Soil type and moisture content alter soil microbial responses to manure from cattle administered antibiotics.

Growing concerns about the global antimicrobial resistance crisis require a better understanding of how antibiotic resistance persists in soil and how antibiotic exposure impacts soil microbial communities. In agroecosystems, these responses are complex because environmental factors may influence how soil microbial communities respond to manure and antibiotic exposure. The study aimed to determine how soil type and moisture alter responses of microbial communities to additions of manure from cattle treated with antibiotics. Soil microcosms were constructed using two soil types at 15, 30, or 45% moisture. Microcosms received biweekly additions of manure from cattle given cephapirin or pirlimycin, antibiotic-free manure, or no manure. While soil type and moisture had the largest effects on microbiome structure, impacts of manure treatments on community structure and individual ARG abundances were observed across varying soil conditions. Activity was also affected, as respiration increased in the cephapirin treatment but decreased with pirlimycin. Manure from cattle antibiotics also increased NH4+ and decreased NO3- availability in some scenarios, but the effects were heavily influenced by soil type and moisture. Overall, this work demonstrates that environmental conditions can alter how manure from cattle administered antibiotics impact the soil microbiome. A nuanced approach that considers environmental variability may benefit the long-term management of antibiotic resistance in soil systems.

Campylobacter Colonization and Diversity in Young Turkeys in the Context of Gastrointestinal Distress and Antimicrobial Treatment.

Young turkeys are vulnerable to undifferentiated gastrointestinal distress, including "irritable and crabby syndrome" (ICS), which compromises flock performance and is typically treated with a combination of penicillin and gentamicin (P/G). However, the effects of ICS and P/G treatment on Campylobacter remain poorly understood. We investigated the impact of ICS and P/G treatment on Campylobacter levels and diversity in four flocks from three turkey farms. Cecum and jejunum samples were analyzed weekly from day of hatch to week 4-5. All four flocks became colonized with multidrug resistant (MDR) Campylobacter jejuni and C. coli by week 2-3, and two developed ICS. ICS and P/G treatment did not significantly impact total Campylobacter levels or strain genotypes but impacted species and antimicrobial resistance (AMR) profiles. One flock was raised under antibiotic-free (ABF) conditions while another flock at the same farm was raised conventionally. The ABF flock did not develop ICS while its counterpart did. However, Campylobacter strains, AMR profiles and sequence types were generally shared between these two flocks. Our findings suggest that ICS and P/G treatment impacted Campylobacter population dynamics in commercial young turkey flocks, and that ABF flocks may become readily colonized by MDR strains from non-ABF flocks at the same farm.

Moisture-driven divergence in mineral-associated soil carbon persistence.

Mineral stabilization of soil organic matter is an important regulator of the global carbon (C) cycle. However, the vulnerability of mineral-stabilized organic matter (OM) to climate change is currently unknown. We examined soil profiles from 34 sites across the conterminous USA to investigate how the abundance and persistence of mineral-associated organic C varied with climate at the continental scale. Using a novel combination of radiocarbon and molecular composition measurements, we show that the relationship between the abundance and persistence of mineral-associated organic matter (MAOM) appears to be driven by moisture availability. In wetter climates where precipitation exceeds evapotranspiration, excess moisture leads to deeper and more prolonged periods of wetness, creating conditions which favor greater root abundance and also allow for greater diffusion and interaction of inputs with MAOM. In these humid soils, mineral-associated soil organic C concentration and persistence are strongly linked, whereas this relationship is absent in drier climates. In arid soils, root abundance is lower, and interaction of inputs with mineral surfaces is limited by shallower and briefer periods of moisture, resulting in a disconnect between concentration and persistence. Data suggest a tipping point in the cycling of mineral-associated C at a climate threshold where precipitation equals evaporation. As climate patterns shift, our findings emphasize that divergence in the mechanisms of OM persistence associated with historical climate legacies need to be considered in process-based models.

Stream bacterial diversity peaks at intermediate freshwater salinity and varies by salt type.

Anthropogenic freshwater salinization is an emerging and widespread water quality stressor that increases salt concentrations of freshwater, where specific upland land-uses produce distinct ionic profiles. In-situ studies find salinization in disturbed landscapes is correlated with declines in stream bacterial diversity, but cannot isolate the effects of salinization from multiple co-occurring stressors. By manipulating salt concentration and type in controlled microcosm studies, we identified direct and complex effects of freshwater salinization on bacterial diversity in the absence of other stressors common in field studies using chloride salts. Changes in both salt concentration and cation produced distinct bacterial communities. Bacterial richness, or the total number of amplicon sequence variants (ASVs) detected, increased at conductivities as low as 350 µS cm-1, which is opposite the observations from field studies. Richness remained elevated at conductivities as high as 1500 µS cm-1 in communities exposed to a mixture of Ca, Mg, and K chloride salts, but decreased in communities exposed to NaCl, revealing a classic subsidy-stress response. Exposure to different chloride salts at the same conductivity resulted in distinct bacterial community structure, further supporting that salt type modulates responses of bacterial communities to freshwater salinization. Community variability peaked at 125-350 µS cm-1 and was more similar at lower and upper conductivities suggesting possible shifts in deterministic vs. stochastic assembly mechanisms across freshwater salinity gradients. Based on these results, we hypothesize that modest freshwater salinization (125-350 µS cm-1) lessens hypo-osmotic stress, reducing the importance of salinity as an environmental filter at intermediate freshwater ranges but effects of higher salinities at the upper freshwater range differ based on salt type. Our results also support previous findings that ~300 µS cm-1 is a biological effect concentration and effective salt management strategies may need to consider variable effects of different salt types associated with land-use.

Horsenettle (Solanum carolinense) fruit bacterial communities are not variable across fine spatial scales.

Fruit house microbial communities that are unique from the rest of the plant. While symbiotic microbial communities complete important functions for their hosts, the fruit microbiome is often understudied compared to other plant organs. Fruits are reproductive tissues that house, protect, and facilitate the dispersal of seeds, and thus they are directly tied to plant fitness. Fruit microbial communities may, therefore, also impact plant fitness. In this study, we assessed how bacterial communities associated with fruit of Solanum carolinense, a native herbaceous perennial weed, vary at fine spatial scales (<0.5 km). A majority of the studies conducted on plant microbial communities have been done at large spatial scales and have observed microbial community variation across these large spatial scales. However, both the environment and pollinators play a role in shaping plant microbial communities and likely have impacts on the plant microbiome at fine scales. We collected fruit samples from eight sampling locations, ranging from 2 to 450 m apart, and assessed the fruit bacterial communities using 16S rRNA gene amplicon sequencing. Overall, we found no differences in observed richness or microbial community composition among sampling locations. Bacterial community structure of fruits collected near one another were not more different than those that were farther apart at the scales we examined. These fine spatial scales are important to obligate out-crossing plant species such as S. carolinense because they are ecologically relevant to pollinators. Thus, our results could imply that pollinators serve to homogenize fruit bacterial communities across these smaller scales.

Emergent properties of microbial communities drive accelerated biogeochemical cycling in disturbed temperate forests.

Despite ever-increasing availability of detailed information about microbial community structure, relationships of microbial diversity with ecosystem functioning remain unclear. We investigated these relationships at the Coweeta Hydrologic Laboratory, where past forest disturbances (e.g., clear-cut) have altered both ecosystem processes (e.g., increased N export) and microbial communities (e.g., increased bacterial diversity). We sampled soils from disturbed and adjacent reference forests, characterized resident microbial communities, and measured several microbial C-cycle and N-cycle process rates. Microbial communities from historically disturbed soils exhibited altered ecosystem functioning, including generally higher rates of C- and N-cycle processes. Disturbed soil microbial communities also exhibited altered ecosystem multifunctionality, a composite variable consisting of all measured process rates as well as extracellular enzyme activities. Although we found few relationships between ecosystem functions and microbial alpha diversity, all functions were correlated with microbial community composition metrics, particularly r:K strategist ratios of bacterial phyla. Additionally, for both ecosystem multifunctionality and specific processes (i.e., C- and N-mineralization), microbial metrics significantly improved models seeking to explain variation in process rates. Our work sheds light on the links between microbial communities and ecosystem functioning and identifies specific microbial metrics important for modeling ecosystem responses to environmental change.

Historical forest disturbance mediates soil microbial community responses to drought.

Despite the abundance of studies demonstrating the effects of drought on soil microbial communities, the role of land use legacies in mediating these drought effects is unclear. To assess historical land use influences on microbial drought responses, we conducted a drought-rewetting experiment in soils from two adjacent and currently forested watersheds with distinct land use histories: an undisturbed 'reference' site and a 'disturbed' site that was clear-cut and converted to agriculture ~60 years prior. We incubated intact soil cores at either constant moisture or under a drought-rewet treatment and characterized bacterial and fungal communities using amplicon sequencing throughout the experiment. Bacterial alpha diversity decreased following drought-rewetting while fungal diversity increased. Bacterial beta diversity also changed markedly following drought-rewetting, especially in historically disturbed soils, while fungal beta diversity exhibited little response. Additionally, bacterial beta diversity in disturbed soils recovered less from drought-rewetting compared with reference soils. Disturbed soil communities also exhibited notable reductions in nitrifying taxa, increases in putative r-selected bacteria, and reductions in network connectivity following drought-rewetting. Overall, our study reveals historical land use to be important in mediating responses of soil bacterial communities to drought, which will influence the ecosystem-scale trajectories of these environments under ongoing and future climate change.

Increasing Freshwater Salinity Impacts Aerosolized Bacteria.

Increases in the salt concentration of freshwater result in detrimental impacts on water quality and ecosystem biodiversity. Biodiversity effects include freshwater microbiota, as increasing salinity can induce shifts in the structure of native freshwater bacterial communities, which could disturb their role in mediating basal ecosystem services. Moreover, salinity affects the wave breaking and bubble-bursting mechanisms via which water-to-air dispersal of bacteria occurs. Given this dual effect of freshwater salinity on waterborne bacterial communities and their aerosolization mechanism, further effects on aerosolized bacterial diversity and abundance are anticipated. Cumulative salt additions in the freshwater-euhaline continuum (0-35 g/kg) were administered to a freshwater sample aerosolized inside a breaking wave analogue tank. Waterborne and corresponding airborne bacteria were sampled at each salinity treatment and later analyzed for diversity and abundance. Results demonstrated that the airborne bacterial community was significantly different (PERMANOVA; F1,22 = 155.1, r2 = 0.38, p < 0.001) from the waterborne community. The relative aerosolization factor (r-AF), defined as the air-to-water relative abundance ratio, revealed that different bacterial families exhibited either an enhanced (r-AF ≫ 1), neutral (r-AF ∼ 1), or diminished (r-AF ≪ 1) transfer to the aerosol phase throughout the salinization gradient. Going from freshwater to euhaline conditions, aerosolized bacterial abundance exhibited a nonmonotonic response with a maximum peak at lower oligohaline conditions (0.5-1 g/kg), a decline at higher oligohaline conditions (5 g/kg), and a moderate increase at polyhaline-euhaline conditions (15-35 g/kg). Our results demonstrate that increases in freshwater salinity are likely to influence the abundance and diversity of aerosolized bacteria. These shifts in aerosolized bacterial communities might have broader implications on public health by increasing exposure to airborne pathogens via inhalation. Impacts on regional climate, related to changes in biological ice-nucleating particles (INPs) emission from freshwater, are also expected.

Freshwater salinization increases survival of Escherichia coli and risk of bacterial impairment.

Elevated levels of Escherichia coli (E. coli) are responsible for more designated freshwater stream impairments than any other contaminant in the United States. E. coli are intentionally used as a sentinel of fecal contamination for freshwaters because previous research indicates that salt concentrations in brackish or marine waters reduce E. coli survival, rendering it a less effective indicator of public health risks. Given increasing evidence of freshwater salinization associated with upland anthropogenic land-use, understanding the effects on fecal indicators is critical; however, changes in E. coli survival along the freshwater salinity range (≤ 1500 µS cm-1) have not been previously examined. Through a series of controlled mesocosm experiments, we provide direct evidence that salinization causes E. coli survival rates in freshwater to increase at conductivities as low as 350 µS cm-1 and peak at 1500 µS cm-1, revealing a subsidy-stress response across the freshwater-marine continuum. Furthermore, specific base cations affect E. coli survival differently, with Mg2+ increasing E. coli survival rates relative to other chloride salts. Further investigation of the mechanisms by which freshwater salinization increases susceptibility to or exacerbates bacterial water quality impairments is recommended. Addressing salinization with nuanced approaches that consider salt sources and chemistry could assist in prioritizing and addressing bacterial water quality management.

Effects of bacterial inoculation and nitrogen loading on bacterial-algal consortium composition and functions in an integrated photobioelectrochemical system.

An integrated photo-bioelectrochemical system (IPB) for wastewater treatment combines a microbial fuel cell with an algal bioreactor, eliminating requirements for aeration, promoting electricity generation, remediating nutrients and producing algal biomass for conversion into biofuel or other bioproducts. To examine strategies for improving IPB functions of electrochemical output and nutrient removal efficiency, this study tested effects of cathode bacterial inoculation and nitrogen loading on cathode microbial community and IPB performance. IPB cathodes were inoculated with the green alga Chlorella vulgaris, in combination with nitrite-oxidizing bacteria (NOB) Nitrobacter winogradskyi, and/or ammonium-oxidizing bacteria (AOB) Nitrosomonas europaea. IPB performance was examined before and after nitrifying bacteria inoculations and under three ammonium loading concentrations in the wastewater medium. Bacterial communities in the cathode suspension and biofilm were examined by 16S rRNA gene sequence analysis. Relative to the algae only control, cathode inoculation with NOB and/or AOB improved net nutrient removal, but resulted in reduced dissolved oxygen availability, which impaired electricity generation. Higher ammonium loading increased electricity production and nutrient removal, possibly by overcoming algal-bacterial competition. Inoculation with nitrifying bacteria resulted in minor changes to total bacterial composition and AOB or NOB comprised <3% of total sequences after 1 month. Community composition changed more dramatically following increase in ammonium-N concentration from 40 to 80 mg L-1. Manipulation of N loading could be a useful strategy to improve IPB performance, while inoculation of AOB or NOB may be beneficial for treatment of water with high ammonium loading when N removal is the primary system goal.

Prolonged exposure to manure from livestock-administered antibiotics decreases ecosystem carbon-use efficiency and alters nitrogen cycling.

Microbial communities drive soil ecosystem function but are also susceptible to environmental disturbances. We investigated whether exposure to manure sourced from cattle either administered or not administered antibiotics affected microbially mediated terrestrial ecosystem function. We quantified changes in microbial community composition via amplicon sequencing, and terrestrial elemental cycling via a stable isotope pulse-chase. Exposure to manure from antibiotic-treated cattle caused: (i) changes in microbial community structure; and (ii) alterations in elemental cycling throughout the terrestrial system. This exposure caused changes in fungal : bacterial ratios, as well as changes in bacterial community structure. Additionally, exposure to manure from cattle treated with pirlimycin resulted in an approximate two-fold increase in ecosystem respiration of recently fixed-carbon, and a greater proportion of recently added nitrogen in plant and soil pools compared to the control manure. Manure from antibiotic-treated cattle therefore affects terrestrial ecosystem function via the soil microbiome, causing decreased ecosystem carbon use efficiency, and altered nitrogen cycling.

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.

Amphibian skin fungal communities vary across host species and do not correlate with infection by a pathogenic fungus.

Amphibian population declines caused by the fungus Batrachochytrium dendrobatidis (Bd) have prompted studies on the bacterial community that resides on amphibian skin. However, studies addressing the fungal portion of these symbiont communities have lagged behind. Using ITS1 amplicon sequencing, we examined the fungal portion of the skin microbiome of temperate and tropical amphibian species currently coexisting with Bd in nature. We assessed cooccurrence patterns between bacterial and fungal OTUs using a subset of samples for which bacterial 16S rRNA gene amplicon data were also available. We determined that fungal communities were dominated by members of the phyla Ascomycota and Basidiomycota, and also by Chytridiomycota in the most aquatic amphibian species. Alpha diversity of the fungal communities differed across host species, and fungal community structure differed across species and regions. However, we did not find a correlation between fungal diversity/community structure and Bd infection, though we did identify significant correlations between Bd and specific OTUs. Moreover, positive bacterial-fungal cooccurrences suggest that positive interactions between these organisms occur in the skin microbiome. Understanding the ecology of amphibian skin fungi, and their interactions with bacteria will complement our knowledge of the factors influencing community assembly and the overall function of these symbiont communities.

Fecal indicator dynamics at the watershed scale: Variable relationships with land use, season, and water chemistry.

Tracking fecal contamination in surface waters is critical to remediating water quality; however, general and source-specific fecal indicators often provide conflicting results. To understand the spatial and temporal dynamics of multiple fecal indicators and the sources they represent, we measured weekly concentrations of two general fecal indicator bacteria (FIB), a genetic indicator of human-associated Bacteroides (HF183), and surface water chemistry in nine mixed land-use watersheds in southwest Virginia, USA. At the watershed scale, general and source-specific indicators were decoupled, with distinct spatial, temporal, and chemical patterns. Random Forest analysis of individual sample variability identified temperature, watershed, nutrients, and cations as top predictors of indicator concentrations. However, these patterns - and the specific nutrients and cations identified - varied by indicator type. Among watersheds, FIB increased with developed land cover and during the summer months, while HF183 increased during the winter and only in urban watersheds. Nutrients generally related poorly to FIB and HF183, except E. coli, which correlated with total nitrogen. In contrast, all fecal indicators showed strong correlations with cations. FIB were more strongly related to calcium, magnesium, and potassium concentrations, while HF183 was related to sodium. These results suggest that, even at the watershed scale, 1) HF183 detects mainly human fecal contamination, while FIB detect broader ecosystem fecal inputs, and 2) poor correlation between specific and generalist fecal indicators is caused by unique spatial, temporal, and transport dynamics of different fecal sources in watersheds.

Soil Bacterial and Fungal Communities Exhibit Distinct Long-Term Responses to Disturbance in Temperate Forests.

In Appalachian ecosystems, forest disturbance has long-term effects on microbially driven biogeochemical processes such as nitrogen (N) cycling. However, little is known regarding long-term responses of forest soil microbial communities to disturbance in the region. We used 16S and ITS sequencing to characterize soil bacterial (16S) and fungal (ITS) communities across forested watersheds with a range of past disturbance regimes and adjacent reference forests at the Coweeta Hydrologic Laboratory in the Appalachian mountains of North Carolina. Bacterial communities in previously disturbed forests exhibited consistent responses, including increased alpha diversity and increased abundance of copiotrophic (e.g., Proteobacteria) and N-cycling (e.g., Nitrospirae) bacterial phyla. Fungal community composition also showed disturbance effects, particularly in mycorrhizal taxa. However, disturbance did not affect fungal alpha diversity, and disturbance effects were not consistent at the fungal class level. Co-occurrence networks constructed for bacteria and fungi showed that disturbed communities were characterized by more connected and tightly clustered network topologies, indicating that disturbance alters not only community composition but also potential ecological interactions among taxa. Although bacteria and fungi displayed different long-term responses to forest disturbance, our results demonstrate clear responses of important bacterial and fungal functional groups (e.g., nitrifying bacteria and mycorrhizal fungi), and suggest that both microbial groups play key roles in the long-term alterations to biogeochemical processes observed following forest disturbance in the region.

Engineered and Environmental Controls of Microbial Denitrification in Established Bioretention Cells.

Bioretention cells (BRCs) are effective tools for treating urban stormwater, but nitrogen removal by these systems is highly variable. Improvements in nitrogen removal are hampered by a lack of data directly quantifying the abundance or activity of denitrifying microorganisms in BRCs and how they are controlled by original BRC design characteristics. We analyzed denitrifiers in twenty-three BRCs of different designs across three mid-Atlantic states (MD, VA, and NC) by quantifying two bacterial denitrification genes ( nirK and nosZ) and potential enzymatic denitrification rates within the soil medium. Overall, we found that BRC design factors, rather than local environmental variables, had the greatest effects on variation in denitrifier abundance and activity. Specifically, denitrifying populations and denitrification potential increased with organic carbon and inorganic nitrogen concentrations in the soil media and decreased in BRCs planted with grass compared to other types of vegetation. Furthermore, the top layers of BRCs consistently contained greater concentrations and activity of denitrifying bacteria than bottom layers, despite longer periods of saturation and the presence of permanently saturated zones designed to promote denitrification at lower depths. These findings suggest that there is still considerable potential for design improvements when constructing BRCs that could increase denitrification and mitigate nitrogen export to receiving waters.

Mainstream upflow nitritation-anammox system with hybrid anaerobic pretreatment: Long-term performance and microbial community dynamics.

Mainstream nitritation-anammox is of strong interest to energy- and resource-efficient domestic wastewater treatment. However, there lack in-depth studies of pretreatment, tests of actual wastewater, and examination of long-term performance. Herein, an upflow nitritation-anammox granular reactor has been investigated to treat primary effluent with a hybrid anaerobic reactor (HAR) as pretreatment for more than 300 days. This system achieved 92% of COD removal, 75% of which was accomplished by the HAR, and had an average final effluent COD concentration of 22 mg L-1. More than 90% of ammonium was removed in the nitritation-anammox reactor, achieving a nitrogen removal rate of 81.0 g N m-3 d-1 in the last stage. The accumulation of sulfate-reducing bacteria in the HAR evidenced the effect of sulfate on COD removal and subsequent nitrogen removal. Anammox bacteria (predominantly Ca. Jettenia asiatica) accounted for up to 40.2% of total granular communities, but their abundance decreased over time in the suspended communities. The dynamics of major metabolisms and functional genes involved in nitrogen conversion were predicted by PICRUSt based on the taxonomic data, providing more insights into the functions of the microbial communities. These results have demonstrated the effectiveness and importance of anaerobic pretreatment to successful mainstream nitritation-anammox.

Unravelling and Reconstructing the Nexus of Salinity, Electricity, and Microbial Ecology for Bioelectrochemical Desalination.

Microbial desalination cells (MDCs) are an emerging concept for simultaneous water/wastewater treatment and energy recovery. The key to developing MDCs is to understand fundamental problems, such as the effects of salinity on system performance and the role of microbial community and functional dynamics. Herein, a tubular MDC was operated under a wide range of salt concentrations (0.05-4 M), and the salinity effects were comprehensively examined. The MDC generated higher current with higher salt concentrations in the desalination chamber. When fed with 4 M NaCl, the MDC achieve a current density of 300 A m-3 (anode volume), which was one of the highest among bioelectrochemical system studies. Community analysis and electrochemical measurements suggested that electrochemically active bacteria Pseudomonas and Acinetobacter transferred electrons extracellularly via electron shuttles, and the consequent ion migration led to high anode salinities and conductivity that favored their dominance. Predictive functional dynamics and Bayesian networks implied that the taxa putatively not capable of extracellular electron transfer (e.g., Bacteroidales and Clostridiales) might indirectly contribute to bioelectrochemical desalination. By integrating the Bayesian network with logistic regression, current production was successfully predicted from taxonomic data. This study has demonstrated uncompromised system performance under high salinity and thus has highlighted the potential of MDCs as an energy-efficient technology to address water-energy challenges. The statistical modeling approach developed in this study represents a significant step toward understating microbial communities and predicting system performance in engineered biological systems.

Tracking the downstream impacts of inadequate sanitation in central Appalachia.

Poor sanitation in rural infrastructure is often associated with high levels of fecal contamination in adjacent surface waters, which presents a community health risk. Although microbial source tracking techniques have been widely applied to identify primary remediation needs in urban and/or recreational waters, use of human-specific markers has been more limited in rural watersheds. This study quantified the human source tracking marker Bacteroides-HF183, along with more general fecal indicators (i.e. culturable Escherichia coli and a molecular Enterococcus marker), in two Appalachian streams above and below known discharges of untreated household waste. Although E. coli and Enterococcus were consistently recovered in samples collected from both streams, Bacteroides-HF183 was only detected sporadically in one stream. Multiple linear regression analysis demonstrated a positive correlation between the concentration of E. coli and the proximity and number of known waste discharge points upstream; this correlation was not significant with respect to Bacteroides-HF183, likely due to the low number of quantifiable samples. These findings suggest that, while the application of more advanced source targeting strategies can be useful in confirming the influence of substandard sanitation on surface waters to justify infrastructure improvements, they may be of limited use without concurrent traditional monitoring targets and on-the-ground sanitation surveys.

Stormwater loadings of antibiotic resistance genes in an urban stream.

Antibiotic resistance presents a critical public health challenge and the transmission of antibiotic resistance via environmental pathways continues to gain attention. Factors driving the spread of antibiotic resistance genes (ARGs) in surface water and sources of ARGs in urban stormwater have not been well-characterized. In this study, five ARGs (sul1, sul2, tet(O), tet(W), and erm(F)) were quantified throughout the duration of three storm runoff events in an urban inland stream. Storm loads of all five ARGs were significantly greater than during equivalent background periods. Neither fecal indicator bacteria measured (E. coli or enterococci) was significantly correlated with sul1, sul2, or erm(F), regardless of whether ARG concentration was absolute or normalized to 16S rRNA levels. Both E. coli and enterococci were correlated with the tetracycline resistance genes, tet(O) and tet(W). Next-generation shotgun metagenomic sequencing was conducted to more thoroughly characterize the resistome (i.e., full complement of ARGs) and profile the occurrence of all ARGs described in current databases in storm runoff in order to inform future watershed monitoring and management. Between 37 and 121 different ARGs were detected in each stream sample, though the ARG profiles differed among storms. This study establishes that storm-driven transport of ARGs comprises a considerable fraction of overall downstream loadings and broadly characterizes the urban stormwater resistome to identify potential marker ARGs indicative of impact.