Cerium oxide nanoparticles (nCeO2) exert minimal adverse effects on microbial communities in soils with and without biosolids amendment.

Increased use of nano-cerium oxide (nCeO2) in an array of industrial applications has raised environmental concerns due to potential increased loadings to the soil environment. This research investigated the potential adverse effects of nCeO2 (10-30 nm) on the soil microbial community in two exposure scenarios: direct application to soil, and indirect application to soil through chemical spiking of biosolids, followed by mixing into soil. Total Ce in test soils without, and with biosolids amendment, ranged from 44 to 770, and 73 to 664 mg Ce kg-1 soil, respectively. In order to help distinguish whether observed effects were elicited by the solid-phase colloids or the activity of dissolved Ce, a soluble Ce salt (Ce (NO3)3) treatment was included in select assays. A suite of tests was used to investigate effects on critical processes: microbial growth (heterotrophic plate count), microbial activity (organic matter (OM) decomposition, enzyme activity and, nitrification) and diversity (structural and functional). Although results showed significant inhibition on microbial growth in soil without biosolids amendment at ≥ 156 mg Ce kg-1 soil by week 5, these results were inconsistent and non-significant thereafter. In general, nCeO2 showed no evidence of consistent adverse effects on OM decomposition, nitrification, soil enzyme activities and functional diversity. Leucine aminopeptidase showed significant (p< 0.05) stimulatory effects over time at ≥ 44 mg Ce kg-1 in soils without biosolids, which was not observed in soils with biosolids amendment. The lack of inhibitory effects of nCeO2 may be attributed to its low solubility; Ce in soil extracts (0.01 M CaCl2) were all below detection (< 0.003 mg kg-1) in the nCeO2-spiked soils, but detectable in the Ce (NO3)3 samples. In contrast, soluble Ce at 359 mg Ce kg-1 showed a significant reduction in OM decomposition and effects on microbial genomic diversity based on the 16S rDNA data in soils with and without biosolids amendment (359 and 690 mg Ce kg-1). The nCeO2 behaviour and effects information described herein are expected to help fulfill data gaps for the characterization of this priority nanomaterial.

Low dose antibiotic ingestion potentiates systemic and microbiome changes induced by silver nanoparticles.

Changes in the mammalian gut microbiome are linked to the impairment of immunological function and numerous other pathologies. Antimicrobial silver nanoparticles (AgNPs) are incorporated into numerous consumer products (e.g., clothing, cosmetics, food packaging), which may directly impact the gut microbiome through ingestion. The human health impact of chronic AgNP ingestion is still uncertain, but evidence from exposure to other antimicrobials provides a strong rationale to assess AgNP effects on organ function, immunity, metabolism, and gut-associated microbiota. To investigate this, mice were gavaged daily for 5 weeks with saline, AgNPs, antibiotics (ciprofloxacin and metronidazole), or AgNPs combined with antibiotics. Animals were weighed daily, assessed for glucose tolerance, organ function, tissue and blood cytokine and leukocyte levels. At the end of the study, we used 16S rDNA amplicon and whole-metagenome shotgun sequencing to assess changes in the gut microbiome. In mice exposed to both AgNPs and antibiotics, silver was found in the stomach, and small and large intestines, but negligible amounts were present in other organs examined. Mice exposed to AgNPs alone showed minimal tissue silver levels. Antibiotics, but not AgNPs, altered glucose metabolism. Mice given AgNPs and antibiotics together demonstrated slower weight gain, reduced peripheral lymphocytes, and elevated splenic, but not circulatory markers of inflammation. 16S rDNA profiling of cecum and feces and metagenomic sequencing of fecal DNA demonstrated that combined AgNP-antibiotic treatment also significantly altered the structure and function of the gut microbiota, including depletion of the indicator species Akkermansia muciniphila. This study provides evidence for possible biological effects from repeated ingestion of AgNP-containing consumer products when antibiotics are also being used and raises concern that an impaired gut microbiome (e.g., through antibiotic use) can potentiate the harm from chemical exposures such as AgNPs.

The Ecobiomics project: Advancing metagenomics assessment of soil health and freshwater quality in Canada.

Transformative advances in metagenomics are providing an unprecedented ability to characterize the enormous diversity of microorganisms and invertebrates sustaining soil health and water quality. These advances are enabling a better recognition of the ecological linkages between soil and water, and the biodiversity exchanges between these two reservoirs. They are also providing new perspectives for understanding microorganisms and invertebrates as part of interacting communities (i.e. microbiomes and zoobiomes), and considering plants, animals, and humans as holobionts comprised of their own cells as well as diverse microorganisms and invertebrates often acquired from soil and water. The Government of Canada's Genomics Research and Development Initiative (GRDI) launched the Ecobiomics Project to coordinate metagenomics capacity building across federal departments, and to apply metagenomics to better characterize microbial and invertebrate biodiversity for advancing environmental assessment, monitoring, and remediation activities. The Project has adopted standard methods for soil, water, and invertebrate sampling, collection and provenance of metadata, and nucleic acid extraction. High-throughput sequencing is located at a centralized sequencing facility. A centralized Bioinformatics Platform was established to enable a novel government-wide approach to harmonize metagenomics data collection, storage and bioinformatics analyses. Sixteen research projects were initiated under Soil Microbiome, Aquatic Microbiome, and Invertebrate Zoobiome Themes. Genomic observatories were established at long-term environmental monitoring sites for providing more comprehensive biodiversity reference points to assess environmental change.

Soil exposed to silver nanoparticles reveals significant changes in community structure and altered microbial transcriptional profiles.

Anthropogenic activities can disrupt soil ecosystems, normally resulting in reduced soil microbial health. Regulatory agencies need to determine the effects of uncharacterized substances on soil microbial health to establish the safety of these chemicals if they end up in the environment. Previous work has focused on measuring traditional ecotoxicologial endpoints within the categories of microbial biomass, activity, and community structure/diversity. Because these tests can be labor intensive, lengthy to conduct, and cannot measure changes in individual gene functions, we wanted to establish whether metatranscriptomics could be used as a more sensitive endpoint and provide a perspective on community function that is more informative than taxonomic identification of microbes alone. We spiked a freshly collected sandy loam soil (Vulcan, Alberta, Canada) with 0, 60, 145, 347, 833, and 2000 mg kg-1 of silver nanoparticles (AgNPs), a known antagonist of microorganisms due to its propensity for dissolution of toxic silver ions. Assessments performed in our previous work using traditional tests demonstrated the toxicity of AgNPs on soil microbial processes. We expanded this analysis with genomics-based tests by measuring changes in community taxonomic structure and function using 16S rDNA profiling and metatranscriptomics. In addition to identifying bacterial taxa affected by AgNPs, we found that genes involved in heavy metal resistance (e.g., the CzcA efflux pump) and other toxicity response pathways were highly upregulated in the presence of silver. Dose-response analysis using BMDExpress2 software successfully modeled many physiologically relevant genes responding to low concentrations of AgNPs. We found that the transcriptomic point of departure (BMD50) was lower than the IC50s calculated using the traditional tests in our previous work. These results suggest that dose-response modeling of metatranscriptomic gene expression is a useful tool in soil microbial health assessment. SUMMARY: Genomics-based endpoints for the assessment of soil microbial health can be used to perform quantitative dose-response modeling, and soil-based RNAseq adds functional insights.

Draft Genome Sequence of the Industrially Significant Bacterium Bacillus amyloliquefaciens NRRL 942.

Bacillus amyloliquefaciens NRRL 942 is a Gram-positive bacterium with several potential industrial uses. We have sequenced the whole genome of this organism to assist in understanding the biological mechanisms that might modulate human health or environmental risk in the event of its release into the environment.

Comparison of Methods to Identify Pathogens and Associated Virulence Functional Genes in Biosolids from Two Different Wastewater Treatment Facilities in Canada.

The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.

Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil.

In this study, we show that noncoding sequences from amplified fragment length polymorphisms (AFLPs) can provide robust and sensitive genetic markers suitable for PCR-based discrimination of closely related strains of Bacillus and Paenibacillus, and quantitative PCR (qPCR)-based tracking of the strains in complex natural systems like soil. Quantitative PCR was accurate in the approximately 1 x 10(9) to approximately 1 x 10(4) colony forming units (CFU)/g soil range. The detection limit was improved to approximately 1 x 10(2) CFU/g when amplicons were analyzed by gel electrophoresis. Studies with laboratory-contained intact soil-core microcosms indicated that environmental persistence trends vary among different strains. For example, Bacillus circulans ATCC 9500, Bacillus amyloliquefaciens DSL 13563-0, Bacillus licheniformis ATCC 12713, Paenibacillus polymyxa NRRL B-4317, and 3 Bacillus subtilisstrains (ATCC 6051A, ATCC 55405, and NRRL B-941) died down to below the 1 x 10(2) CFU/g detection limit by days 28-105. In contrast, over a 105-day period, B. licheniformis ATCC 55406, Bacillus megaterium NRRL B-14308, and P. polymyxa strains ATCC 55407 and DSL 13540-4 died down but persisted at levels just above the detection limit, whereas Bacillus thuringiensis ATCC 13367 experienced a less than 10-fold decrease in cell numbers.

Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens.

Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10(3)A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater.

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR.

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).

Methods of studying soil microbial diversity.

Soil microorganisms, such as bacteria and fungi, play central roles in soil fertility and promoting plant health. This review examines and compares the various methods used to study microbial diversity in soil.

Simulated microgravity (SMG) and bacteria.

This past century has been a scientific revolution in the understanding of the cell as the basic unit of life. However an immense paucity of knowledge exists on microbial growth, survival, function and structure in space. However, there are significant constraints placed on conducting biological research in space such as time, available stowage space, trained personnel, power requirements, weight and the possibility of accidental microbiological contamination. One Earth-based approach is to use a modification of a clinostat known as a HARV (high-aspect-ratio-vessel; Synthecon Inc., Houston, Texas, USA) to conduct this research. In this note we describe the use of the HARV to examine the effects of randomized microgravity (RMG) on bacterial growth and membrane polarization.