Metagenomic Profiling of Microbial Composition and Antibiotic Resistance Determinants in Puget Sound

<div><p>Human-health relevant impacts on marine ecosystems are increasing on both spatial and temporal scales. Traditional indicators for environmental health monitoring and microbial risk assessment have relied primarily on single species analyses and have provided only limited spatial and temporal information. More high-throughput, broad-scale approaches to evaluate these impacts are therefore needed to provide a platform for informing public health. This study uses shotgun metagenomics to survey the taxonomic composition and antibiotic resistance determinant content of surface water bacterial communities in the Puget Sound estuary. Metagenomic DNA was collected at six sites in Puget Sound in addition to one wastewater treatment plant (WWTP) that discharges into the Sound and pyrosequenced. A total of ∼550 Mbp (1.4 million reads) were obtained, 22 Mbp of which could be assembled into contigs. While the taxonomic and resistance determinant profiles across the open Sound samples were similar, unique signatures were identified when comparing these profiles across the open Sound, a nearshore marina and WWTP effluent. The open Sound was dominated by α-Proteobacteria (in particular <em>Rhodobacterales sp.</em>), γ-Proteobacteria and Bacteroidetes while the marina and effluent had increased abundances of Actinobacteria, β-Proteobacteria and Firmicutes. There was a significant increase in the antibiotic resistance gene signal from the open Sound to marina to WWTP effluent, suggestive of a potential link to human impacts. Mobile genetic elements associated with environmental and pathogenic bacteria were also differentially abundant across the samples. This study is the first comparative metagenomic survey of Puget Sound and provides baseline data for further assessments of community composition and antibiotic resistance determinants in the environment using next generation sequencing technologies. In addition, these genomic signals of potential human impact can be used to guide initial public health monitoring as well as more targeted and functionally-based investigations.</p> </div

Recruitment plot of the unassembled Puget Sound reads to the alpha-Proteobacterium HTCC2255 genome assembly.

<p>Open Sound and Marina metagenomic sequence reads were recruited to the 2.23 MB assembly (GI number: 211594581) using BLASTN and an E-value cutoff of 10⁻⁵. Over 91% of the assembly was matched at greater than 95% identity, equaling approximately 7X coverage.</p

Sampling locations and summary statistics for the Puget Sound metagenomes.

a<p>NA, not applicable.</p>b<p>Based on observational data.</p

Locations of sampling sites in Puget Sound.

<p>Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048000#pone-0048000-t001" target="_blank">Table 1</a> for the geographic coordinates of the sampling stations. WWTP, Wastewater treatment plant.</p

Relative abundance of major taxonomic groups in the Puget Sound samples and other selected metagenomes.

<p>Taxonomic groupings were based on BLASTX comparison to the NCBI taxonomy using MG-Rast <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048000#pone.0048000-Meyer1" target="_blank">[39]</a> and ≥50% identity and an alignment length ≥50 amino acids. The ‘other’ category includes bacteria taxa present in <1% of sequences in all samples, eukaryotes and viruses. Shading is proportional to the relative abundance of each taxon within a metagenome. The cladogram was displayed using hierarchical clustering and the Euclidian distance metric. See Materials and Methods for references describing the additional metagenomes used for comparison. The open Sound locations cluster with other more saline environments including open and coastal ocean samples while the Marina and WWTP effluent samples cluster within a larger clade containing other freshwater and freshwater-impacted metagenomes.</p

Over- and under-representation of predominant taxa in selected biomes relative to the open Puget Sound metagenome.

<p>(A) Overall taxa, (B) α-Proteobacteria, (C) β-Proteobacteria, (D) Bacteroidetes and (E) Actinobacteria. The difference in proportions refers to the percent difference in the relative abundances of a given taxa between two locations. Only taxa with a difference of proportions >1% are shown. Negative values indicate higher relative abundance in the Puget Sound (p<10⁻¹⁵ for all comparisons). See Materials and Methods for references describing the additional metagenomes used for comparison. A unique taxonomic signature was identified for the Puget Sound consisting of an over-representation of α-Proteobacteria, γ-Proteobacteria and Bacteriodetes and an under-representation of β-Proteobacteria and Actinobacteria.</p

Prevalence of mobile genetic elements in the Puget Sound metagenomes.

<p>(A) Plasmid sequences, (B) transposable elements using a BLAST search against GenBank, (C) transposable elements using a hidden markov model (HMM) search against the Pfam database and (D) top ten most abundant transposable elements from the WWTP effluent by percent abundance. Bars represent 95% confidence intervals for binomial proportions. P-values are relative to the open Sound and are corrected for multiple testing. For open Sound vs. Marina and WWTP, *p<0.05 and **p<0.005. For Marina vs. WWTP, ⁺p<0.05 and ⁺⁺p<0.005. Bars represent 95% confidence intervals for binomial proportions. The dashed line represents the mean percent for the open Sound. Similar profiles for mobile genetic elements were seen for the open Sound samples while significant differences emerged when comparing to the Marina and effluent.</p

Occupational exposure limit for silver nanoparticles: considerations on the derivation of a general health-based value

<p>With the increased production and widespread commercial use of silver nanoparticles (AgNPs), human and environmental exposures to silver nanoparticles are inevitably increasing. In particular, persons manufacturing and handling silver nanoparticles and silver nanoparticle containing products are at risk of exposure, potentially resulting in health hazards. While silver dusts, consisting of micro-sized particles and soluble compounds have established occupational exposure limits (OELs), silver nanoparticles exhibit different physicochemical properties from bulk materials. Therefore, we assessed silver nanoparticle exposure and related health hazards in order to determine whether an additional OEL may be needed. Dosimetric evaluations in our study identified the liver as the most sensitive target organ following inhalation exposure, and as such serves as the critical target organ for setting an occupational exposure standard for airborne silver nanoparticles. This study proposes an OEL of 0.19 μg/m³ for silver nanoparticles derived from benchmark concentrations (BMCs) from subchronic rat inhalation toxicity assessments and the human equivalent concentration (HEC) with kinetic considerations and additional uncertainty factors. It is anticipated that this level will protect workers from potential health hazards, including lung, liver, and skin damage.</p

Variability in metagenomic samples from the Puget Sound: Relationship to temporal and anthropogenic impacts

<div><p>Whole-metagenome sequencing (WMS) has emerged as a powerful tool to assess potential public health risks in marine environments by measuring changes in microbial community structure and function in uncultured bacteria. In addition to monitoring public health risks such as antibiotic resistance determinants, it is essential to measure predictors of microbial variation in order to identify natural versus anthropogenic factors as well as to evaluate reproducibility of metagenomic measurements.This study expands our previous metagenomic characterization of Puget Sound by sampling new nearshore environments including the Duwamish River, an EPA superfund site, and the Hood Canal, an area characterized by highly variable oxygen levels. We also resampled a wastewater treatment plant, nearshore and open ocean sites introducing a longitudinal component measuring seasonal and locational variations and establishing metagenomics sampling reproducibility. Microbial composition from samples collected in the open sound were highly similar within the same season and location across different years, while nearshore samples revealed multi-fold seasonal variation in microbial composition and diversity. Comparisons with recently sequenced predominant marine bacterial genomes helped provide much greater species level taxonomic detail compared to our previous study. Antibiotic resistance determinants and pollution and detoxification indicators largely grouped by location showing minor seasonal differences. Metal resistance, oxidative stress and detoxification systems showed no increase in samples proximal to an EPA superfund site indicating a lack of ecosystem adaptation to anthropogenic impacts. Taxonomic analysis of common sewage influent families showed a surprising similarity between wastewater treatment plant and open sound samples suggesting a low-level but pervasive sewage influent signature in Puget Sound surface waters. Our study shows reproducibility of metagenomic data sampling in multiple Puget Sound locations while establishing baseline measurements of antibiotic resistance determinants, pollution and detoxification systems. Combining seasonal and longitudinal data across these locations provides a foundation for evaluating variation in future studies.</p></div

Environmental metadata for 11metagenomic samples.

<p>Environmental metadata for 11metagenomic samples.</p