Alterations in the Endophyte-Enriched Root-Associated Microbiome of Rice Receiving Growth-Promoting Treatments of Urea Fertilizer and Rhizobium Biofertilizer.

We examined the bacterial endophyte-enriched root-associated microbiome within rice (Oryza sativa) 55 days after growth in soil with and without urea fertilizer and/or biofertilization with a growth-promotive bacterial strain (Rhizobium leguminosarum bv. trifolii E11). After treatment to deplete rhizosphere/rhizoplane communities, washed roots were macerated and their endophyte-enriched communities were analyzed by 16S ribosomal DNA 454 amplicon pyrosequencing. This analysis clustered 99,990 valid sequence reads into 1105 operational taxonomic units (OTUs) with 97% sequence identity, 133 of which represented a consolidated core assemblage representing 12.04% of the fully detected OTU richness. Taxonomic affiliations indicated Proteobacteria as the most abundant phylum (especially α- and γ-Proteobacteria classes), followed by Firmicutes, Bacteroidetes, Verrucomicrobia, Actinobacteria, and several other phyla. Dominant genera included Rheinheimera, unclassified Rhodospirillaceae, Pseudomonas, Asticcacaulis, Sphingomonas, and Rhizobium. Several OTUs had close taxonomic affiliation to genera of diazotrophic rhizobacteria, including Rhizobium, unclassified Rhizobiales, Azospirillum, Azoarcus, unclassified Rhizobiaceae, Bradyrhizobium, Azonexus, Mesorhizobium, Devosia, Azovibrio, Azospira, Azomonas, and Azotobacter. The endophyte-enriched microbiome was restructured within roots receiving growth-promoting treatments. Compared to the untreated control, endophyte-enriched communities receiving urea and/or biofertilizer treatments were significantly reduced in OTU richness and relative read abundances. Several unique OTUs were enriched in each of the treatment communities. These alterations in structure of root-associated communities suggest dynamic interactions in the host plant microbiome, some of which may influence the well-documented positive synergistic impact of rhizobial biofertilizer inoculation plus low doses of urea-N fertilizer on growth promotion of rice, considered as one of the world's most important food crops.

Abundance of Chlorinated Solvent and 1,4-Dioxane Degrading Microorganisms at Five Chlorinated Solvent Contaminated Sites Determined via Shotgun Sequencing.

Shotgun sequencing was used for the quantification of taxonomic and functional biomarkers associated with chlorinated solvent bioremediation in 20 groundwater samples (five sites), following bioaugmentation with SDC-9. The analysis determined the abundance of (1) genera associated with chlorinated solvent degradation, (2) reductive dehalogenase (RDases) genes, (3) genes associated with 1,4-dioxane removal, (4) genes associated with aerobic chlorinated solvent degradation, and (5) D. mccartyi genes associated with hydrogen and corrinoid metabolism. The taxonomic analysis revealed numerous genera previously linked to chlorinated solvent degradation, including Dehalococcoides, Desulfitobacterium, and Dehalogenimonas. The functional gene analysis indicated vcrA and tceA from D. mccartyi were the RDases with the highest relative abundance. Reads aligning with both aerobic and anaerobic biomarkers were observed across all sites. Aerobic solvent degradation genes, etnC or etnE, were detected in at least one sample from each site, as were pmoA and mmoX. The most abundant 1,4-dioxane biomarker detected was Methylosinus trichosporium OB3b mmoX. Reads aligning to thmA or Pseudonocardia were not found. The work illustrates the importance of shotgun sequencing to provide a more complete picture of the functional abilities of microbial communities. The approach is advantageous over current methods because an unlimited number of functional genes can be quantified.

Long-Term Effect of Different Fertilization and Cropping Systems on the Soil Antibiotic Resistome.

Different fertilization and cropping systems may influence short- and long-term residues of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in soil. Soils from dryland (peanut) and paddy (rice) fields, which originated from the same nonagricultural land (forested), were treated with either chemical fertilizer, composted manure, or no fertilizer for 26 years before sampling, which occurred one year after the last applications. ARGs and MGEs were investigated using highly parallel qPCR and high-throughput sequencing. Six of the 11 antibiotics measured by LC-MS/MS were detected in the manure applied soil, but not in the nonmanured soils, indicating their source was from previous manure applications. Compared to the unfertilized control, manure application did not show a large accumulation of ARGs in either cropping system but there were some minor effects of soil management on indigenous ARGs. Paddy soil showed higher accumulation of these ARGs, which corresponded to higher microbial biomass than the dryland soil. Chemical fertilizer increased relative abundance of these ARGs in dryland soil but decreased their relative abundance in paddy soil. These results show how long-term common soil management practices affect the abundance and type of ARGs and MGEs in two very different soil environments, one aerobic and the other primarily anaerobic.

Antimicrobial Resistance in the Environment.

This review su mmarizes selected publications from 2017 highlighting the occurrence of antimicrobial resistance (AMR) genes in the environment with emphasis on the aquatic environment. The review also covers different treatment technologies being developed for AMR genes as an environmental contaminant. The progress made in the area of AMR gene databases and tools is also reviewed. Besides a brief introduction, the content is categorized into three main sections: i) Occurrence of AMR in the Environment, ii) Treatment technologies for AMR, and iii) AMR databases and tools.

Quantification of microRNAs directly from body fluids using a base-stacking isothermal amplification method in a point-of-care device.

MicroRNAs have been proposed to be a class of biomarkers of disease as expression levels are significantly altered in various tissues and body fluids when compared to healthy controls. As such, the detection and quantification of microRNAs is imperative. While many methods have been established for quantification of microRNAs, they typically rely on time consuming handling such as RNA extraction, purification, or ligation. Here we describe a novel method for quantification of microRNAs using direct amplification in body fluids without upstream sample preparation. Tested with a point-of-care device (termed Gene-Z), the presence of microRNA promotes base-stacking hybridization, and subsequent amplification between two universal strands. The base-stacking approach, which was achieved in <60 min, provided a sensitivity of 1.4 fmol per reaction. Tested in various percentages of whole blood, plasma, and faeces, precision (coefficient of variation = 2.6%) was maintained and comparable to amplification in pristine samples. Overall, the developed method represents a significant step towards rapid, one-step detection of microRNAs.

Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp. reductive dehalogenase genes from groundwater.

TaqMan probe-based quantitative polymerase chain reaction (qPCR) specific to the biomarker reductive dehalogenase (RDase) genes is a widely accepted molecular biological tool (MBT) for determining the abundance of Dehalococcoides sp. in groundwater samples from chlorinated solvent-contaminated sites. However, there are significant costs associated with this MBT. In this study, we describe an approach that requires only low-cost laboratory equipment (a bench top centrifuge and a water bath) and requires less time and resources compared to qPCR. The method involves the concentration of biomass from groundwater, without DNA extraction, and loop-mediated isothermal amplification (LAMP) of the cell templates. The amplification products are detected by a simple visual color change (orange/green). The detection limits of the assay were determined using groundwater from a contaminated site. In addition, the assay was tested with groundwater from three additional contaminated sites. The final approach to detect RDase genes, without DNA extraction or a thermal cycler, was successful to 1.8 × 105 gene copies per L for vcrA and 1.3 × 105 gene copies per L for tceA. Both values are below the threshold recommended for effective in situ dechlorination.

TCDD administered on activated carbon eliminates bioavailability and subsequent shifts to a key murine gut commensal.

Activated carbon (AC) is an increasingly attractive remediation alternative for the sequestration of dioxins at contaminated sites globally. However, the potential for AC to reduce the bioavailability of dioxins in mammals and the residing gut microbiota has received less attention. This question was partially answered in a recent study examining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hallmark toxic responses in mice administered with TCDD sequestered by AC or freely available in corn oil by oral gavage. Results from that study support the use of AC to significantly reduce the bioavailability of TCDD to the host. Herein, we examined the bioavailability of TCDD sequestered to AC on a key murine gut commensal and the influence of AC on the community structure of the gut microbiota. The analysis included qPCR to quantify the expression of segmented filamentous bacteria (SFB) in the mouse ileum, which has responded to TCDD-induced host toxicity in previous studies and community structure via sequencing the 16S ribosomal RNA (rRNA) gene. The expression of SFB 16S rRNA gene and functional genes significantly increased with TCDD administered with corn oil vehicle. Such a response was absent when TCDD was sequestered by AC. In addition, AC appeared to have a minimal influence on murine gut community structure and diversity, affecting only the relative abundance of Lactobacillaceae and two other groups. Results of this study further support the remedial use of AC for eliminating bioavailability of TCDD to host and subsequent influence on the gut microbiome.

Antimicrobial Resistance in the Environment.

This review summarizes selected publications of 2016 with emphasis on occurrence and treatment of antibiotic resistance genes and bacteria in the aquatic environment and wastewater and drinking water treatment plants. The review is conducted with emphasis on fate, modeling, risk assessment and data analysis methodologies for characterizing abundance. After providing a brief introduction, the review is divided into the following four sections: i) Occurrence of AMR in the Environment, ii) Treatment Technologies for AMR, iii) Modeling of Fate, Risk, and Environmental Impact of AMR, and iv) ARG Databases and Pipelines.

Early detection monitoring for aquatic non-indigenous species: Optimizing surveillance, incorporating advanced technologies, and identifying research needs.

Following decades of ecologic and economic impacts from a growing list of nonindigenous and invasive species, government and management entities are committing to systematic early- detection monitoring (EDM). This has reinvigorated investment in the science underpinning such monitoring, as well as the need to convey that science in practical terms to those tasked with EDM implementation. Using the context of nonindigenous species in the North American Great Lakes, this article summarizes the current scientific tools and knowledge - including limitations, research needs, and likely future developments - relevant to various aspects of planning and conducting comprehensive EDM. We begin with the scope of the effort, contrasting target-species with broad-spectrum monitoring, reviewing information to support prioritization based on species and locations, and exploring the challenge of moving beyond individual surveys towards a coordinated monitoring network. Next, we discuss survey design, including effort to expend and its allocation over space and time. A section on sample collection and analysis overviews the merits of collecting actual organisms versus shed DNA, reviews the capabilities and limitations of identification by morphology, DNA target markers, or DNA barcoding, and examines best practices for sample handling and data verification. We end with a section addressing the analysis of monitoring data, including methods to evaluate survey performance and characterize and communicate uncertainty. Although the body of science supporting EDM implementation is already substantial, research and information needs (many already actively being addressed) include: better data to support risk assessments that guide choice of taxa and locations to monitor; improved understanding of spatiotemporal scales for sample collection; further development of DNA target markers, reference barcodes, genomic workflows, and synergies between DNA-based and morphology-based taxonomy; and tools and information management systems for better evaluating and communicating survey outcomes and uncertainty.

Isothermal assay targeting class 1 integrase gene for environmental surveillance of antibiotic resistance markers.

Antimicrobial resistance genes (ARGs) present in the environment pose a risk to human health due to potential for transfer to human pathogens. Surveillance is an integral part of mitigating environmental dissemination. Quantification of the mobile genetic element class 1 integron-integrase gene (intI1) has been proposed as a surrogate to measuring multiple ARGs. Measurement of such indicator genes can be further simplified by adopting emerging nucleic acids methods such as loop mediated isothermal amplification (LAMP). In this study, LAMP assays were designed and tested for estimating relative abundance of the intI1 gene, which included design of a universal bacteria 16S rRNA gene assay. Following validation of sensitivity and specificity with known bacterial strains, the assays were tested using DNA extracted from river and lake samples. Results showed a significant Pearson correlation (R2 = 0.8) between the intI1 gene LAMP assay and ARG relative abundance (measured via qPCR). To demonstrate the ruggedness of the LAMP assays, experiments were also run in the hands of relatively "untrained" personnel by volunteer undergraduate students at a local community college using a hand-held real-time DNA analysis device - Gene-Z. Overall, results support use of the intI1 gene as an indicator of ARGs and the LAMP assays exhibit the opportunity for volunteers to monitor environmental samples for anthropogenic pollution outside of a specialized laboratory.

Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9.

Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures.

Influence of Soil Characteristics and Proximity to Antarctic Research Stations on Abundance of Antibiotic Resistance Genes in Soils.

Soil is an important environmental reservoir of antibiotic resistance genes (ARGs), which are increasingly recognized as environmental contaminants. Methods to assess the risks associated with the acquisition or transfer of resistance mechanisms are still underdeveloped. Quantification of background levels of antibiotic resistance genes and what alters those is a first step in understanding our environmental resistome. Toward this goal, 62 samples were collected over 3 years from soils near the 30-year old Gondwana Research Station and for 4 years before and during development of the new Jang Bogo Research Station, both at Terra Nova Bay in Antarctica. These sites reflect limited and more extensive human impact, respectively. A qPCR array with 384 primer sets targeting antibiotic resistance genes and mobile genetic elements (MGEs) was used to detect and quantify these genes. A total of 73 ARGs and MGEs encompassing eight major antibiotic resistance gene categories were detected, but most at very low levels. Antarctic soil appeared to be a common reservoir for seven ARGs since they were present in most samples (42%-88%). If the seven widespread genes were removed, there was a correlation between the relative abundance of MGEs and ARGs, more typical of contaminated sites. There was a relationship between ARG content and distance from both research stations, with a significant effect at the Jang Bogo Station especially when excluding the seven widespread genes; however, the relative abundance of ARGs did not increase over the 4 year period. Silt, clay, total organic carbon, and SiO2 were the top edaphic factors that correlated with ARG abundance. Overall, this study identifies that human activity and certain soil characteristics correlate with antibiotic resistance genes in these oligotrophic Antarctic soils and provides a baseline of ARGs and MGEs for future comparisons.

Diverse and abundant antibiotic resistance genes in Chinese swine farms.

Antibiotic resistance genes (ARGs) are emerging contaminants posing a potential worldwide human health risk. Intensive animal husbandry is believed to be a major contributor to the increased environmental burden of ARGs. Despite the volume of antibiotics used in China, little information is available regarding the corresponding ARGs associated with animal farms. We assessed type and concentrations of ARGs at three stages of manure processing to land disposal at three large-scale (10,000 animals per year) commercial swine farms in China. In-feed or therapeutic antibiotics used on these farms include all major classes of antibiotics except vancomycins. High-capacity quantitative PCR arrays detected 149 unique resistance genes among all of the farm samples, the top 63 ARGs being enriched 192-fold (median) up to 28,000-fold (maximum) compared with their respective antibiotic-free manure or soil controls. Antibiotics and heavy metals used as feed supplements were elevated in the manures, suggesting the potential for coselection of resistance traits. The potential for horizontal transfer of ARGs because of transposon-specific ARGs is implicated by the enrichment of transposases--the top six alleles being enriched 189-fold (median) up to 90,000-fold in manure--as well as the high correlation (r(2) = 0.96) between ARG and transposase abundance. In addition, abundance of ARGs correlated directly with antibiotic and metal concentrations, indicating their importance in selection of resistance genes. Diverse, abundant, and potentially mobile ARGs in farm samples suggest that unmonitored use of antibiotics and metals is causing the emergence and release of ARGs to the environment.

Antimicrobial Resistance in the Environment.

This review summarizes important publications from 2015 pertaining to the occurrence of antimicrobial resistance (AMR) in the environment. Emphasis is placed on sources of antibiotic resistance in the aquatic environment including wastewater treatment plants, hospitals, and agriculture, treatment and mitigation techniques, and surveillance and analysis methodologies for characterizing abundance data. As such, this review is organized into the following sections: i) occurrence of AMR in the environment, including surface waters, aquaculture, and wastewater ii) treatment technologies, and iii) technologies for rapid surveillance of AMR, iv) transmission between matrices, v) databases and analysis methods, and vi) gaps in AMR understanding.

Static self-directed sample dispensing into a series of reaction wells on a microfluidic card for parallel genetic detection of microbial pathogens.

A microfluidic card is described for simultaneous and rapid genetic detection of multiple microbial pathogens. The hydrophobic surface of native acrylic and a novel microfluidic mechanism termed "airlock" were used to dispense sample into a series of 64 reaction wells without the use of valves, external pumping peripherals, multiple layers, or vacuum assistance. This airlock mechanism was tested with dilutions of whole human blood, saliva, and urine, along with mock samples of varying viscosities and surface tensions. Samples spiked with genomic DNA (gDNA) or crude lysates from clinical bacterial isolates were tested with loop mediated isothermal amplification assays (LAMP) designed to target virulence and antibiotic resistance genes. Reactions were monitored in real time using the Gene-Z, which is a portable smartphone-driven system. Samples loaded correctly into the microfluidic card in 99.3% of instances. Amplification results confirmed no carryover of pre-dispensed primer between wells during sample loading, and no observable diffusion between adjacent wells during the 60 to 90 min isothermal reaction. Sensitivity was comparable between LAMP reactions tested within the microfluidic card and in conventional vials. Tests demonstrate that the airlock card works with various sample types, manufacturing techniques, and can potentially be used in many point-of-care diagnostics applications.

Thirty-minute screening of antibiotic resistance genes in bacterial isolates with minimal sample preparation in static self-dispensing 64 and 384 assay cards.

In a clinical setting, molecular assays such as polymerase chain reaction offer a rapid means to infer or confirm identity and therapeutic decisions. Accordingly, a number of molecular assays targeting identity and antibiotic resistance (AR) genes have been developed; however, these methods can be technically complex and relatively expensive. Herein, we describe a diagnostic concept utilizing isothermal amplification technology with non-purified heat-lysed cells and self-dispensing cards for testing multiple primers in parallel. This proof-of-concept study, performed with Staphylococcus aureus isolates and associated AR genes, was compared with culture-based susceptibility and quantitative PCR (qPCR). Results demonstrate reduced sample processing steps resulting in a turnaround time (starting from bacterial culture to ending in the antibiotic resistance gene profile) in less than 30 min. For antibiotics tested in which an associated AR gene was targeted on the Gene-Z card, 69% (18/26) of culture-based resistance events were positive for related AR genes. A comparison of loop-mediated isothermal amplification (LAMP) and qPCR assays targeting the same antibiotic resistance genes showed a 98.2% agreement in terms of presence and absence calls. Identity-based discrepancies between conventional (phenotypic) and molecular (genotypic) results were further resolved, and we were able to demonstrate higher accuracy in identification with the molecular analysis.

In-feed antibiotic effects on the swine intestinal microbiome.

Antibiotics have been administered to agricultural animals for disease treatment, disease prevention, and growth promotion for over 50 y. The impact of such antibiotic use on the treatment of human diseases is hotly debated. We raised pigs in a highly controlled environment, with one portion of the littermates receiving a diet containing performance-enhancing antibiotics [chlortetracycline, sulfamethazine, and penicillin (known as ASP250)] and the other portion receiving the same diet but without the antibiotics. We used phylogenetic, metagenomic, and quantitative PCR-based approaches to address the impact of antibiotics on the swine gut microbiota. Bacterial phylotypes shifted after 14 d of antibiotic treatment, with the medicated pigs showing an increase in Proteobacteria (1-11%) compared with nonmedicated pigs at the same time point. This shift was driven by an increase in Escherichia coli populations. Analysis of the metagenomes showed that microbial functional genes relating to energy production and conversion were increased in the antibiotic-fed pigs. The results also indicate that antibiotic resistance genes increased in abundance and diversity in the medicated swine microbiome despite a high background of resistance genes in nonmedicated swine. Some enriched genes, such as aminoglycoside O-phosphotransferases, confer resistance to antibiotics that were not administered in this study, demonstrating the potential for indirect selection of resistance to classes of antibiotics not fed. The collateral effects of feeding subtherapeutic doses of antibiotics to agricultural animals are apparent and must be considered in cost-benefit analyses.

Detection and Occurrence of Indicator Organisms and Pathogens.

This review summarizes the literature pertaining to the occurrence and detection of indicator organisms and pathogens published during 2014. It is organized into the following sections: i) detection and quantification of fecal indicators and waterborne pathogens, ii) microbial source tracking (MST) using genotypic and phenotypic methods, iii) antibiotic resistant bacteria (ARB), iv) live vs. dead cell differentiation methods, and v) next generation sequencing (NGS).

DNA extraction-free quantification of Dehalococcoides spp. in groundwater using a hand-held device.

Nucleic acid amplification of biomarkers is increasingly used to measure microbial activity and predict remedial performance in sites with trichloroethene (TCE) contamination. Field-based genetic quantification of microorganisms associated with bioremediation may help increase accuracy that is diminished through transport and processing of groundwater samples. Sterivex cartridges and a previously undescribed mechanism for eluting biomass was used to concentrate cells. DNA extraction-free loop mediated isothermal amplification (LAMP) was monitored in real-time with a point of use device (termed Gene-Z). A detection limit of 10(5) cells L(­1) was obtained, corresponding to sensitivity between 10 to 100 genomic copies per reaction for assays targeting the Dehalococcoides spp. specific 16S rRNA gene and vcrA gene, respectively. The quantity of Dehalococcoides spp. genomic copies measured from two TCE contaminated groundwater samples with conventional means of quantification including filtration, DNA extraction, purification, and qPCR was comparable to the field ready technique. Overall, this method of measuring Dehalococcoides spp. and vcrA genes in groundwater via direct amplification without intentional DNA extraction and purification is demonstrated, which may provide a more accurate mechanism of predicting remediation rates.

Characteristics of bacterial community and extracellular enzymes in response to atrazine application in black soil.

The ecological functioning of black soil largely depends on the activities of various groups of microorganisms. However, little is known about how atrazine, a widely used herbicide with known harmful effects on the environment, influences the microbial ecology of black soil, and the extracellular enzymes related to the carbon, nitrogen and phosphorus cycles. Here, we evaluated the change in extracellular enzymes and bacterial community characteristics in black soil after exposure to various concentrations of atrazine. Low concentrations of applied atrazine (10 - 20 mg kg-1) were almost completely degraded after 120 days. At high concentrations (80 - 100 mg kg-1), about 95% of the applied atrazine was degraded over the same period. Additionally, linear fitting of data indicated that the total enzymatic activity index (TEI) and bacterial α-diversity index were negatively correlated with atrazine applied concentration. The atrazine had a greater effect on bacterial beta diversity after 120 days, which differentiated species clusters treated with low and high atrazine concentrations. Soil bacterial community structure and function were affected by atrazine, especially at high atrazine concentrations (80 - 100 mg kg-1). Key microorganisms such as Sphingomonas and Nocardioides were identified as biomarkers for atrazine dissipation. Functional prediction indicated that most metabolic pathways might be involved in atrazine dissipation. Overall, the findings enhance our understanding of the factors driving atrazine degradation in black soil and supports the use of biomarkers as indicators of atrazine dissipation.