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

Relative abundances of top 10 pathogenic genera for 11 metagenomic samples.

<p>Increasing taxonomic abundance is indicated by darker blue boxes. The horizontal color bar at the top reflects sample collection location (WWTP, nearshore or open sound samples).</p

Alpha-diversity for 11 metagenomic samples.

<p>Alpha-diversity for 11 metagenomic samples.</p

Environmental metadata for 11metagenomic samples.

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

Relative abundances for the 10 most common bacterial species in 5 nearshore and 4 open sound samples.

<p>Increasing taxonomic abundance is indicated by darker blue boxes. Nearshore samples (blue bar) clustered by sampling season and open sound samples (orange bar) clustered by sampling location over different sampling years.</p

Class rank beta-diversity for 11 Puget Sound samples.

<p>Nearshore marina samples showed the largest phylogenetic distance for repeat samples in different seasons.</p

Potential anthropogenic determinants and detoxification systems related to public health.

<p>(a) Percent of MEGAN LCA family taxonomic rank assignments for sewage influent bacterial families. (b) Percent of MEGAN taxonomic assignments for 28 human bacterial pathogenic genera. (c) Combined percentage of reads assigned to antibiotic resistance determinant elements, polycyclic aromatic hydrocarbon (PAH) degradation and detoxification systems Hidden Markov models.</p

Regional and seasonal community composition differences in Puget Sound showing relative taxonomic abundances for 11 metagenomic samples.

<p>Increasing taxonomic abundance is indicated by darker blue boxes. The horizontal color bar at the top reflects season of sample collection. Seattle Shilshole marina nearshore samples (Marina-1 and Marina-2) showed multi-fold seasonal differences between winter and summer measurements in Actinobacteria, Betaproteobacteria, Flavobacteriia and Alphaproteobacteria classes. Archaea were most prominent in offshore P28 and P32 samples and comprised as much as 17% (P28-2) in relative abundance.</p

Neighbor-joining tree representation of phylogenetic distances for sewage associated bacterial families.

<p>Open sound samples were taxonomically closer in relative abundance to WWTP samples than Marina and Herring’s House Park samples although smaller in absolute abundance as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192412#pone.0192412.g005" target="_blank">Fig 5a</a>.</p

Map of sample sites for Puget1 and Puget2 projects.

<p>Map of sample sites for Puget1 and Puget2 projects.</p