Microbial communities of the Laurentian Great Lakes reflect connectivity and local biogeochemistry.

The Laurentian Great Lakes are a vast, interconnected freshwater system spanning strong physicochemical gradients, thus constituting a powerful natural laboratory for addressing fundamental questions about microbial ecology and evolution. We present a comparative analysis of pelagic microbial communities across all five Laurentian Great Lakes, focusing on Bacterial and Archaeal picoplankton characterized via 16S rRNA amplicon sequencing. We collected samples throughout the water column from the major basins of each lake in spring and summer over 2 years. Two oligotypes, classified as LD12 (Alphaproteobacteria) and acI-B1 (Actinobacteria), were among the most abundant in every sample. At the same time, microbial communities showed distinct patterns with depth during summer stratification. Deep hypolimnion samples were frequently dominated by a Chloroflexi oligotype that reached up to 19% relative abundance. Stratified surface communities differed between the colder, less productive upper lakes (Superior, Michigan, Huron) and warmer, more productive lower lakes (Erie, Ontario), in part due to an Actinobacteria oligotype (acI-C2) that averaged 7.7% of sequences in the lower lakes but <0.2% in the upper lakes. Together, our findings suggest that both hydrologic connectivity and local selective pressures shape microbial communities in the Great Lakes and establish a framework for future investigations.

Analysis of the gull fecal microbial community reveals the dominance of Catellicoccus marimammalium in relation to culturable Enterococci.

Gulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB. Enterococcaceae and Enterobacteriaceae were the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealed Catellicoccus marimammalium as the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcus indoxyl-ß-D-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified as Enterococcus spp., 1.2% were identified as Streptococcus spp., and none were identified as C. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions of C. marimammalium 16S rRNA gene sequences (>50-fold) relative to typical mEI culturable Enterococcus spp. C. marimammalium therefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment.

First Report and Characterization of a Plasmid-Encoded blaSFO-1 in a Multi-Drug-Resistant Aeromonas hydrophila Clinical Isolate.

Antibiotic resistance remains one of the most pressing public health issues facing the world today. At the forefront of this battle lies the ever-increasing identification of extended-spectrum beta-lactamases and carbapenemases within human pathogens, conferring resistance towards broad-spectrum and last-resort antimicrobials. This study was prompted due to the identification of a pathogenic Aeromonas hydrophila isolate (strain MAH-4) collected from abdominal fluid, which presented a robust resistance pattern against second-, third-, and fourth-generation cephalosporins, ertapenem, ciprofloxacin, gentamicin, levofloxacin and moxifloxacin, and beta lactam/beta-lactamase inhibitor combinations. Whole genome sequencing was performed and identified a 328 kb plasmid (pMAH4) encoding 10 antibiotic resistance genes, including blaSFO-1, blaTEM-1, and blaOXA-1 of A. hydrophia MAH-4. This is the first report of beta-lactamase SFO-1 within a clinical strain of Aeromonas. Due to the remarkable sequence identity of pMAH4 to plasmids associated with Enterobacterales genera like Klebsiella and the extensive capabilities of Aeromonas for horizontal gene transfer, our identification of a clinical isolate encoding SFO-1 on a plasmid suggests antibiotic resistance gene mobility between Enterobacterales and non-Enterobacterales species.

Anthropogenic particle concentrations and fluxes in an urban river are temporally variable and impacted by storm events.

Anthropogenic particles (AP), which include microplastics and other synthetic, semisynthetic, and anthropogenically modified materials, are pollutants of concern in aquatic ecosystems worldwide. Rivers are important conduits and retention sites for AP, and time series data on the movement of these particles in lotic ecosystems are needed to assess the role of rivers in the global AP cycle. Much research assessing AP pollution extrapolates stream loads based on single time point measurements, but lotic ecosystems are highly variable over time (e.g., seasonality and storm events). The accuracy of models describing AP dynamics in rivers is constrained by the limited studies that examine how frequent changes in discharge drive particle retention and transport. This study addressed this knowledge gap by using automated, high-resolution sampling to track AP concentrations and fluxes during multiple storm events in an urban river (Milwaukee River) and comparing these measurements to commonly monitored water quality metrics. AP concentrations and fluxes varied significantly across four storm events, highlighting the temporal variability of AP dynamics. When data from the sampling periods were pooled, there were increases in particle concentration and flux during the early phases of the storms, suggesting that floods may flush AP into the river and/or resuspend particles from the benthic zone. AP flux was closely linked to river discharge, suggesting large loads of AP are delivered downstream during storms. Unexpectedly, AP concentrations were not correlated with other simultaneously measured water quality metrics, including total suspended solids, fecal coliforms, chloride, nitrate, and sulfate, indicating that these metrics cannot be used to estimate AP. These data will contribute to more accurate models of particle dynamics in rivers and global plastic export to oceans. PRACTITIONER POINTS: Anthropogenic particle (AP) concentrations and fluxes in an urban river varied across four storm events. AP concentrations and fluxes were the highest during the early phases of the storms. Storms increased AP transport downstream compared with baseflow. AP concentrations did not correlate with other water quality metrics during storms.

Sewage reflects the distribution of human faecal Lachnospiraceae.

Faecal pollution contains a rich and diverse community of bacteria derived from animals and humans, many of which might serve as alternatives to the traditional enterococci and Escherichia coli faecal indicators. We used massively parallel sequencing (MPS) of the 16S rRNA gene to characterize microbial communities from wastewater treatment plant (WWTP) influent sewage from 12 cities geographically distributed across the USA. We examined members of the Clostridiales, which included the families Clostridiaceae, Lachnospiraceae and Ruminococcaceae for their potential as sewage indicators. Lachnospiraceae was one of the most abundant groups of faecal bacteria in sewage, and several Lachnospiraceae high-abundance sewage pyrotags occurred in at least 46 of 48 human faecal samples. Clone libraries targeting Clostridium coccoides (C. coccoides) in sewage samples demonstrated that Lachnospiraceae-annotated V6 pyrotags encompassed the previously reported C. coccoides group. We used oligotyping to profile the genus Blautia within Lachnospiraceae and found oligotypes comprised of 24 entropy components that showed patterns of host specificity. These findings suggest that indicators based on Blautia might have the capacity to discriminate between different faecal pollution sources. Development of source-specific alternative indicators would enhance water quality assessments, which leads to improved ecosystem health and reduced human health risk due to waterborne disease.

Comparison of the microbial community structures of untreated wastewaters from different geographic locales.

Microbial sewage communities consist of a combination of human fecal microorganisms and nonfecal microorganisms, which may be residents of urban sewer infrastructure or flowthrough originating from gray water or rainwater inputs. Together, these different microorganism sources form an identifiable community structure that may serve as a signature for sewage discharges and as candidates for alternative indicators specific for human fecal pollution. However, the structure and variability of this community across geographic space remains uncharacterized. We used massively parallel 454 pyrosequencing of the V6 region in 16S rRNA genes to profile microbial communities from 13 untreated sewage influent samples collected from a wide range of geographic locations in the United States. We obtained a total of 380,175 high-quality sequences for sequence-based clustering, taxonomic analyses, and profile comparisons. The sewage profile included a discernible core human fecal signature made up of several abundant taxonomic groups within Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. DNA sequences were also classified into fecal, sewage infrastructure (i.e., nonfecal), and transient groups based on data comparisons with fecal samples. Across all sewage samples, an estimated 12.1% of sequences were fecal in origin, while 81.4% were consistently associated with the sewage infrastructure. The composition of feces-derived operational taxonomic units remained congruent across all sewage samples regardless of geographic locale; however, the sewage infrastructure community composition varied among cities, with city latitude best explaining this variation. Together, these results suggest that untreated sewage microbial communities harbor a core group of fecal bacteria across geographically dispersed wastewater sewage lines and that ambient water quality indicators targeting these select core microorganisms may perform well across the United States.

A microbial signature approach to identify fecal pollution in the waters off an urbanized coast of Lake Michigan.

Urban coasts receive watershed drainage from ecosystems that include highly developed lands with sewer and stormwater infrastructure. In these complex ecosystems, coastal waters are often contaminated with fecal pollution, where multiple delivery mechanisms that often contain multiple fecal sources make it difficult to mitigate the pollution. Here, we exploit bacterial community sequencing of the V6 and V6V4 hypervariable regions of the bacterial 16S rRNA gene to identify bacterial distributions that signal the presence of sewer, fecal, and human fecal pollution. The sequences classified to three sewer infrastructure-associated bacterial genera, Acinetobacter, Arcobacter, and Trichococcus, and five fecal-associated bacterial families, Bacteroidaceae, Porphyromonadaceae, Clostridiaceae, Lachnospiraceae, and Ruminococcaceae, served as signatures of sewer and fecal contamination, respectively. The human fecal signature was determined with the Bayesian source estimation program SourceTracker, which we applied to a set of 40 sewage influent samples collected in Milwaukee, WI, USA to identify operational taxonomic units (≥ 97 % identity) that were most likely of human fecal origin. During periods of dry weather, the magnitudes of all three signatures were relatively low in Milwaukee's urban rivers and harbor and nearly zero in Lake Michigan. However, the relative contribution of the sewer and fecal signature frequently increased to > 2 % of the measured surface water communities following sewer overflows. Also during combined sewer overflows, the ratio of the human fecal pollution signature to the fecal pollution signature in surface waters was generally close to that of sewage, but this ratio decreased dramatically during dry weather and rain events, suggesting that nonhuman fecal pollution was the dominant source during these weather-driven scenarios. The qPCR detection of two human fecal indicators, human Bacteroides and Lachno2, confirmed the urban fecal footprint in this ecosystem extends to at least 8 km offshore.

An inexpensive, reproducible method to quantify activated sludge foaming potential: Validation through lab-scale studies and year-long full-scale sampling campaign.

Activated sludge is a conventional treatment process for biochemical oxygen demand (BOD) and total suspended solids (TSS) removal at water resource recovery facilities (WRRFs). Foaming events are a common operational issue in activated sludge and can lead to decreased treatment efficiency, maintenance issues, and potential environmental health risks. Stable foaming events are caused by biological and chemical drivers (i.e., microbes and surfactants) during the aeration process. However, foaming events are difficult to predict and quantify. We present an inexpensive and easy-to-use method that can be applied at WRRFs to quantify foaming potential. Subsequently, the method was applied over a year-long full-scale study while data on microbial community composition and functional parameters associated with foaming potential were collected from activated sludge samples at South Shore Water Reclamation Facility (WRF) (Oak Creek, WI). Results from the development of the foaming potential method using linear alkylbenzene sulfonate (LAS) showed that the method was reproducible (relative standard deviation <20%) and able to capture changes in foam-inducing constituents. Using full-scale activated sludge samples, higher relative abundance values for the following genera were associated with foaming events: Zoogloea, Flavobacterium, Variovorax, and Bdellovibrio. This is the first report that Variovorx and Bdellovibrio relative abundance was correlated with foaming events in activated sludge. Furthermore, the foaming potential positively correlated (ρ = 0.24) with soluble total nitrogen. Characterizing foaming events through frequent sampling and monitoring of specific genera and functional parameters may allow for predictions and preemptive mitigation efforts to avoid negative consequences in the future. PRACTITIONER POINTS: A reproducible method to measure foaming potential in activated sludge is available. Genera Zoogloea, Flavobacterium, Variovorax, and Bdellovibrio correlated with foaming events. A year-long sampling campaign of activated sludge measuring foaming potential and microbial community composition was conducted at South Shore Water Reclamation Facility in Oak Creek, WI. More research at other facilities with this method is needed to understand links between microbes and foaming.

Impact of corrosion inhibitors on antibiotic resistance, metal resistance, and microbial communities in drinking water.

Corrosion inhibitors, including zinc orthophosphate, sodium orthophosphate, and sodium silicate, are commonly used to prevent the corrosion of drinking water infrastructure. Metals such as zinc are known stressors for antibiotic resistance selection, and phosphates can increase microbial growth in drinking water distribution systems (DWDS). Yet, the influence of corrosion inhibitor type on antimicrobial resistance in DWDS is unknown. Here, we show that sodium silicates can decrease antibiotic resistant bacteria (ARB) and antibiotic-resistance genes (ARGs), while zinc orthophosphate increases ARB and ARGs in source water microbial communities. Based on controlled bench-scale studies, zinc orthophosphate addition significantly increased the abundance of ARB resistant to ciprofloxacin, sulfonamides, trimethoprim, and vancomycin, as well as the genes sul1, qacEΔ1, an indication of resistance to quaternary ammonium compounds, and the integron-integrase gene intI1. In contrast, sodium silicate dosage at 10 mg/L resulted in decreased bacterial growth and antibiotic resistance selection compared to the other corrosion inhibitor additions. Source water collected from the drinking water treatment plant intake pipe resulted in less significant changes in ARB and ARG abundance due to corrosion inhibitor addition compared to source water collected from the pier at the recreational beach. In tandem with the antibiotic resistance shifts, significant microbial community composition changes also occurred. Overall, the corrosion inhibitor sodium silicate resulted in the least selection for antibiotic resistance, which suggests it is the preferred corrosion inhibitor option for minimizing antibiotic resistance proliferation in DWDS. However, the selection of an appropriate corrosion inhibitor must also be appropriate for the water chemistry of the system (e.g., pH, alkalinity) to minimize metal leaching first and foremost and to adhere to the lead and copper rule. IMPORTANCE Antibiotic resistance is a growing public health concern across the globe and was recently labeled the silent pandemic. Scientists aim to identify the source of antibiotic resistance and control points to mitigate the spread of antibiotic resistance. Drinking water is a direct exposure route to humans and contains antibiotic-resistant bacteria and associated resistance genes. Corrosion inhibitors are added to prevent metallic pipes in distribution systems from corroding, and the type of corrosion inhibitor selected could also have implications on antibiotic resistance. Indeed, we found that sodium silicate can minimize selection of antibiotic resistance while phosphate-based corrosion inhibitors can promote antibiotic resistance. These findings indicate that sodium silicate is a preferred corrosion inhibitor choice for mitigation of antibiotic resistance.

One Health and Global Health View of Antimicrobial Susceptibility through the "Eye" of Aeromonas: Systematic Review and Meta-Analysis.

Antimicrobial resistance (AMR) is one of the most pressing public health concerns; therefore, it is imperative to advance our understanding of the factors influencing AMR from Global and One Health perspectives. To address this, Aeromonas populations were identified using 16S rRNA gene libraries among human, agriculture, aquaculture, drinking water, surface water, and wastewater samples, supporting its use as indicator bacteria to study AMR. A systematic review and meta-analysis was then performed from Global and One Health perspectives, including data from 221 articles describing 15 891 isolates from 57 countries. The interconnectedness of different environments was evident as minimal differences were identified between sectors among 21 different antimicrobials. However, resistance to critically important antibiotics (aztreonam and cefepime) was significantly higher among wastewater populations compared with clinical isolates. Additionally, isolates from untreated wastewater typically exhibited increased AMR compared with those from treated wastewater. Furthermore, aquaculture was associated with increased AMR to ciprofloxacin and tetracycline compared with wild-caught seafood. Using the World Health Organization AWaRe classifications, countries with lower consumption of "Access" compared to "Watch" drugs from 2000 to 2015 demonstrated higher AMR levels. The current analysis revealed negative correlations between AMR and anthropogenic factors, such as environmental performance indices and socioeconomic standing. Environmental health and sanitation were two of the environmental factors most strongly correlated with AMR. The current analysis highlights the negative impacts of "Watch" drug overconsumption, anthropogenic activity, absence of wastewater infrastructure, and aquaculture on AMR, thus stressing the need for proper infrastructure and global regulations to combat this growing problem.

Full-Length 16S rRNA Gene Sequences from Raw Sewage Samples Spanning Geographic and Seasonal Gradients in Conveyance Systems across the United States.

Wastewater microbiome research often relies on sequencing of hypervariable regions of 16S rRNA genes, which are difficult to classify at refined taxonomic levels. Here, we introduce a data set of near-full-length 16S rRNA genes from samples designed to capture known geographic and seasonal variations in municipal wastewater microbial communities.

Guts of the Urban Ecosystem: Microbial Ecology of Sewer Infrastructure.

Microbes have inhabited the oceans and soils for millions of years and are uniquely adapted to their habitat. In contrast, sewer infrastructure in modern cities dates back only ~150 years. Sewer pipes transport human waste and provide a view into public health, but the resident organisms that likely modulate these features are relatively unexplored. Here, we show that the bacterial assemblages sequenced from untreated wastewater in 71 U.S. cities were highly coherent at a fine sequence level, suggesting that urban infrastructure separated by great spatial distances can give rise to strikingly similar communities. Within the overall microbial community structure, temperature had a discernible impact on the distribution patterns of closely related amplicon sequence variants, resulting in warm and cold ecotypes. Two bacterial genera were dominant in most cities regardless of their size or geographic location; on average, Arcobacter accounted for 11% and Acinetobacter 10% of the entire community. Metagenomic analysis of six cities revealed these highly abundant resident organisms carry clinically important antibiotic resistant genes blaCTX-M, blaOXA, and blaTEM. In contrast, human fecal bacteria account for only ~13% of the community; therefore, antibiotic resistance gene inputs from human sources to the sewer system could be comparatively small, which will impact measurement capabilities when monitoring human populations using wastewater. With growing awareness of the metabolic potential of microbes within these vast networks of pipes and the ability to examine the health of human populations, it is timely to increase our understanding of the ecology of these systems. IMPORTANCE Sewer infrastructure is a relatively new habitat comprised of thousands of kilometers of pipes beneath cities. These wastewater conveyance systems contain large reservoirs of microbial biomass with a wide range of metabolic potential and are significant reservoirs of antibiotic resistant organisms; however, we lack an adequate understanding of the ecology or activity of these communities beyond wastewater treatment plants. The striking coherence of the sewer microbiome across the United States demonstrates that the sewer environment is highly selective for a particular microbial community composition. Therefore, results from more in-depth studies or proven engineering controls in one system could be extrapolated more broadly. Understanding the complex ecology of sewer infrastructure is critical for not only improving our ability to treat human waste and increasing the sustainability of our cities but also to create scalable and effective sewage microbial observatories, which are inevitable investments of the future to monitor health in human populations.

Genome Streamlining, Proteorhodopsin, and Organic Nitrogen Metabolism in Freshwater Nitrifiers.

Microbial nitrification is a critical process governing nitrogen availability in aquatic systems. Freshwater nitrifiers have received little attention, leaving many unanswered questions about their taxonomic distribution, functional potential, and ecological interactions. Here, we reconstructed genomes to infer the metabolism and ecology of free-living picoplanktonic nitrifiers across the Laurentian Great Lakes, a connected series of five of Earth's largest lakes. Surprisingly, ammonia-oxidizing bacteria (AOB) related to Nitrosospira dominated over ammonia-oxidizing archaea (AOA) at nearly all stations, with distinct ecotypes prevailing in the transparent, oligotrophic upper lakes compared to Lakes Erie and Ontario. Unexpectedly, one ecotype of Nitrosospira encodes proteorhodopsin, which could enhance survival under conditions where ammonia oxidation is inhibited or substrate limited. Nitrite-oxidizing bacteria (NOB) "Candidatus Nitrotoga" and Nitrospira fluctuated in dominance, with the latter prevailing in deeper, less-productive basins. Genome reconstructions reveal highly reduced genomes and features consistent with genome streamlining, along with diverse adaptations to sunlight and oxidative stress and widespread capacity for organic nitrogen use. Our findings expand the known functional diversity of nitrifiers and establish their ecological genomics in large lake ecosystems. By elucidating links between microbial biodiversity and biogeochemical cycling, our work also informs ecosystem models of the Laurentian Great Lakes, a critical freshwater resource experiencing rapid environmental change. IMPORTANCE Microorganisms play critical roles in Earth's nitrogen cycle. In lakes, microorganisms called nitrifiers derive energy from reduced nitrogen compounds. In doing so, they transform nitrogen into a form that can ultimately be lost to the atmosphere by a process called denitrification, which helps mitigate nitrogen pollution from fertilizer runoff and sewage. Despite their importance, freshwater nitrifiers are virtually unexplored. To understand their diversity and function, we reconstructed genomes of freshwater nitrifiers across some of Earth's largest freshwater lakes, the Laurentian Great Lakes. We discovered several new species of nitrifiers specialized for clear low-nutrient waters and distinct species in comparatively turbid Lake Erie. Surprisingly, one species may be able to harness light energy by using a protein called proteorhodopsin, despite the fact that nitrifiers typically live in deep dark water. Our work reveals the unique biodiversity of the Great Lakes and fills key gaps in our knowledge of an important microbial group, the nitrifiers.

Chronic exposure to high-density polyethylene microplastic through feeding alters the nutrient metabolism of juvenile yellow perch (Perca flavescens).

Microplastics are emergent contaminants threatening aquatic organisms including aquacultured fish. This study investigated the effects of high-density polyethylene (HDPE, 100 to 125 µm) on yellow perch (Perca flavescens) based on integrative evaluation including growth performance, nutritional status, nutrient metabolism, fish health, and gut microbial community. Five test diets (0, 1, 2, 4, or 8 g HDPE/100 g diet) containing 41% protein and 10.5% lipid were fed to juvenile perch (average body weight, 25.9 ± 0.2 g; n = 15) at a feeding rate of 1.5% to 2.0% body weight daily. The feeding trial was conducted in a flow-through water system for 9 wk with 3 tanks per treatment and 15 yellow perch per tank. No mortality or HDPE accumulation in the fish was found in any treatments. Weight gain and condition factor of fish were not significantly impacted by HDPE (P > 0.05). Compared to the control group, fish fed the 8% HDPE diet had significantly decreased levels of protein and ash (P < 0.05). In response to the increasing levels of HDPE exposure, the hepatosomatic index value, hepatocyte size, and liver glycogen level were increased, but lipid content was reduced in the liver tissues. Compared to the control treatment, fish fed the 8% HDPE diet had significant accumulations of total bile acids and different metabolism pathways such as bile acid biosynthesis, pyruvate metabolism, and carnitine synthesis. Significant enterocyte necrosis was documented in the foregut of fish fed the 2% or 8% HDPE diet; and significant cell sloughing was observed in the midgut and hindgut of fish fed the 8% HDPE diet. Fish fed the 2% HDPE diet harbored different microbiota communities compared to the control fish. This study demonstrates that HDPE ranging from 100 to 125 µm in feed can be evacuated by yellow perch with no impact on growth. However, dietary exposure to HDPE decreased whole fish nutrition quality, altered nutrient metabolism and the intestinal histopathology as well as microbiota community of yellow perch. The results indicate that extended exposure may pose a risk to fish health and jeopardize the nutrition quality of aquacultured end product. This hypothesis remains to be investigated further.

Seasonal differences in bacterial community composition following nutrient additions in a eutrophic lake.

We examined the effects of nutrient amendments on epilimnetic freshwater bacteria during three distinct periods in the eutrophic Lake Mendota's seasonal cycle (spring overturn, summer stratification and autumn overturn). Microcosm treatments enriched solely with phosphorus containing compounds did not result in a large bacterial community composition (BCC) change or community activity response (assessed via alkaline phosphatase activity, APA) relative to the controls during any season. Treatments enriched with carbon- and nitrogen-containing compounds resulted in a dramatic BCC change and a large APA increase in the autumn and spring seasons, but only treatments receiving carbon, nitrogen and phosphorus (CNP) exhibited similar responses in the summer season. Despite the fact that the amendments created similar CNP concentration conditions across seasons, the BCC following amendment greatly varied among seasons. 16S rRNA gene sequence analysis indicated that many common freshwater bacterial lineages from the Alpha- and Betaproteobacteria class and Bacteroidetes phylum were favoured following nutrient (CNP) addition, but individual taxa were generally not favoured across all seasons. Targeted quantitative PCR analysis revealed that the abundance of the Actinobacteria acIB1 cluster decreased in all microcosms during all three seasons, while the Flavobacterium aquatile (spring) and ME-B0 (summer) clusters of Bacteroidetes increased following CNP addition. These results suggest a particular bacterial group is not universally favoured by increased nutrient loads to a lake; therefore, efforts to predict which bacteria are involved in nutrient cycling during these periods must take into account the seasonality of freshwater bacterial communities.

Lachnospiraceae and Bacteroidales alternative fecal indicators reveal chronic human sewage contamination in an urban harbor.

The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the Lachnospiraceae family, which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci and the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.

Community structures of fecal bacteria in cattle from different animal feeding operations.

The fecal microbiome of cattle plays a critical role not only in animal health and productivity but also in food safety, pathogen shedding, and the performance of fecal pollution detection methods. Unfortunately, most published molecular surveys fail to provide adequate detail about variability in the community structures of fecal bacteria within and across cattle populations. Using massively parallel pyrosequencing of a hypervariable region of the rRNA coding region, we profiled the fecal microbial communities of cattle from six different feeding operations where cattle were subjected to consistent management practices for a minimum of 90 days. We obtained a total of 633,877 high-quality sequences from the fecal samples of 30 adult beef cattle (5 individuals per operation). Sequence-based clustering and taxonomic analyses indicate less variability within a population than between populations. Overall, bacterial community composition correlated significantly with fecal starch concentrations, largely reflected in changes in the Bacteroidetes, Proteobacteria, and Firmicutes populations. In addition, network analysis demonstrated that annotated sequences clustered by management practice and fecal starch concentration, suggesting that the structures of bovine fecal bacterial communities can be dramatically different in different animal feeding operations, even at the phylum and family taxonomic levels, and that the feeding operation is a more important determinant of the cattle microbiome than is the geographic location of the feedlot.

Urban wastewater bacterial communities assemble into seasonal steady states.

BACKGROUND: Microorganisms in urban sanitary sewers exhibit community properties that suggest sewers are a novel ecosystem. Sewer microorganisms present both an opportunity as a control point for wastewater treatment and a risk to human health. If treatment processes are to be improved and health risks quantified, then it is necessary to understand microbial distributions and dynamics within this community. Here, we use 16S rRNA gene sequencing to characterize raw influent wastewater bacterial communities in a 5-year time series from two wastewater treatment plants in Milwaukee, WI; influent wastewater from 77 treatment plants across the USA; and wastewater in 12 Milwaukee residential sewers. RESULTS: In Milwaukee, we find that in transit from residences to treatment plants, the human bacterial component of wastewater decreases in proportion and exhibits stochastic temporal variation. In contrast, the resident sewer community increases in abundance during transit and cycles seasonally according to changes in wastewater temperature. The result is a bacterial community that assembles into two distinct community states each year according to the extremes in wastewater temperature. Wastewater bacterial communities from other northern US cities follow temporal trends that mirror those in Milwaukee, but southern US cities have distinct community compositions and differ in their seasonal patterns. CONCLUSIONS: Our findings provide evidence that environmental conditions associated with seasonal change and climatic differences related to geography predictably structure the bacterial communities residing in below-ground sewer pipes. Video abstract.

Human Fecal Contamination Corresponds to Changes in the Freshwater Bacterial Communities of a Large River Basin.

Microbial water quality is generally monitored by culturable fecal indicator bacteria (FIB), which are intended to signal human health risk due to fecal pollution. However, FIB have limited utility in most urbanized watersheds as they do not discriminate among fecal pollution sources, tend to make up a small fraction of the total microbial community, and do not inform on pollution impacts on the native ecosystem. To move beyond these limitations, we assessed entire bacterial communities and investigated how bacterial diversity relates to traditional ecological and human health-relevant water quality indicators throughout the Milwaukee River Basin. Samples were collected from 16 sites on 5 days during the summer, including both wet and dry weather events, and were processed by 16S rRNA gene amplicon sequencing. Historical water quality at each sampling location, as opposed to upstream land use, was associated significantly with bacterial community alpha diversity. Source partitioning the sequence data was important for determining water quality relationships. Sewage-associated bacterial sequences were detected in all samples, and the relative abundance of sewage sequences was strongly associated with the human Bacteroides fecal marker. From this relationship, we developed a preliminary threshold for human sewage pollution when using bacterial community sequence data. Certain abundant freshwater bacterial sequences were also associated with human fecal pollution, suggesting their possible utility in water quality monitoring. This study sheds light on how bacterial community analysis can be used to supplement current water quality monitoring techniques to better understand interactions between ecological water quality and human health indicators. IMPORTANCE Surface waters in highly developed mixed-use watersheds are frequently impacted by a wide variety of pollutants, leading to a range of impairments that must be monitored and remediated. With advancing technologies, microbial community sequencing may soon become a feasible method for routine evaluation of the ecological quality and human health risk of a water body. In this study, we partnered with a local citizen science organization to evaluate the utility of microbial community sequencing for identifying pollution sources and ecological impairments in a large mixed-use watershed. We show that changes in microbial community diversity and composition are indicative of both long-term ecological impairments and short-term fecal pollution impacts. By source partitioning the sequence data, we also estimate a threshold target for human sewage pollution, which may be useful as a starting point for future development of sequencing-based water quality monitoring techniques.