Successful Treatment of Recurrent Clostridioides difficile Infection Using a Novel, Drinkable, Oral Formulation of Fecal Microbiota.

BACKGROUND: Fecal microbiota transplants can be administered orally in encapsulated form or require invasive procedures to administer liquid formulations. There is a need for an oral liquid formulation of fecal microbiota for patients who are unable to swallow capsules, especially if they require multiple, repeated administrations. AIMS: These studies were conducted to develop a protocol to manufacture an organoleptically acceptable powdered fecal microbiota formulation that can be suspended in a liquid carrier and used for fecal microbiota transplantation. METHODS: Several processing steps were investigated, including extra washes of microbiota prior to lyophilization and an addition of a flavoring agent. The viability of bacteria in the transplant formulation was tested using live/dead microscopy staining and engraftment into antibiotic-treated mice. After development of a clinical protocol for suspension of the powdered microbiota, the new formulation was tested in three elderly patients with recurrent Clostridioides difficile infections and who have difficulties in swallowing capsules. Changes in the microbial community structure in one of the patients were characterized using 16S rRNA gene profiling and engraftment analysis. RESULTS: The processing steps used to produce an organoleptically acceptable suspension of powdered fecal microbiota did not result in loss of its viability. The powder could be easily suspended in a liquid carrier. The use of the new formulation was associated with abrogation of the cycle of C. difficile infection recurrences in the three patients. CONCLUSION: We developed a novel organoleptically acceptable liquid formulation of fecal microbiota that is suitable for use in clinical trials for patients with difficulties in swallowing capsules.

Structure and Functional Properties of Bacterial Communities in Surface Sediments of the Recently Declared Nutrient-Saturated Lake Villarrica in Southern Chile.

Lake Villarrica, one of Chile's main freshwater water bodies, was recently declared a nutrient-saturated lake due to increased phosphorus (P) and nitrogen (N) levels. Although a decontamination plan based on environmental parameters is being established, it does not consider microbial parameters. Here, we conducted high-throughput DNA sequencing and quantitative polymerase chain reaction (qPCR) analyses to reveal the structure and functional properties of bacterial communities in surface sediments collected from sites with contrasting anthropogenic pressures in Lake Villarrica. Alpha diversity revealed an elevated bacterial richness and diversity in the more anthropogenized sediments. The phylum Proteobacteria, Bacteroidetes, Acidobacteria, and Actinobacteria dominated the community. The principal coordinate analysis (PCoA) and redundancy analysis (RDA) showed significant differences in bacterial communities of sampling sites. Predicted functional analysis showed that N cycling functions (e.g., nitrification and denitrification) were significant. The microbial co-occurrence networks analysis suggested Chitinophagaceae, Caldilineaceae, Planctomycetaceae, and Phycisphaerae families as keystone taxa. Bacterial functional genes related to P (phoC, phoD, and phoX) and N (nifH and nosZ) cycling were detected in all samples by qPCR. In addition, an RDA related to N and P cycling revealed that physicochemical properties and functional genes were positively correlated with several nitrite-oxidizing, ammonia-oxidizing, and N-fixing bacterial genera. Finally, denitrifying gene (nosZ) was the most significant factor influencing the topological characteristics of co-occurrence networks and bacterial interactions. Our results represent one of a few approaches to elucidate the structure and role of bacterial communities in Chilean lake sediments, which might be helpful in conservation and decontamination plans.

p-Cresol Sulfate Is a Sensitive Urinary Marker of Fecal Microbiota Transplantation and Antibiotics Treatments in Human Patients and Mouse Models.

Fecal microbiota transplantation (FMT) has emerged as a highly effective therapy for recurrent Clostridioides difficile infection (rCDI) and also a potential therapy for other diseases associated with dysbiotic gut microbiota. Monitoring metabolic changes in biofluids and excreta is a noninvasive approach to identify the biomarkers of microbial recolonization and to understand the metabolic influences of FMT on the host. In this study, the pre-FMT and post FMT urine samples from 11 rCDI patients were compared through metabolomic analyses for FMT-induced metabolic changes. The results showed that p-cresol sulfate in urine, a microbial metabolite of tyrosine, was rapidly elevated by FMT and much more responsive than other microbial metabolites of aromatic amino acids (AAAs). Because patients were treated with vancomycin prior to FMT, the influence of vancomycin on the microbial metabolism of AAAs was examined in a mouse feeding trial, in which the decreases in p-cresol sulfate, phenylacetylglycine, and indoxyl sulfate in urine were accompanied with significant increases in their AAA precursors in feces. The inhibitory effects of antibiotics and the recovering effects of FMT on the microbial metabolism of AAAs were further validated in a mouse model of FMT. Overall, urinary p-cresol sulfate may function as a sensitive and convenient therapeutic indicator on the effectiveness of antibiotics and FMT for the desired manipulation of gut microbiota in human patients.

Afforestation can lower microbial diversity and functionality in deep soil layers in a semiarid region.

Afforestation is an effective approach to rehabilitate degraded ecosystems, but often depletes deep soil moisture. Presently, it is not known how an afforestation-induced decrease in moisture affects soil microbial community and functionality, hindering our ability to understand the sustainability of the rehabilitated ecosystems. To address this issue, we examined the impacts of 20 years of afforestation on soil bacterial community, co-occurrence pattern, and functionalities along vertical profile (0-500 cm depth) in a semiarid region of China's Loess Plateau. We showed that the effects of afforestation with a deep-rooted legume tree on cropland were greater in deep than that of in top layers, resulting in decreased bacterial beta diversity, more responsive bacterial taxa and functional groups, increased homogeneous selection, and decreased network robustness in deep soils (120-500 cm). Organic carbon and nitrogen decomposition rates and multifunctionality also significantly decreased by afforestation, and microbial carbon limitation significantly increased in deep soils. Moreover, changes in microbial community and functionality in deep layer was largely related to changes in soil moisture. Such negative impacts on deep soils should be fully considered for assessing afforestation's eco-environment effects and for the sustainability of ecosystems because deep soils have important influence on forest ecosystems in semiarid and arid climates.

Bioaugmentation with As-transforming bacteria improves arsenic availability and uptake by the hyperaccumulator plant Pteris vittata (L).

Inorganic arsenic (As) is a toxic and carcinogenic pollutant that has long-term impacts on environmental quality and human health. Pteris vittata plants hyperaccumulate As from soils. Soil bacteria are critical for As-uptake by P. vittata. We examined the use of taxonomically diverse soil bacteria to modulate As speciation in soil and their effect on As-uptake by P. vittata. Aqueous media inoculated with Pseudomonas putida MK800041, P. monteilii MK344656, P. plecoglossicida MK345459, Ochrobactrum intermedium MK346993 or Agrobacterium tumefaciens MK346997 resulted in the oxidation of 5-30% As(III) and a 49-79% reduction of As(V). Soil inoculated with P. monteilii increased extractable As(III) and As(V) from 0.5 and 0.09 in controls to 0.9 and 0.39 mg As kg-1 soil dry weight, respectively. Moreover, and P. vittata plants inoculated with P. monteilii, P. plecoglossicida, O. intermedium strains, and A. tumefaciens strains MK344655, MK346994, MK346997, significantly increased As-uptake by 43, 32, 12, 18, 16, and 14%, respectively, compared to controls. The greatest As-accumulation (1.9 ± 0.04 g kg-1 frond Dwt) and bioconcentration factor (16.3 ± 0.35) was achieved in plants inoculated with P. monteilii. Our findings indicate that the tested bacterial strains can increase As-availability in soils, thus enhancing As-accumulation by P. vittata. Novelty statement Pteris vittata, a well-known As-hyperaccumulator, has the remarkable ability to accumulate higher levels of As in their above-ground biomass. The As-tolerant bacteria-plant interactions play a significant role in bioremediation by mediating As-redox and controlling As-availability and uptake by P. vittata. Our studies indicated that most of the tested bacterial strains isolated from As-impacted soil significantly enhanced As-uptake by P. vittata. P. monteilii oxidized 20% of As(III) and reduced 50% of As(V), increased As-extraction from soils, and increased As-uptake by 43% greater compared with control. Therefore, these strains associated with P. vittata can be used in large-scale field applications to remediate As-contaminated soil.

Composition and predicted functions of the bacterial community in spouting pool sediments from the El Tatio Geyser field in Chile.

The El Tatio Geyser Field (ETGF), located in Northern Chile, is the main geyser field in the southern hemisphere. Despite this, details of its microbial ecology are still unknown. Here, we briefly report on  the composition and predicted functions of the bacterial community in spouting pool sediments from the ETGF as revealed by high-throughput sequencing of 16S rRNA genes. Results of this analysis showed that while there were differences in richness and diversity between samples, bacterial communities were primarily dominated by the phyla Proteobacteria, followed Firmicutes, Bacteroidetes, Acidobacteria, and Chloroflexi. Analyses of predicted functional activity indicated that the functions were mostly attributed to chemoheterotrophy and aerobic chemoheterotrophy, followed by sulfur (respiration of sulfur compounds and sulfate) and nitrogen (nitrate reduction, respiration of nitrogen and nitrate) cycling. Taken together, our results suggest a high diversity in taxonomy and predictive functions of bacterial communities in sediments from spouting pools. This study provides fundamentally important  information on the structure and function  predictive functions of microbiota  communities in spouting pools. Moreover, since the ETGF is intensively visited and impacted by tens of thousands of tourists every year, our results can be used to help guide the design of sustainable conservation strategies.

Influence of Environmental Stressors on the Microbiota of Zebra Mussels (Dreissena polymorpha).

Host-associated microbiota play a critical role in host fitness by providing nutrition, enhancing digestion capabilities, and by providing protection from pathogens. Here, we investigated the effects of two environmental stressors, temperature, and salinity, on the microbiota associated with zebra mussels (ZMs), a highly invasive bivalve in North America. To examine this in detail, lake-collected ZMs were acclimated to laboratory conditions, and subjected to temperature and salinity stress conditions. The impact of these stressors on the diversity, composition, and dynamics of ZM-associated microbiota were assessed by using amplicon- and shotgun-based sequencing, and qPCR-based approaches. Elevated temperature was found to be the primary driver of ZM mortality, although salinity alone also increased its likelihood. Stressor-induced ZM mortality, which ranged between 53 and 100%, was concomitant with significant increases in the relative abundance of several genera of putative opportunistic pathogens including Aeromonas. These genera were only present in low relative abundance in ZMs obtained from the control tank with 0% mortality. Shotgun sequencing and qPCR analyses indicated that the relative and absolute abundances of pathogenic Aeromonas species (particularly A. veronii) were significantly greater in temperature-induced dead ZMs. Taken together, our results show that environmental stress, especially elevated temperature (> 25 °C), is associated with the rapid mortality of ZMs as well as the proliferation of putative opportunistic bacterial pathogens.

Methanogen Abundance Thresholds Capable of Differentiating In Vitro Methane Production in Human Stool Samples.

BACKGROUND: Intestinal methane (CH4) gas production has been associated with a number of clinical conditions and may have important metabolic and physiological effects. AIMS: In this study, taxonomic and functional gene analyses and in vitro CH4 gas measurements were used to determine if molecular markers can potentially serve as clinical tests for colonic CH4 production. METHODS: We performed a cross-sectional study involving full stool samples collected from 33 healthy individuals. In vitro CH4 gas measurements were obtained after 2-h incubation of stool samples and used to characterize samples as CH4 positive (CH4+) and CH4 negative (CH4-; n = 10 and 23, respectively). Next, we characterized the fecal microbiota through high-throughput DNA sequencing with a particular emphasis on archaeal phylum Euryarchaeota. Finally, qPCR analyses, targeting the mcrA gene, were done to determine the ability to differentiate CH4+ versus CH4- samples and to delineate major methanogen species associated with CH4 production. RESULTS: Methanobrevibacter was found to be the most abundant methane producer and its relative abundance provides a clear distinction between CH4+ versus CH4- samples. Its sequencing-based relative abundance detection threshold for CH4 production was calculated to be 0.097%. The qPCR-based detection threshold separating CH4+ versus CH4- samples, based on mcrA gene copies, was 5.2 × 105 copies/g. CONCLUSION: Given the decreased time-burden placed on patients, a qPCR-based test on a fecal sample can become a valuable tool in clinical assessment of CH4 producing status.

Select and resequence reveals relative fitness of bacteria in symbiotic and free-living environments.

Assays to accurately estimate relative fitness of bacteria growing in multistrain communities can advance our understanding of how selection shapes diversity within a lineage. Here, we present a variant of the "evolve and resequence" approach both to estimate relative fitness and to identify genetic variants responsible for fitness variation of symbiotic bacteria in free-living and host environments. We demonstrate the utility of this approach by characterizing selection by two plant hosts and in two free-living environments (sterilized soil and liquid media) acting on synthetic communities of the facultatively symbiotic bacterium Ensifer meliloti We find (i) selection that hosts exert on rhizobial communities depends on competition among strains, (ii) selection is stronger inside hosts than in either free-living environment, and (iii) a positive host-dependent relationship between relative strain fitness in multistrain communities and host benefits provided by strains in single-strain experiments. The greatest changes in allele frequencies in response to plant hosts are in genes associated with motility, regulation of nitrogen fixation, and host/rhizobia signaling. The approach we present provides a powerful complement to experimental evolution and forward genetic screens for characterizing selection in bacterial populations, identifying gene function, and surveying the functional importance of naturally occurring genomic variation.

Source-Associated Gastroenteritis Risk from Swimming Exposure to Aging Fecal Pathogens.

Human contact with fecally contaminated waters often raises public health concern. The infection potential closely relates to the fecal source type and the aging persistence of waterborne pathogens. In this study, the health risk of contracting gastroenteritis from exposure to aging fecal contamination was predicted using source-associated markers. Microbial decay characteristics in typical summer seawater were incorporated into a pathogen dose estimation model for a constant fecal input. Results show that the median illness probability commensurate with the health benchmark of 36/1000 corresponded to the marker concentrations of ∼7.8, ∼6.6, ∼3.7, and ∼3.5 log10 gene copies/100 mL for seagulls, cattle, raw sewage, and treated effluent, respectively. The error in risk estimates due to neglecting microbial decay was linearly correlated to the decay differences between markers and pathogens. Specifically, the health risk associated with nonhuman sources, which was primarily contributed by bacterial and parasitic pathogens, can be substantially overestimated, while that for virus-dominated human sources was insignificantly affected by the differential decay. Additionally, seagulls dominated the Enterococcus concentration in waters with a mixture of the above-mentioned sources, although they posed limited health risk. This study provides an approach to understanding the influence of fecal aging on health risk estimation.

Indigenous soil bacteria and the hyperaccumulator Pteris vittata mediate phytoremediation of soil contaminated with arsenic species.

Arsenic (As) is a pollutant of major concern worldwide, posing as a threat to both human health and the environment. Phytoremediation has been proposed as a viable mechanism to remediate As-contaminated soil environments. Pot experiments were performed to evaluate the phytoextraction efficiency of As by Pteris vittata, a known As hyperaccumulating fern, from soil amended with different concentrations of arsenate [As(V)] and arsenite [As(III)], the more common, inorganic As forms in soil. The greatest accumulation of As (13.3 ± 0.36 g/kg Dwt) was found in fronds of plants grown in soil spiked with 1.0 g As(V)/kg. The maximum As-bioaccumulation factor (27.3 ± 1.9) was achieved by plants grown in soil amended with 0.05 g As(V)/kg. A total of 864 bacterial cultures were isolated and examined for their ability to enhance phytoremediation of As-contaminated soils. Traits examined included tolerance to As (III and V), production of siderophores, and/or ability to solubilize calcium phosphate and indole acetic acid (IAA) production. A culture-based survey shows greater numbers of viable and As-resistant bacteria were found in the rhizosphere of As-grown plants compared to bulk and unplanted soils. The percentage of bacteria resistant to As(V) was greater (P < 0.0001) than those resistant to As(III) in culture medium containing 0.5, 1, 1.5, and 2 g As/L. Higher (P < 0.0001) percentages of siderophore producing (77%) and phosphate solubilizing (61%) bacteria were observed among cultures isolated from unplanted soil. About 5% (44 of 864) of the isolates were highly resistant to both As (III) and As (V) (2 g/L), and were examined for their As-transformation ability and IAA production. A great proportion of the isolates produced IAA (82%) and promoted As (V)-reduction (95%) or As(III)-oxidation (73%), and 71% exhibited dual capacity for both As(V) reduction and As(III) oxidation. Phylogenetic analysis indicated that 67, 23, and 10% of these isolates belonged to Proteobacteria, Actinobacteria, and Firmicutes, respectively. Analysis of the 16S rRNA gene sequences confirmed that these isolates were closely related to 12 genera and 25 species of bacteria and were dominated by members of the genus Pseudomonas (39%). These results show that these isolates could potentially be developed as inocula for enhancing plant uptake during large scale phytoremediation of As-impacted soils.

Antibiotic-induced Disruption of Intestinal Microbiota Contributes to Failure of Vertical Sleeve Gastrectomy.

OBJECTIVE: The aim of this study was to test whether the perioperative composition of intestinal microbiota can contribute to variable outcomes following vertical sleeve gastrectomy (VSG). SUMMARY OF BACKGROUND DATA: Although bariatric surgery is the most effective treatment for obesity, metabolic outcomes are variable. METHODS: Diet-induced obese mice were randomized to VSG or sham surgery, with or without exposure to antibiotics that selectively suppress mainly gram-positive (fidaxomicin, streptomycin) or gram-negative (ceftriaxone) bacteria on postoperative days (POD) 1-4. Fecal microbiota was characterized before surgery and on POD 7 and 28. Mice were metabolically characterized on POD 30-32 and euthanized on POD 35. RESULTS: VSG resulted in weight loss and shifts in the intestinal microbiota composition relative to sham-operated mice. Antibiotic exposure resulted in sustained reductions in alpha (within-sample) diversity of microbiota and shifts in its composition. All antibiotic treatments proved to be detrimental to metabolic VSG outcomes, regardless of antimicrobial specificity of antibiotics. These effects involved functionally distinct pathways. Specifically, fidaxomicin and streptomycin markedly altered hepatic bile acid signaling and lipid metabolism, while ceftriaxone resulted in greater reduction of key antimicrobial peptides. However, VSG mice exposed to antibiotics, regardless of their specificity, had significantly increased subcutaneous adiposity and impaired glucose homeostasis without changes in food intake relative to control VSG mice. CONCLUSION: Dysbiosis induced by brief perioperative antibiotic exposure attenuates weight loss and metabolic improvement following VSG. Potential mechanisms include disruption of bile acid homeostasis and reduction in the production of gut antimicrobial peptides. Results of this study implicate the intestinal microbiota as an important contributor to metabolic homeostasis and a potentially modifiable target influencing clinical outcomes following VSG.

A microfluidic platform for the simultaneous quantification of methanogen populations in anaerobic digestion processes.

Methanogens are a diverse group of archaea that play a critical role in the global carbon cycle. The lack of appropriate molecular tools to simultaneously quantify numerous methanogenic taxa, however, has largely limited our ability to study these communities in a wide variety of habitats, such as anaerobic digesters (ADs). In this study, 34 probe-based quantitative PCR (qPCR) assays were designed to target all known methanogenic genera within the archaeal phylum Euryarchaeota. These qPCR assays were adapted to a high-throughput microfluidic platform, which allowed for the simultaneous detection and absolute quantification of numerous taxa in a single run. The resulting microfluidic qPCR (MFQPCR) platform was successfully used to decipher structure-function relationships among methanogenic communities in four laboratory-scale digesters exposed to a transient organic overload. Twelve of the 34 genera targeted in the MFQPCR were detected in the ADs, similar to results obtained using high-throughput sequencing. The MFQPCR platform and conventional qPCR assays also generated similar quantitative results. The MFQPCR tool developed here will help optimize AD technologies for efficient waste treatment and enhanced biogas production and can facilitate studies that will increase our understanding of methanogenic communities in other environments.

Breastmilk and NICU surfaces are potential sources of fungi for infant mycobiomes.

Surfaces within the neonatal intensive care unit (NICU), especially those handled frequently by hospital staff, provide sources of gut-colonizing bacteria for hospitalized infants, in addition to those acquired perinatally from maternal sources such as breastmilk. In comparison to bacteria, very little is known about potential sources of colonizing fungi in the NICU setting. Thus, the objective of this study was to characterize fungal communities (mycobiomes) of potential colonization sources for neonates hospitalized in a large university NICU. We hypothesized that the unit surfaces would contain different mycobiomes than those of human-associated (breastmilk) sources. We characterized mycobiomes of NICU surfaces of multiple individual patient care areas as well as those of breastmilk samples by sequencing the internal transcribed spacer region 2 (ITS2) of the fungal rDNA locus. We found that, across all samples, Candida and Saccharomyces species were the most prevalent taxa and had the greatest relative abundances. Breastmilk samples had significantly higher fungal alpha-diversities than NICU surface samples and fungal community compositions (beta diversities) differed significantly between the two sample types. Mycobiome compositions were predominantly driven by the relative abundances of three fungal taxa: Candida albicans, Candida parapsilosis, and Saccharomyces cerevisiae. In total, 21 individual fungal taxa showed significantly greater relative abundances in breastmilk as compared to NICU surfaces, with three being of particular interest to human health: Candida glabrata, Candida tropicalis, and Cryptococcus neoformans. Since no fungal DNA was detected when whole breastmilk was used as the DNA template, as opposed to breastmilk subjected to cell lysis during the DNA isolation procedure, our results indicate that DNA is from fungal cells and is not cell-free DNA. In summary, both NICU surfaces and human breastmilk harbor distinct fungal communities that could provide a source of fungi for the developing infant gut mycobiota. In particular, Candida and Saccharomyces species are abundant and prevalent for both of these potential sources that infants are exposed to.

An Alkane Sulfonate Monooxygenase Is Required for Symbiotic Nitrogen Fixation by Bradyrhizobium diazoefficiens (syn. Bradyrhizobium japonicum) USDA110T.

Sulfur (S)-containing molecules play an important role in symbiotic nitrogen fixation and are critical components of nitrogenase and other iron-S proteins. S deficiency inhibits symbiotic nitrogen fixation by rhizobia. However, despite its importance, little is known about the sources of S that rhizobia utilize during symbiosis. We previously showed that Bradyrhizobium diazoefficiens USDA110T can assimilate both inorganic and organic S and that genes involved in organic S utilization are expressed during symbiosis. Here, we show that a B. diazoefficiens USDA110T mutant with a sulfonate monooxygenase (ssuD) insertion is defective in nitrogen fixation. Microscopy analyses revealed that the ΔssuD mutant was defective in root hair infection and that ΔssuD mutant bacteroids showed degradation compared to the wild-type strain. Moreover, the ΔssuD mutant was significantly more sensitive to hydrogen peroxide-mediated oxidative stress than the wild-type strain. Taken together, these results show that the ability of rhizobia to utilize organic S plays an important role in symbiotic nitrogen fixation. Since nodules have been reported to be an important source of reduced S used during symbiosis and nitrogen fixation, further research will be needed to determine the mechanisms involved in the regulation of S assimilation by rhizobia.IMPORTANCE Rhizobia form symbiotic associations with legumes that lead to the formation of nitrogen-fixing nodules. Sulfur-containing molecules play a crucial role in nitrogen fixation; thus, the rhizobia inside nodules require large amounts of sulfur. Rhizobia can assimilate both inorganic (sulfate) and organic (sulfonates) sources of sulfur. However, very little is known about rhizobial sulfur metabolism during symbiosis. In this report, we show that sulfonate utilization by Bradyrhizobium diazoefficiens is important for symbiotic nitrogen fixation in both soybean and cowpea. The symbiotic defect is probably due to increased sensitivity to oxidative stress from sulfur deficiency in the mutant strain defective for sulfonate utilization. The results of this study can be extended to other rhizobium-legume symbioses, as sulfonate utilization genes are widespread in these bacteria.

Impacts of Sampling Design on Estimates of Microbial Community Diversity and Composition in Agricultural Soils.

Soil microbiota play important and diverse roles in agricultural crop nutrition and productivity. Yet, despite increasing efforts to characterize soil bacterial and fungal assemblages, it is challenging to disentangle the influences of sampling design on assessments of communities. Here, we sought to determine whether composite samples-often analyzed as a low cost and effort alternative to replicated individual samples-provide representative summary estimates of microbial communities. At three Minnesota agricultural research sites planted with an oat cover crop, we conducted amplicon sequencing for soil bacterial and fungal communities (16SV4 and ITS2) of replicated individual or homogenized composite soil samples. We compared soil microbiota from within and among plots and then among agricultural sites using both sampling strategies. Results indicated that single or multiple replicated individual samples, or a composite sample from each plot, were sufficient for distinguishing broad site-level macroecological differences among bacterial and fungal communities. Analysis of a single sample per plot captured only a small fraction of the distinct OTUs, diversity, and compositional variability detected in the analysis of multiple individual samples or a single composite sample. Likewise, composite samples captured only a fraction of the diversity represented by the six individual samples from which they were formed, and, on average, analysis of two or three individual samples offered greater compositional coverage (i.e., greater number of OTUs) than a single composite sample. We conclude that sampling design significantly impacts estimates of bacterial and fungal communities even in homogeneously managed agricultural soils, and our findings indicate that while either strategy may be sufficient for broad macroecological investigations, composites may be a poor substitute for replicated samples at finer spatial scales.

Influence of Library Composition on SourceTracker Predictions for Community-Based Microbial Source Tracking.

Community-based microbial source tracking (MST) utilizes high-throughput DNA sequencing to profile and compare the microbial communities in different fecal sources and environmental samples. SourceTracker, a program that compares a library of OTUs from fecal sources (i.e., sources) to those in environmental samples (i.e., sinks) in order to determine sources of fecal contamination, is an emerging tool for community-based MST studies. In this study, we investigated the ability of SourceTracker to determine sources of known fecal contamination in spiked, in situ mesocosms containing different source contributors. We also evaluated how SourceTracker results were impacted by accounting for autochthonous taxa present in the sink environment. While SourceTracker was able to predict most sources present in the in situ mesocosms, fecal source library composition substantially influenced the program's ability to predict source contributions. Moreover, prediction results were most reliable when the library contained only known sources, autochthonous taxa were accounted for and when source profiles had low intragroup variability. Although SourceTracker struggled to differentiate between sources with similar bacterial community structures, it was able to consistently identify abundant and expected sources, suggesting that the SourceTracker program can be a useful tool for community-based MST studies.

Cultivar and phosphorus effects on switchgrass yield and rhizosphere microbial diversity.

Switchgrass (Panicum virgatum L.) is a native perennial grass identified as a promising biofuel crop for production on marginal agricultural lands. As such, research into switchgrass fertility and the switchgrass rhizosphere microbiome has been ongoing in an effort to increase production sustainability. We examined the effects of cultivar and phosphorus (P) fertilization on biomass yield, P removal, and rhizosphere bacterial and fungal community structure in three switchgrass cultivars: Sunburst, Shawnee, and Liberty. The Liberty cv. is the first lowland-type bioenergy switchgrass adapted to USDA hardiness zones 4, 5, and 6. On a medium soil test P clay loam soil, biomass yield response to applied P was linear, increasing 135 kg ha-1 for every kilogram of P applied prior to establishment. Average post-frost biomass yield was 9.6 Mg ha-1 year-1 when unfertilized, and maximum biomass yield was 10.3 Mg ha-1 year-1 when fertilized at 58.6 kg ha-1 P, suggesting that P application on medium soil test P soils is beneficial for switchgrass establishment and early growth. Switchgrass cv. Shawnee was more productive than cvs. Liberty or Sunburst (11.3, 10.2, and 8.6 Mg ha-1 year-1, respectively). Both bacterial and fungal communities were significantly shaped by cultivar. These shifts, while inconsistent between year and cultivar, may reflect a selection of the microbial community from that present in soil to maximize total nutrient uptake, regardless of additional P amendments. Phosphorus fertilization did not affect microbial community structure. Results of this study suggest that the cultivar-associated selection of particular microbial taxa may have implications for increased productivity.

Fecal pollution: new trends and challenges in microbial source tracking using next-generation sequencing.

In this minireview, we expand upon traditional microbial source tracking (MST) methods by discussing two recently developed, next-generation-sequencing (NGS)-based MST approaches to identify sources of fecal pollution in recreational waters. One method defines operational taxonomic units (OTUs) that are specific to a fecal source, e.g., humans and animals or shared among multiple fecal sources to determine the magnitude and likely source association of fecal pollution. The other method uses SourceTracker, a program using a Bayesian algorithm, to determine which OTUs have contributed to an environmental community based on the composition of microbial communities in multiple fecal sources. Contemporary NGS-based MST tools offer a promising avenue to rapidly characterize fecal source contributions for water monitoring and remediation efforts at a broader and more efficient scale than previous molecular MST methods. However, both NGS methods require optimized sequence processing methodologies (e.g. quality filtering and clustering algorithms) and are influenced by primer selection for amplicon sequencing. Therefore, care must be taken when extrapolating data or combining datasets. Furthermore, traditional limitations of library-dependent MST methods, including differential decay of source material in environmental waters and spatiotemporal variation in source communities, remain to be fully understood. Nevertheless, increasing use of these methods, as well as expanding fecal taxon libraries representative of source communities, will help improve the accuracy of these methods and provide promising tools for future MST investigations.

Application of SourceTracker for Accurate Identification of Fecal Pollution in Recreational Freshwater: A Double-Blinded Study.

The efficacy of SourceTracker software to attribute contamination from a variety of fecal sources spiked into ambient freshwater samples was investigated. Double-blinded samples spiked with ≤5 different sources (0.025-10% vol/vol) were evaluated against fecal taxon libraries characterized by next-generation amplicon sequencing. Three libraries, including an initial library (17 nonlocal sources), a blinded source library (5 local sources), and a composite library (local and nonlocal sources), were used with SourceTracker. SourceTracker's predictions of fecal compositions in samples were made, in part, based on distributions of taxa within abundant genera identified as discriminatory by discriminant analyses but also using a large percentage of low abundance taxa. The initial library showed poor ability to characterize blinded samples, but, using local sources, SourceTracker showed 91% accuracy (31/34) at identifying the presence of source contamination, with two false positives for sewage and one for horse. Furthermore, sink predictions of source contamination were positively correlated (Spearman's ρ ≥ 0.88, P < 0.001) with spiked source volumes. Using the composite library did not significantly affect sink predictions ( P > 0.79) compared to those made using the local sources alone. Results of this study indicate that geographically associated fecal samples are required for SourceTracker to assign host sources accurately.