Metagenomic Applications for Environmental Health Surveillance: A One Health Case Study from the Pacific Northwest Ecosystem1

YOUNGBLOOD, Jessicaa, WALLANCE, Jamesb, PORT, Jessec, CULLEN, Alisond and FAUSTMAN, Elainee

a University of Washington, Seattle, United States. E-mail:
b University of Washington, Seattle, United States. E-mail:
c University of Washington, Seattle, United States. E-mail:
d University of Washington, Seattle, United States. E-mail:
e University of Washington, Seattle, United States. E-mail:

Abstract The marine environment is the largest, most diverse and influential ecosystem on Earth. Still largely unexplored, the foundation for further ocean exploration begins with the most abundant and productive life forms in the ocean, the microbial community. Microbes are essential to all life and play an intimate role in ecosystem function and environmental health. Microbial community composition and function are important metrics that can be used to monitor and predict environmental changes highly relevant to global health. Standard lab techniques used for environmental microbial assessment are limited in scope and high throughput, comprehensive approaches offer a tremendous opportunity to expand our estimates and monitoring of microbial diversity. Metagenomics in combination with 454-pyrosequencing, marine metadata and bioinformatics analysis offers a sensitive approach to evaluate intact community genomes for the novel detection and characterization of microbial populations. Metagenomic studies reveal community composition, functional potential and environmental preferences that suggest key species and roles necessary in sustaining a functioning, healthy environment. In addition to its ecological relevance, metagenomic profiling creates translational research opportunities for monitoring environmentally hosted, human health determinants. In this case study based in Washington State’s Puget Sound estuary revealed the high reproducibility and discriminatory capabilities of metagenomic profiling and a comparative analysis of metagenomes exposed significant differences in microbial diversity and antibiotic resistance determinants across a gradient of anthropogenic impact. Our results demonstrate the power of metagenomics for understanding one health.

Keywords Metagenomics, Environmental Health, Antibiotic Resistance, Microbial Community, Marine Environment

1  Introduction

As the largest, most diverse and influential ecosystem on Earth, the marine environment plays a critical role in human health and well-being. Worldwide, an unprecedented amount of marine environments are altered by environmental changes, such as climate change and urbanization, causing severe changes in ocean productivity, resource availability, ocean acidification, glacial melting, sea-level rise, increased intensity and frequency of storms, seasonal weather pattern disruption and reduced freshwater supply and quality (Herr and Galland 2009). Given the magnitude of our marine environments and the unprecedented rate of global environmental change, these profound consequences will continue to pressure our diverse populations with threats to food, habitat and human health security, mandating the necessity for further research to understand potential environmental health consequences. The foundation for evaluating future marine impacts begins with the most abundant and productive life forms in the environment, the microbial community (Gianoulis, Raes et al. 2009: 1374-1379).

1.1  Microbial Community

Microbes are essential to all life and play an intimate role in ecosystem function, health and productivity. Microbial populations are sensitive to environmental and anthropogenic impacts and their resilience can be measured through spatial and temporal profiling of taxonomic and functional composition (Nogales, Lanfranconi et al. 2011: 275-298). Their strong influence on global processes (i.e. carbon and nitrogen biogeochemical cycles), immediate responses to environmental changes and rapid reproducibility allow for the sensitive and specific qualitative and quantitative assessments that are needed to inform sustainable environmental decisions. Current metrics and standard laboratory based culture and isolation techniques used for microbial assessment and are limited in detection, quantification and prevention strategies, and it is estimated that >99% of environmental organisms have not been cultured (Amann, Ludwig et al. 1995: 143-169). Efforts to understand and effectively quantify microbial communities are essential for the wellbeing and sustainability of our marine ecosystems and future management and policy decisions will call for the integration of comprehensive based assessment approaches. Major advancements in the field of environmental genomics in tandem with next generation sequencing technology allow for high-throughput and culture-independent investigations into the composition of marine microbial communities. One of the challenges is to identify at what region and where do we take action? What do we need to know? What do we do for surveillance? Our work is centered on a One Health Approach and integrates the call for multidimensional consideration to create translational research for preventable and sustainable human, animal and environmental health.

1.2  Metagenomics

Metagenomics, also known as environmental genomics, is a culture and isolation independent method of studying microbial genetic material directly recovered from an environment. The DNA is then linked with genes that are associated to microbial taxonomy and function providing the unique ability to characterize the microbial community as it exists in nature. With this approach, compositional diversity and change can provide a sensitive tool for detection, identification and prediction of environmental conditions over space and time and in response to anthropogenic impacts. Using a metagenomic platform offers a tremendous opportunity to expand our estimates and monitoring of microbial communities and their imperative functions and as a result of these unique benefits it has become a popular tool for assessing marine microbial communities with over 100 publicly available datasets. Metagenomic projects from all over the world have already shown that different environments (e.g. ocean, freshwater, soil, human, air) have unique taxonomic and functional profiles and that these profiles can change with environmental or human perturbations (Tringe, von Mering et al. 2005: 554-557; Rusch, Halpern et al. 2007: e77; Turnbaugh, Ley et al. 2007: 804-810). As these environments continue to change, the effects on the microbial community are hypothesized to lead to loss of microbial diversity, changes in microbial community function and health ultimately decreasing our ecosystems quality and resilience with threats of increasing tolerance, persistence and geographic range of toxins, antibiotic resistance and opportunistic pathogens. Of particular interest are bacterial pathogens, as they impose a heavy burden of disease and an alarming number of treatment options are being compromised by bacterial acquisition, evolution and progression of antibiotic resistance genes (ARGs). Figure 1 shows how the benefits of metagenomics can be applied to promote One Health concepts.

Figure 1: The metagenomic profile is comprised of the taxonomic composition and functional potential of the community. From the metagenomic data we can identify environmental and human health determinants to form a One Health approach.

1.3  Public Health Relevance

As environmental sequencing technologies are becoming more affordable and resourceful, additional opportunities for metagenomics are developing and include a spectrum of applications. These opportunities have the potential to reveal significant insight into marine process drivers and capacity. They also offer the opportunity for integration into new methods and framework for environmental health decisions and management. In addition to ecological relevance, advanced metagenomic surveillance of environments allows for translational research for human health by increasing the limits of detection and quantification of human health determinates. In this study we use the broad scale, metagenomic screening approach of the Antibiotic Resistance Determinants (ARD) Index established by Port et al. (2013) to look for public health relevant antibiotic resistant determinants from intact marine communities and wastewater treatment plants genomes and to provide an example for Public Health Translation(Port, Cullen et al. 2013).

1.3.1  Antibiotic Resistance Determinants (ARD) Index

The ARD Index provides a method of characterizing the presence and dissemination of ARGs in the environment and is a key component in the translation for of incorporating environmental genomic data into environmental health decisions and management (Port, Cullen et al. 2013). Factors that are included in the ARD Index and that we have identified as risk factors for antibiotic resistance are classified into three categories that provide the ecological context and etiology for resistance potential. These categories include: antibiotic resistance gene potential, gene-transfer potential and pathogenicity potential and are further defined by sub-categories: metal resistance genes (antibiotic resistance gene potential), plasmids, transposable elements and phages (gene-transfer potential) and pathogenic bacteria (pathogenicity potential) (Figure 2).

Figure 2: The conceptual framework for the ARD index(Port, Cullen et al. 2013). Human health determinants were evaluated based on the potential for antibiotic resistance, gene transfer and pathogenicity. This framework helps link changes in the microbial community with human health. The bioinformatic criteria for the identification of ARDs are shown in the bottom right-hand corner. The criteria are based on percent sequence match and a minimum amino acid (aa) or basepair (bp) length.

2  The Puget Sound of Washington State

The Puget Sound estuary is the second largest estuary in the United States and is an inlet of the Pacific Ocean connected by the Strait of Juan de Fuca. This estuary is characterized as a fjord system of flooded glacial valleys, containing expansive areas of deep open water to shallow bays and numerous inlets resulting in high seasonal freshwater input from the Olympic and Cascade Mountain watersheds. The Sound covers around 2,500 miles of shoreline and the Puget Sound Region provides homes to approximately 67% of Washington State’s population (Babson, Kawase et al. 2006). These waterways are an integral part of the Washington community and contribute substantially to the economic stability and growth of the Pacific Northwest. We have used this estuary as a case study because the aquatic ecosystem is closely surrounded by urban development, providing an excellent opportunity for One Health context.

2.1  Longitudinal Study

This field-based study is the second generation of metagenomic analysis of the Puget Sound Estuary and includes the addition and characterization of seven metagenomes, comprising a total of 14 samples from 10 different locations including a proximal wastewater treatment plant that discharges effluent into the Puget Sound (Port, Wallace et al. 2012: e48000). The baseline framework and design of this study is based on the methods by Port et al. (2012). The objective of this study was to further define the Puget Sound metagenome by more effectively addressing coastal areas and their environmental signals of human impact and environmental health relevance. The primary variables investigated were the taxonomic composition, function potential and the environmental and human health determinants of surface water bacterial communities. This longitudinal study used 454 next generation sequencing, field metadata, and bioinformatics analysis to profile the surface water bacterial communities of the Puget Sound, both temporally and spatially. Particular attention was focused on our Nearshore marine areas that are greatly affected by anthropogenic and environmental influences such as: sewer, agricultural, and industrial run-off and their contributing role in the evolution, progression and persistence of human health determinants in the Puget Sound.

3  Results

This project leveraged off our longitudinal metagenomic samples from the Puget Sound as well as the abundance of publicly accessible data from around the world to assess the potential effect and response of microbial communities to global environmental changes. Our samples have been collected over time and across diverse marine environments, including highly impacted near shore environments, hazardous waste sites and wastewater treatment effluents. Using these samples we have validated the reproducible and discriminatory capabilities of metagenomics revealing the significant differences in microbial community composition and functions. One example of this is illustrated in our assessment at the bacterial phyla level. Longitudinal samples taken in the Open Sound (P28 and P32) approximately a year apart exhibited highly similar compositional structure, while longitudinal samples taken from the Marina and WWTP effluent during different seasons displayed considerable microbial compositional differences(most at the anthropogenic regions of the Puget Sound). Factors that were correlated with these differences were revealed across temperature and salinity gradients (Youngblood 2013). In our 2012 samples 58% of the sequences were assigned to the Domain level. Bacteria accounted for 94% and ranged from 88-98% with the remaining portion comprised of Archaea 1.5%, Eukaryota (3.4%) and viruses (1.1%). Proteobacteria had the highest representation across all samples and ranged from 54-76%, followed by Bacteroidetes in all samples, except the waste-water treatment plant effluent. When we used just the 16S rRNA to determine the phyla, 1031 sequences (0.09%) were uploaded to the RDP classifier with approximately 96% of those sequences annotated at the phylum level. This provides evidence of our ability to integrate the signal. In this study we used the broad scale screening approach of the ARD Index to look for environmental health relevant antibiotic resistant determinants. Using community level surveillance, we assessed the putative levels of antibiotic resistant determinants across all Puget Sound and WWTP metagenomes using the following determinant categories: gene transfer potential, antibiotic resistance genes and pathogenicity potential. Samples were grouped according to proximal location (Open Sound, Nearshore, WWTP) and preliminary results revealed an increase in averaged relative abundance across a gradient of anthropogenic impact and environmental health relevance between the Open Sound, Nearshore and WWTP effluent samples in all antibiotic resistant determinants, with the exception of the sub-category phages which exhibited the highest representation in the Nearshore samples (Figure 3). Additionally, discernible differences were also observed in the significant environmental and human health determinants such as the causative agent in Bacterial Cold Water disease (Flavobacterium psychrophilum), a severe fish disease as well as several pathogenic species (examples include Escherichia coli and Vibrio cholerae) affiliated with well-known gastrointestinal illnesses and infectious disease in humans (Youngblood 2013).

4  Added Value to the One Health Approach

Our case study presented here illustrates a metagenomic approach to profile the surface water bacterial communities of the Puget Sound to study the relationships and interactions between the marine environment, the bacterial community and human health. This case study allows us to further discover the roles and influence these system have on each other allowing us to better understand the marine impacts on human health and human impacts on marine ecosystems.

5  Conclusions

Metagenomics, in combination with next generation sequencing and bioinformatics, offers a powerful and sensitive approach to evaluate intact community genomes for the novel detection and characterization of microbial communities strengthening our knowledge and depth of our most influential surroundings and building capacity towards preventative environmental management decisions. In our investigation of Puget Sound we were able to identify taxonomic composition, functional potential and environmental health determinants of the surface water bacterial communities and to address coastal areas and their environmental signals of human impact and environmental health relevance. We revealed a high level of reproducibility and discriminatory capability of metagenomic profiling and propose metagenomics as a high-throughput method to assess the taxonomic characterization and functional potentials of microbial populations. Comparative analysis of all metagenomes revealed significant differences in both microbial diversity and human health determinants across a gradient of anthropogenic impact further illustrating human impacts on marine ecosystems and marine impacts on human health. Advanced characterization of environments through annotated metagenomic data and marine associated metadata can help define the physical, chemical and biological parameters that play a contributing role in the community diversity necessarily to sustain a flourishing environment. Our results are the first to demonstrate the successful characterization of the Puget Sound, as well as the future applications and significance of metagenomic analyses. This data will inform microbial monitoring of Puget Sound and provide innovative, translational research to further characterize and expand environmental monitoring, policy and global health impact and awareness.


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Youngblood, J.; Wallace, J.; Port, J.; Cullen, A.; Faustman, E.(2014):Metagenomic Applications for Environmental Health Surveillance: A One Health Case Study from the Pacific Northwest Ecosystem. In: Planet@Risk, 2(4), Special Issue on One Health: 281-284, Davos: Global Risk Forum GRF Davos.

This article is based on a presentation given during the 2nd GRF Davos One Health Summit 2013, held 17-20 November 2013 in Davos, Switzerland (