665 before to 17 during intervention; and a significant reduction in the density of a gull-associated marker was observed (p < 0.001). Enterococcus spp. and Escherichia coli densities were also significantly reduced during gull control (p < 0.001 and p = 0.012, respectively for culture methods; p = 0.012 and p = 0.034, respectively for qPCR). Linear regression results indicate that a 50% reduction in gulls was associated with a 38% and 29% decrease in Enterococcus spp. and E. coli densities, respectively. Potentially human pathogenic bacteria were detected on 64% of days prior to gull control and absent during gull intervention, a significant reduction (p = 0.005). This study demonstrates that gull removal can be a highly successful beach remedial action to improve microbial water quality.

A cross comparison of QPCR to agar-based or defined substrate test methods for the determination of Escherichia coli and enterococci in municipal water quality monitoring programs.

Molecular methods such as quantitative, real-time polymerase chain reaction (QPCR) are intended to shorten the period between sampling and publicly available results. Cross comparison studies in Racine, WI, USA evaluated QPCR against agar-based (US EPA Method 1600) and defined substrate (IDEXX Colilert-18) methods for the detection and quantification of Escherichia coli and enterococci in a variety of aqueous environments (wastewater, stormwater, and surface water). Regulatory outcomes were also compared based on choice of indicator and method. Positive correlation was seen between QPCR cell equivalents and viable cells through the wastewater treatment process and in all surface water samples (river or freshwater bathing beach) but not in direct stormwater discharge. For surface water samples, correlation improved with the application of a site-specific corrective factor, with regulatory action correctly predicted 98% of the time at bathing beaches. This study suggests the potential utility of QPCR for certain water quality monitoring applications.

Bacteroidales diversity in ring-billed gulls (Laurus delawarensis) residing at Lake Michigan beaches.

This study investigated the occurrence and diversity of Bacteroidales fecal bacteria in gulls residing in the Great Lakes region. Members of this bacterial order have been widely employed as human and bovine host-specific markers of fecal pollution; however, few studies have focused on gulls, which can be a major source of fecal indicator bacteria and pathogens at beaches. We found a low but consistent occurrence of Bacteroidales in gulls at five beaches in three different counties spanning the Wisconsin shoreline of Lake Michigan. The percentages of gulls positive for Bacteroidales were 4 to 8% at beaches in the southern part of the state and 8 to 50% at beaches in the north. Sequencing of 931 clones from seven gull Bacteroidales 16S rRNA gene libraries revealed a large amount of diversity in both individual and pooled gull fecal samples. Two libraries constructed from pooled gull fecal samples (n = 5 and n = 6) did not have a greater richness of sequences than individual samples, suggesting that even within a single gull diversity is high and an extensive sequencing effort is needed to characterize the populations. Estimates of the numbers of operational taxonomic units (OTUs) for the libraries obtained using different similarity levels revealed a large amount of microdiveristy with a limited number of OTUs at the 95% similarity level. Gull sequences were clustered by the beach from which they were collected, suggesting that there were geographic effects on the distribution of Bacteriodales. More than 53% of the 16S rRNA gene sequences from gulls at the southern beaches were associated with the family Porphyromonadaceae, primarily the genus Parabacteroides, whereas sequences from gulls at the northern beaches were comprised of Bacteroidaceae and Prevotellaceae sequences. Comparison of gull sequences with sequences from goose, canine, raccoon, and sewage sources revealed distinct clusters of closely related gull sequences; however, these sequences were widely dispersed across a dendrogram that included all other sources, including previously characterized gull Bacteroidales from other studies, suggesting that geographic influence or simply sample representation plays a greater role in the observed population structure than strictly the host gut environment.