Bacterial assemblages of urban microbiomes mobilized by runoff waters match land use typologies and harbor core species involved in pollutant degradation and opportunistic human infections.

Cities are patchworks of urban catchments divided into functional units according to their commercial, residential and industrial activities, and socio-urbanistic patterns. The hypothesis of city surface microbiomes being structured by socio-urbanistic variables leading to an emergence of synurbic taxa was tested. According to the r/K microbial ecology theory, a gradient of well-adapted synurbic K-strategists and of opportunistic -r-strategists should occur over city surfaces. K-strategists would be core components while r-ones would be transiently detected. To resolve these patterns, sub-catchments (n = 21) of an area of high commercial and industrial activities were investigated over three time periods covering one year. The sub-catchments' land use patterns and associated human behaviors were converted into socio-urbanistic variables and groupings. Bacterial cells mobilized by runoffs per sub-catchment were recovered, and analyzed by classical approaches, microbial source tracking DNA assays and DNA meta-barcoding approaches. Relationships between these datasets, the runoff physico-chemical properties, and descriptors of the socio-urbanistic groupings were investigated. 16S rRNA meta-barcoding analyses showed evidence of the occurrence of K- and r-like strategists. Twenty-eight core genera were identified, and correlation networks revealed large bacterial modules organized around actinobacterial taxa involved in hydrocarbon degradation processes. Other bacterial networks were related to the occurrences of hygienic wastes, and involved bacteria originating from fecal contaminations. Several r-strategists like Sulfurospirillum were recorded and found associated to point source pollutions. The tpm-metabarcoding approach deciphered these r / K strategists at the species level among more than ten genera. Nine core K-like Pseudomomas species were identified. The P. aeruginosa human opportunistic pathogen and P. syringae phytopathogens were part of these K-strategists. Other tpm-harboring bacterial pathogens showed r-like opportunistic distribution patterns. Correlation network analyses indicated a strong incidence of hygienic wastes and hydrocarbon-pollutions on tpm-harboring bacteria. These analyses demonstrated the occurrence of core synurbic bacterial K-strategists over city surfaces.

Spatio-temporal variations in chemical pollutants found among urban deposits match changes in thiopurine S-methyltransferase-harboring bacteria tracked by the tpm metabarcoding approach.

The bTPMT (bacterial thiopurine S-methyltransferase), encoded by the tpm gene, can detoxify metalloid-containing oxyanions and xenobiotics. The hypothesis of significant relationships between tpm distribution patterns and chemical pollutants found in urban deposits was investigated. The tpm gene was found conserved among eight bacterial phyla with no sign of horizontal gene transfers but a predominance among gammaproteobacteria. A DNA metabarcoding approach was designed for tracking tpm-harboring bacteria among polluted urban deposits and sediments recovered for more than six years in a detention basin (DB). This DB recovers runoff waters and sediments from a zone of high commercial activities. The PCR products from DB samples led to more than 540,000 tpm reads after DADA2 or MOTHUR bio-informatic manipulations that were allocated to more than 88 and less than 634 sequence variants per sample. The tpm community patterns were significantly different between the recent urban deposits and those that had accumulated for more than 2 years in the DB, and between those of the DB surface and the DB settling pit. These groups of samples had distinct mixture of priority pollutants. Significant relationships between tpm ordination patterns, sediment accumulation time periods and location, and concentrations in PAH, chlorpyrifos, and 4-nonylphenols (NP) were observed. These correlations matched the higher occurrences of, among others, Aeromonas, Pseudomonas, and Xanthomonas tpm-harboring bacteria in recent urban DB deposits more contaminated with chrysene and alkylphenol ethoxylates. Highly significant drops in tpm reads allocated to Aeromonas species were recorded in the oldest DB sediments accumulating naphthalene and metallic pollutants. Degraders of urban pollutants such as P. aeruginosa and P. putida showed conserved distribution patterns over time but P. syringae phytopathogens were more abundant in the oldest sediments. TPMT-harboring bacteria can be used to assess the incidence of high risk priority pollutants on environmental systems.

Bacteriome genetic structures of urban deposits are indicative of their origin and impacted by chemical pollutants.

Urban activities generate surface deposits over impervious surfaces that can represent ecological and health hazards. Bacteriome genetic structures of deposits washed off during rainfall events, over an urban industrial watershed, were inferred from 16 S rRNA gene (rrs) sequences generated by high throughput sequencing. Deposits were sampled over a 4 year-period from a detention basin (DB). Major shifts, matching key management practices, in the structure of these urban bacteriomes, were recorded. Correlation analyses of rrs similarities between samples and their respective concentrations in chemical pollutants, markers of human fecal contaminations (HF183) and antimicrobial resistances (integrons), were performed. Harsher environmental constraints building up in the older deposits led to an increase number of rrs reads from extremophiles such as Acidibacter and Haliangium. Deposits accumulating in the decantation pit of the DB showed an increase in rrs reads from warm blooded intestinal tract bacteria such as Bacteroides and Prevotella. This enrichment matched higher concentrations of Bacteroides HF183 genotypes normally restricted to humans. Bacteriomes of urban deposits appeared good indicators of human-driven environmental changes. Their composition was found representative of their origin. Soil particles and rain appeared to be major contributors of the inferred bacterial taxa recovered from recent deposits.

Strong incidence of Pseudomonas aeruginosa on bacterial rrs and ITS genetic structures of cystic fibrosis sputa.

Cystic fibrosis (CF) lungs harbor a complex community of interacting microbes, including pathogens like Pseudomonas aeruginosa. Meta-taxogenomic analysis based on V5-V6 rrs PCR products of 52 P. aeruginosa-positive (Pp) and 52 P. aeruginosa-negative (Pn) pooled DNA extracts from CF sputa suggested positive associations between P. aeruginosa and Stenotrophomonas and Prevotella, but negative ones with Haemophilus, Neisseria and Burkholderia. Internal Transcribed Spacer analyses (RISA) from individual DNA extracts identified three significant genetic structures within the CF cohorts, and indicated an impact of P. aeruginosa. RISA clusters Ip and IIIp contained CF sputa with a P. aeruginosa prevalence above 93%, and of 24.2% in cluster IIp. Clusters Ip and IIIp showed lower RISA genetic diversity and richness than IIp. Highly similar cluster IIp RISA profiles were obtained from two patients harboring isolates of a same P. aeruginosa clone, suggesting convergent evolution in the structure of their microbiota. CF patients of cluster IIp had received significantly less antibiotics than patients of clusters Ip and IIIp but harbored the most resistant P. aeruginosa strains. Patients of cluster IIIp were older than those of Ip. The effects of P. aeruginosa on the RISA structures could not be fully dissociated from the above two confounding factors but several trends in these datasets support the conclusion of a strong incidence of P. aeruginosa on the genetic structure of CF lung microbiota.

Human-Driven Microbiological Contamination of Benthic and Hyporheic Sediments of an Intermittent Peri-Urban River Assessed from MST and 16S rRNA Genetic Structure Analyses.

Rivers are often challenged by fecal contaminations. The barrier effect of sediments against fecal bacteria was investigated through the use of a microbial source tracking (MST) toolbox, and by Next Generation Sequencing (NGS) of V5-V6 16S rRNA gene (rrs) sequences. Non-metric multi-dimensional scaling analysis of V5-V6 16S rRNA gene sequences differentiated bacteriomes according to their compartment of origin i.e., surface water against benthic and hyporheic sediments. Classification of these reads showed the most prevalent operating taxonomic units (OTU) to be allocated to Flavobacterium and Aquabacterium. Relative numbers of Gaiella, Haliangium, and Thermoleophilum OTU matched the observed differentiation of bacteriomes according to river compartments. OTU patterns were found impacted by combined sewer overflows (CSO) through an observed increase in diversity from the sewer to the hyporheic sediments. These changes appeared driven by direct transfers of bacterial contaminants from wastewaters but also by organic inputs favoring previously undetectable bacterial groups among sediments. These NGS datasets appeared more sensitive at tracking community changes than MST markers. The human-specific MST marker HF183 was strictly detected among CSO-impacted surface waters and not river bed sediments. The ruminant-specific DNA marker was more broadly distributed but intense bovine pollution was required to detect transfers from surface water to benthic and hyporheic sediments. Some OTU showed distribution patterns in line with these MST datasets such as those allocated to the Aeromonas, Acinetobacter, and Pseudomonas. Fecal indicators (Escherichia coli and total thermotolerant coliforms) were detected all over the river course but their concentrations were not correlated with MST ones. Overall, MST and NGS datasets suggested a poor colonization of river sediments by bovine and sewer bacterial contaminants. No environmental outbreak of these bacterial contaminants was detected.

Impact of dairy manure pre-application treatment on manure composition, soil dynamics of antibiotic resistance genes, and abundance of antibiotic-resistance genes on vegetables at harvest.

Manuring ground used for crop production is an important agricultural practice. Should antibiotic-resistant enteric bacteria carried in the manure be transferred to crops that are consumed raw, their consumption by humans or animals will represent a route of exposure to antibiotic resistance genes. Treatment of manures prior to land application is a potential management option to reduce the abundance of antibiotic resistance genes entrained with manure application. In this study, dairy manure that was untreated, anaerobically digested, mechanically dewatered or composted was applied to field plots that were then cropped to lettuce, carrots and radishes. The impact of treatment on manure composition, persistence of antibiotic resistance gene targets in soil following application, and distribution of antibiotic resistance genes and bacteria on vegetables at harvest was determined. Composted manure had the lowest abundance of antibiotic resistance gene targets compared to the other manures. There was no significant difference in the persistence characteristics of antibiotic resistance genes following land application of the various manures. Compared to unmanured soil, antibiotic resistance genes were detected more frequently in soil receiving raw or digested manure, whereas they were not in soil receiving composted manure. The present study suggests that vegetables grown in ground receiving raw or digested manure are at risk of contamination with manure-borne antibiotic resistant bacteria, whereas vegetables grown in ground receiving composted manure are less so.

Persistence of antibiotic resistance and plasmid-associated genes in soil following application of sewage sludge and abundance on vegetables at harvest.

Sewage sludge recovered from wastewater treatment plants contains antibiotic residues and is rich in antibiotic resistance genes, selected for and enriched in the digestive tracts of human using antibiotics. The use of sewage sludge as a crop fertilizer constitutes a potential route of human exposure to antibiotic resistance genes through consumption of contaminated crops. Several gene targets associated with antibiotic resistance (catA1, catB3, ereA, ereB, erm(B), str(A), str(B), qnrD, sul1, and mphA), mobile genetic elements (int1, mobA, IncW repA, IncP1 groups -α, -ß, -δ, -γ, -ε), and bacterial 16S rRNA (rrnS) were quantified by qPCR from soil and vegetable samples obtained from unamended and sludge-amended plots at an experimental field in London, Ontario. The qPCR data reveals an increase in abundance of gene targets in the soil and vegetables samples, indicating that there is potential for additional crop exposure to antibiotic resistance genes carried within sewage sludge following field application. It is therefore advisable to allow an appropriate delay period before harvesting of vegetables for human consumption.

Genome Sequences of Three Strains of the Pseudomonas aeruginosa PA7 Clade.

Draft genome sequences of three P. aeruginosa strains from the PA7 clade are presented here. Their lengths are 6.36 (EML528), 6.44 (EML545), and 6.33 Mb (EML548). Comparisons with the PA7 genome showed 5,113 conserved coding sequences (CDSs), and significant numbers of strain-specific CDSs. Their analysis will improve our understanding of this highly divergent clade.

Impact of fertilizing with raw or anaerobically digested sewage sludge on the abundance of antibiotic-resistant coliforms, antibiotic resistance genes, and pathogenic bacteria in soil and on vegetables at harvest.

The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic-resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes by using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish, and tomatoes) were sown into field plots fertilized inorganically or with class B biosolids or untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria by plate counts did not reveal significant treatment effects of fertilization with class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated by using quantitative PCR (qPCR) were considerably more abundant on vegetables harvested from sewage sludge-treated plots than on vegetables from control plots in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, the results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pretreatment will result in an additional burden of antibiotic resistance genes on harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.

Long-term monitoring of waterborne pathogens and microbial source tracking markers in paired agricultural watersheds under controlled and conventional tile drainage management.

Surface waters from paired agricultural watersheds under controlled tile drainage (CTD) and uncontrolled tile drainage (UCTD) were monitored over 7 years in order to determine if there was an effect of CTD (imposed during the growing season) on occurrences and loadings of bacterial and viral pathogens, coliphages, and microbial source tracking markers. There were significantly lower occurrences of human, ruminant, and livestock (ruminant plus pig) Bacteroidales markers in the CTD watershed in relation to the UCTD watershed. As for pathogens, there were significantly lower occurrences of Salmonella spp. and Arcobacter spp. in the CTD watershed. There were no instances where there were significantly higher quantitative loadings of any microbial target in the CTD watershed, except for F-specific DNA (F-DNA) and F-RNA coliphages, perhaps as a result of fecal inputs from a hobby farm independent of the drainage practice treatments. There was lower loading of the ruminant marker in the CTD watershed in relation to the UCTD system, and results were significant at the level P = 0.06. The odds of Salmonella spp. occurring increased when a ruminant marker was present relative to when the ruminant marker was absent, yet for Arcobacter spp., the odds of this pathogen occurring significantly decreased when a ruminant marker was present relative to when the ruminant marker was absent (but increased when a wildlife marker was present relative to when the wildlife marker was absent). Interestingly, the odds of norovirus GII (associated with human and swine) occurring in water increased significantly when a ruminant marker was present relative to when a ruminant marker was absent. Overall, this study suggests that fecal pollution from tile-drained fields to stream could be reduced by CTD utilization.

Safely coupling livestock and crop production systems: how rapidly do antibiotic resistance genes dissipate in soil following a commercial application of swine or dairy manure?

Animal manures recycled onto crop production land carry antibiotic-resistant bacteria. The present study evaluated the fate in soil of selected genes associated with antibiotic resistance or genetic mobility in field plots cropped to vegetables and managed according to normal farming practice. Referenced to unmanured soil, fertilization with swine or dairy manure increased the relative abundance of the gene targets sul1, erm(B), str(B), int1, and IncW repA. Following manure application in the spring of 2012, gene copy number decayed exponentially, reaching background levels by the fall of 2012. In contrast, gene copy number following manure application in the fall of 2012 or spring of 2013 increased significantly in the weeks following application and then declined. In both cases, the relative abundance of gene copy numbers had not returned to background levels by the fall of 2013. Overall, these results suggest that under conditions characteristic of agriculture in a humid continental climate, a 1-year period following a commercial application of raw manure is sufficient to ensure that an additional soil burden of antibiotic resistance genes approaches background. The relative abundance of several gene targets exceeded background during the growing season following a spring application or an application done the previous fall. Results from the present study reinforce the advisability of treating manure prior to use in crop production systems.

Draft Genome Sequence of the Sulfonamide Antibiotic-Degrading Microbacterium sp. Strain C448.

We report the draft genome sequence of Microbacterium sp. strain C448, isolated from agricultural soil with a decade of exposure to veterinary antibiotics on the basis of using sulfamethazine and other antibiotics as the sole sources of carbon. The genome sequence revealed that strain C448 harbors several antibiotic resistance genes, including sulI.

Using SWAT, Bacteroidales microbial source tracking markers, and fecal indicator bacteria to predict waterborne pathogen occurrence in an agricultural watershed.

Developing the capability to predict pathogens in surface water is important for reducing the risk that such organisms pose to human health. In this study, three primary data source scenarios (measured stream flow and water quality, modelled stream flow and water quality, and host-associated Bacteroidales) are investigated within a Classification and Regression Tree Analysis (CART) framework for classifying pathogen (Escherichia coli 0157:H7, Salmonella, Campylobacter, Cryptosporidium, and Giardia) presence and absence (P/A) for a 178 km(2) agricultural watershed. To provide modelled data, a Soil Water Assessment Tool (SWAT) model was developed to predict stream flow, total suspended solids (TSS), total N and total P, and fecal indicator bacteria loads; however, the model was only successful for flow and total N and total P simulations, and did not accurately simulate TSS and indicator bacteria transport. Also, the SWAT model was not sensitive to an observed reduction in the cattle population within the watershed that may have resulted in significant reduction in E. coli concentrations and Salmonella detections. Results show that when combined with air temperature and precipitation, SWAT modelled stream flow and total P concentrations were useful for classifying pathogen P/A using CART methodology. From a suite of host-associated Bacteroidales markers used as independent variables in CART analysis, the ruminant marker was found to be the best initial classifier of pathogen P/A. Of the measured sources of independent variables, air temperature, precipitation, stream flow, and total P were found to be the most important variables for classifying pathogen P/A. Results indicate a close relationship between cattle pollution and pathogen occurrence in this watershed, and an especially strong link between the cattle population and Salmonella detections.

Assessment of a new Bacteroidales marker targeting North American beaver (Castor canadensis) fecal pollution by real-time PCR.

In many settings wildlife can be a significant source of fecal pathogen input into surface water. The North American beaver (Castor canadensis) is a zoonotic reservoir for several human pathogens including Cryptosporidium spp. and Giardia spp. In order to specifically detect fecal pollution by beavers, we have developed and validated a beaver-specific Bacteroidales marker, designated Beapol01, based on the 16S rRNA gene. The marker is suitable for quantifying pollution using real-time PCR. The specificity and sensitivity of the marker was excellent, Beaver signal was detected in water of a mixed-activity watershed harbouring this rodent. Overall, Beapol01 will be useful for a better understanding of fecal source inputs in drainage basins inhabited by the beaver.

Coherence among different microbial source tracking markers in a small agricultural stream with or without livestock exclusion practices.

Over 1,400 water samples were collected biweekly over 6 years from an intermittent stream protected and unprotected from pasturing cattle. The samples were monitored for host-specific Bacteroidales markers, Cryptosporidium species/genotypes, viruses and coliphages associated with humans or animals, and bacterial zoonotic pathogens. Ruminant Bacteroidales markers did not increase within the restricted cattle access reach of the stream, whereas the ruminant Bacteroidales marker increased significantly in the unrestricted cattle access reach. Human Bacteroidales markers significantly increased downstream of homes where septic issues were documented. Wildlife Bacteroidales markers were detected downstream of the cattle exclusion practice where stream and riparian habitat was protected, but detections decreased after the unrestricted pasture, where the stream and riparian zone was unprotected from livestock. Detection of a large number of human viruses was shown to increase downstream of homes, and similar trends were observed for the human Bacteroidales marker. There was considerable interplay among biomarkers with stream flow, season, and the cattle exclusion practices. There were no to very weak associations with Bacteroidales markers and bacterial, viral, and parasitic pathogens. Overall, discrete sample-by-sample coherence among the different microbial source tracking markers that expressed a similar microbial source was minimal, but spatial trends were physically meaningful in terms of land use (e.g., beneficial management practice) effects on sources of fecal pollution.

Impact of manure fertilization on the abundance of antibiotic-resistant bacteria and frequency of detection of antibiotic resistance genes in soil and on vegetables at harvest.

Consumption of vegetables represents a route of direct human exposure to bacteria found in soil. The present study evaluated the complement of bacteria resistant to various antibiotics on vegetables often eaten raw (tomato, cucumber, pepper, carrot, radish, lettuce) and how this might vary with growth in soil fertilized inorganically or with dairy or swine manure. Vegetables were sown into field plots immediately following fertilization and harvested when of marketable quality. Vegetable and soil samples were evaluated for viable antibiotic-resistant bacteria by plate count on Chromocult medium supplemented with antibiotics at clinical breakpoint concentrations. DNA was extracted from soil and vegetables and evaluated by PCR for the presence of 46 gene targets associated with plasmid incompatibility groups, integrons, or antibiotic resistance genes. Soil receiving manure was enriched in antibiotic-resistant bacteria and various antibiotic resistance determinants. There was no coherent corresponding increase in the abundance of antibiotic-resistant bacteria enumerated from any vegetable grown in manure-fertilized soil. Numerous antibiotic resistance determinants were detected in DNA extracted from vegetables grown in unmanured soil. A smaller number of determinants were additionally detected on vegetables grown only in manured and not in unmanured soil. Overall, consumption of raw vegetables represents a route of human exposure to antibiotic-resistant bacteria and resistance determinants naturally present in soil. However, the detection of some determinants on vegetables grown only in freshly manured soil reinforces the advisability of pretreating manure through composting or other stabilization processes or mandating offset times between manuring and harvesting vegetables for human consumption.

Accelerated Biodegradation of Veterinary Antibiotics in Agricultural Soil following Long-Term Exposure, and Isolation of a Sulfamethazine-degrading sp.

The World Health Organization has identified antibiotic resistance as one of the top three threats to global health. There is concern that the use of antibiotics as growth promoting agents in livestock production contributes to the increasingly problematic development of antibiotic resistance. Many antibiotics are excreted at high rates, and the land application of animal manures represents a significant source of environmental exposure to these agents. To evaluate the long-term effects of antibiotic exposure on soil microbial populations, a series of field plots were established in 1999 that have since received annual applications of a mixture of sulfamethazine (SMZ), tylosin (TYL), and chlortetracycline (CTC). During the first 6 yr (1999-2004) soils were treated at concentrations of 0, 0.01 0.1, and 1.0 mg kg soil, in subsequent years at concentrations of 0, 0.1, 1.0, and 10 mg kg soil. The lower end of this concentration range is within that which would result from an annual application of manure from medicated swine. Following ten annual applications, the fate of the drugs in the soil was evaluated. Residues of SMZ and TYL, but not CTC were removed much more rapidly in soil with a history of exposure to 10 mg/kg drugs than in untreated control soil. Residues of C-SMZ were rapidly and thoroughly mineralized to CO in the historically treated soils, but not in the untreated soil. A SMZ-degrading sp. was isolated from the treated soil. Overall, these results indicate that soil bacteria adapt to long-term exposure to some veterinary antibiotics resulting in sharply reduced persistence. Accelerated biodegradation of antibiotics in matrices exposed to agricultural, wastewater, or pharmaceutical manufacturing effluents would attenuate environmental exposure to antibiotics, and merits investigation in the context of assessing potential risks of antibiotic resistance development in environmental matrices.

Quantitative multi-year elucidation of fecal sources of waterborne pathogen contamination in the South Nation River basin using bacteroidales microbial source tracking markers.

Over a seven-year period (2004-2010) 1095 water samples were obtained from the South Nation River basin at multiple watershed monitoring sites (Ontario, Canada). Real-time PCR using Bacteroidales specific markers was used to identify the origin (human (10% prevalence), ruminant (22%), pig (~2%), Canada goose (4%) and muskrat (7%)) of fecal pollution. In parallel, the distribution of fecal indicator bacteria and waterborne pathogens (Cryptosporidium oocysts, Giardia cysts, Escherichia coli O157:H7, Salmonella enterica and Campylobacter spp.) was evaluated. Associations between the detection of specific Bacteroidales markers and the presence of fecal indicator bacteria, pathogens, and distinct land use or environmental variables were evaluated. Linear correlations between Bacteroidales markers and fecal indicator bacteria were weak. However, mean marker densities, and the presence and absence of markers could be discriminated on the basis of threshold fecal indicator densities. The ruminant-specific Bacteroidales marker was the most frequently detected marker in water, consistent with the large number of dairy farms in the study area. Detection of the human or the ruminant markers were associated with a slightly higher risk of detecting S. enterica. Detection of the muskrat marker was related to more frequent Campylobacter spp. detections. Important positive associations between markers and pathogens were found among: i) total Bacteroidales and Cryptosporidium and Giardia, ii) ruminant marker and S. enterica, and iii) muskrat and Campylobacter spp.

Development and validation of a microbial source tracking marker for the detection of fecal pollution by muskrats.

Surface waters in mixed-activity watersheds can be compromised by fecal pollution from livestock production, sewage outflows or leaking septic systems, or avian or mammalian wildlife. While undertaking water quality research in Eastern Ontario Canada, we observed an abundance of muskrats (Ondatra zibethicus) in some waterways, and sought to determine their significance with respect to fecal pollution loads. To that end, we developed and validated here a sensitive and specific marker, designated MuBa01, based on the 16S rRNA gene that distinguishes muskrat feces from human, livestock, or other wildlife sources. DNA from waterways inhabited by muskrats carried the marker. The widely used pig-specific marker Pig-2-Bac yielded a product with some muskrat feces, suggesting that it lacks specificity required to establish the significance of swine production in areas inhabited by muskrats. Overall, the MuBa01 marker will be useful for distinguishing the significance of this wide-ranging semi-aquatic mammal in the context of other fecal pollution sources.

Microbial and chemical markers: runoff transfer in animal manure-amended soils.

Fecal contamination of water resources is evaluated by the enumeration of the fecal coliforms and Enterococci. However, the enumeration of these indicators does not allow us to differentiate between the sources of fecal contamination. Therefore, it is important to use alternative indicators of fecal contamination to identify livestock contamination in surface waters. The concentration of fecal indicators (, enteroccoci, and F-specific bacteriophages), microbiological markers (Rum-2-bac, Pig-2-bac, and ), and chemical fingerprints (sterols and stanols and other chemical compounds analyzed by 3D-fluorescence excitation-matrix spectroscopy) were determined in runoff waters generated by an artificial rainfall simulator. Three replicate plot experiments were conducted with swine slurry and cattle manure at agronomic nitrogen application rates. Low amounts of bacterial indicators (1.9-4.7%) are released in runoff water from swine-slurry-amended soils, whereas greater amounts (1.1-28.3%) of these indicators are released in runoff water from cattle-manure-amended soils. Microbial and chemical markers from animal manure were transferred to runoff water, allowing discrimination between swine and cattle fecal contamination in the environment via runoff after manure spreading. Host-specific bacterial and chemical markers were quantified for the first time in runoff waters samples after the experimental spreading of swine slurry or cattle manure.