by David Levine, writer
Microbes are everywhere—in the water we drink, the food we eat and in our natural and built environments. The invention of microscopes in the 18th century made them visible to us, and laboratory cultivation methods have enabled us to study them in detail. While these studies have provided immense value, all study has been done in ideal conditions—within a laboratory. Metagenomics—the direct sequencing of clinical specimens and environmental samples—makes it possible to investigate microbes within the complex communities where they live with precise interpretation in a short period of time, compared to traditional methods which can take days or even weeks.
Metagenomics is also being used to study water, soil, stools and food, human and animal guts and has been used to study the spread as well as the predominance of pathogens in a particular locality or specimen. While metagenomics is being used extensively in academia for medical research, and by government organizations such as the US Food and Drug Administration (FDA) and the US Centers for Disease Control and Prevention (CDC), what does metagenomics look like within a public health laboratory? An environmental laboratory within a public health laboratory? A standalone environmental laboratory? An agricultural laboratory? The quick answer is…it’s complicated.
The Public Health Impact of Metagenomics
Christopher J. Grim, PhD, research microbiologist at FDA’s Center for Food Safety and Applied Nutrition (CFSAN) Office of Regulatory Science said that one way FDA/CFSAN is using metagenomics is in the evaluation and improvement of culture-based bacterial analytical methods. Even though most methods are based on commodity-pathogen pairings and have good sensitivity, they can be laborious and tedious, and not every method is optimal.
“Metagenomics has allowed us to evaluate the food associated microbiome, not just the target pathogen, at each step of the culture enrichment process to understand the limitations and advantages of many of the variables that can be manipulated to optimize pathogen recovery. More sensitive methods lead to better analytical results.”
According to Mia Mattioli, PhD, an environmental engineer in CDC’s Environmental Microbiology and Engineering (EME) Laboratory of the Waterborne Disease Prevention Branch, the COVID-19 pandemic not only showed the value of metagenomics, but accelerated its use across government agencies.
“Metagenomics was used to identify SARS-CoV-2 in the community where traditional testing has faced barriers such as correction facilities, college dorms and nursing homes. We know that not everyone can or is willing to be tested for the virus, but everyone goes to the bathroom! So, we can test for SARS-CoV-2 in water samples and assess its impact in community settings.”
While the science is still rapidly developing and not yet available for implementation, CDC envisions one future use of metagenomics in outbreak response, alongside other next generation sequencing tools, to conduct pathogen source tracking from wastewater samples. For example, early in the pandemic SARS-CoV-2 genomes shed into wastewater on the West Coast may have been more closely genetically related to SARS-CoV-2 strains originating from China, while genomes in wastewater from the Northeast may have been more closely related to strains circulating in Europe. Together this metagenomic application could have the potential to inform transmission dynamics and develop more effective community-level mitigation strategies.
Jeffrey W. Mercante, PhD, a research microbiologist in the EME Laboratory, notes that metagenomic exploration of environmental water is a fascinating area of study for their laboratory.
“One way that we have applied metagenomic methods is through characterization of microbial contamination in flood waters. These studies serve to identify microbial hazards that accompany natural events and provide information that public health agencies and professionals can use to reduce potential exposure to disease-causing microorganisms.”
A Cross-Disciplinary Approach
Metagenomics is a widely applicable tool that can capture a lot of information. As such, its usage commonly spans multiple disciplines, according to Padmini Ramachandran MA, researcher at FDA/CFSAN.
“Metagenomics can tell us at high resolution which microbes are present in a setting, how dangerous they are and other traits, and what functions they are performing, just to name a few. As such, its usage commonly spans multiple disciplines.”
FDA/CFSAN has engaged with several stakeholders in regions in the West as an example of public-private partnerships outlined in FDA’s New Era of Smarter Food Safety and as a component of FDA’s Leafy Greens STEC Action Plan to understand environmental or landscape factors that are impactful in the persistence and spread of foodborne pathogens.
“It has been important to engage with all stakeholders, including growers, academic collaborators, our internal partners and other state and federal partners, in order to take a holistic, One Health approach to this continuing problem.” said Ramachandran.
Mercante reflected that metagenomics works best when used in conjunction with other next-generation sequencing approaches or with traditional methods of microbial characterization.
“Our laboratory has explored combining metagenomics with other approaches. I would like to expand and evaluate using combined approaches for some of our US-based and international activities where it has not yet been employed.”
“Ultimately our goal at FDA is to be able to use metagenomics in some capacity in our regulatory decision making,” Grim observed. “We see metagenomics and related culture independent sequencing-based methods as the next progression from isolate whole genome sequencing.” FDA’s GenomeTrakr initiative has been extremely important in outbreak investigations, providing increased resolution in microbial forensics. But this technique would still require the pathogen to be cultured from a food sample. Metagenomics could circumvent the dependence on culture enrichment, which could shorten turnaround time considerably. But the levels of foodborne pathogens at the harvest and retail stage are often quite low compared to the intrinsic microbiota.
“This challenge needs to be solved,” said Grim.
Challenges and Barriers
Enoma Omoregie, PhD, associate director of Environmental Sciences at the New York City Department of Health and Mental Hygiene, has used metagenomic techniques to look at New York City’s water microbiome and detect pathogens in people’s blood.
“Our water, food and built environment can provide a pathway for pathogen transmissions. Traditional environmental microbiological tests are limited to specific pathogens or indicators. Metagenomics methods allow the rapid and simultaneous detection of many pathogens.” However, Omoregie notes that besides proof-of-concept studies, metagenomics is not being used in New York City at this time.
Mark Pandori, PhD, director of the Nevada State Public Health Laboratory (NSPHL) agrees.
“I don’t know of any public health laboratory actively using metagenomics. They either don’t have the capacity, or the personnel trained to do it.” Pandori is advancing NSPHL’s research and collaboration opportunities throughout Nevada by facilitating partnerships with CDC, the Federal Bureau of Investigation, state and federal agencies, hospitals, state and county health districts and departments. Omoregie says the biggest barrier to implementing metagenomics in public laboratories is the lack of standards and protocols.
“How much sequencing do you need before you are confident about results? We need a set of criteria, and the guidelines and recommendations don’t exist.” He also cites a need for bioinformatic knowledge and some public health laboratories may not have the necessary expertise.
“Although the concept of running one set on a sample—whether human, water or waste—is valuable, it requires advanced bioinformatic analyses and/or significant biochemical modifications to the preparation process,” said Pandori.
Joel Sevinsky, PhD, founder of Theiagen Genomics and former member of the Molecular Science Laboratory at the Colorado Department of Public Health and Environment, said because of their size and limitations, some public health laboratories may be much better off using metabarcoding metagenomics rather than shotgun metagenomics. Metabarcoding is the barcoding of DNA or eDNA (environmental DNA) that allows for simultaneous identification of many taxa within the same environmental sample, however often within the same organism group.
“Through DNA metabarcoding, you can use high-throughput sequencing and circumvent the sorting and isolation of the thousands of individuals in bulk mixed samples of organisms,” Sevinsky said. He also notes that metabarcoding is higher throughput and less costly than shotgun metagenomics, which he calls a valuable tool but not practical for public health laboratories due to its expense, the time it takes to sequence and the staff. Grim said FDA is fortunate that there have been few barriers or challenges in implementing these systems.
“One of the challenges to maintaining these systems are the frequent hardware and software upgrades from vendors and integrating those upgrades with our internal scientific system security configurations. Most vendors are working towards keeping the government scientific systems securities in mind when implementing the upgrades.” In contrast, the FDA has automated platforms and instrumentation that they utilize for all sequencing workflows.
“Our Regulatory Genomics team utilizes a Genomics LIMS system to coordinate all our foodborne isolate genomics workflows,” said Grim. “We are working to integrate metagenomic sequencing data collection and analysis workflows into the same system. Additionally, we have recently developed a metadata standard for essential contextual data collection.” In terms of materials utilized (e.g., sputum, wastewater, etc.) Grim said they include DNA extracted from environmental samples (water, soil, air, compost, animal scat), several food matrices (leafy greens, flour, low moisture food, high moisture foods), and culture-based bacterial enrichments that are pathogen-specific but paired with the commodity (quasi metagenomics). For diagnostics and industry applications, FDA is using metagenomic-based approaches and results for research-only purposes for evaluation and improvement of culture-based bacterial analytical methods. As for data privacy on samples, especially when more than one condition is found, FDA/CFSAN is using metagenomic-based approaches for evaluation only and not for regulatory decision making. In addition, it does not process or analyze human clinical samples, so data privacy in that regard does not apply, according to Grim.
“For research studies, such as our longitudinal research studies in Yuma and California, we keep participant identifiers private, including when the research work is published.”
Consider the Data
Since sequencing technologies have rapidly increased their throughput and reduced the cost of sequencing per base pair, a great amount of data is being generated and a careful evaluation regarding their use, analysis and storage is needed.
“Currently, there is no unique consensus regarding the best assembly strategy, since it has been noticed that the assembler performances depend on both biological and technical factors, and different assemblers work better on different datasets,” Grim said. “And since the data obtained with metagenomic sequencing is complex, created pipelines should offer a comprehensive view of genomes of all the microbes present in the sample.” This approach also requires high sequence coverage, so that taxa in small proportion are represented in the data. The large amount of data that are generated require additional data storage and specialized analysis pipelines to efficiently discern the sequences.
Metagenomics is clearly the way of the future and, due to the pandemic, has shown its worth in promoting public health. The challenge is implementing it within the public health laboratory system. As Omoregie and others noted, today’s metagenomics is not being used in public health laboratories due to multiple factors. How to make the transition to use metagenomics in public health laboratories is the next step researchers will be studying in the next few years.