Toward Accurate and Robust Environmental Surveillance Using Metagenomics

Abstract

Environmental surveillance is a critical tool for combatting public health threats represented by the global COVID-19 pandemic and the continuous increase of antibiotic resistance in pathogens. With its power to detect entire microbial communities, metagenomics-based methods stand out in addressing the need. However, several hurdles remain to be overcome in order to generate actionable interpretations from metagenomic sequencing data for infection prevention. Conceptually and technically, we focus on viability assessment, taxonomic resolution, and quantitative metagenomics, and discuss their current advancements, necessary precautions and directions to further development. We highlight the importance of building solid conceptual frameworks and identifying rational limits to facilitate the application of techniques. We also propose the usage of internal standards as a promising approach to overcome analytical bottlenecks introduced by low biomass samples and the inherent lack of quantitation in metagenomics. Taken together, we hope this perspective will contribute to bringing accurate and consistent metagenomics-based environmental surveillance to the ground.

An improved workflow for accurate and robust healthcare environmental surveillance using metagenomics

“…Although sequence-based environmental surveillance of microbial communities for better management of public health has been appealed for and utilized, best practices of the workflow have not been systematically studied to ensure proper interpretations of sequencing results to aid in infection risk assessment 13,14 . This study introduces a well-structured and informed metagenomics-based workflow towards the goal of being appropriate for low-biomass, viability, quantification, and resource estimation.…”

“…We examined the efficiency of PMA treatment coupled with metagenomic sequencing (PMA-MetaSeq) on hospital-associated surface samples with the ZymoBIOMICS Microbial Community spike-in as the internal standard. The Zymo community consists of 8 bacterial species and 2 yeasts, which presumably will function more comprehensively and accurately regarding bias correction and quality control than a single-species standard 13,23,24,43 . Sequencing outcomes were compared with cultivation results for benchmarking, as cultivation is the gold standard for determining microbial viability.…”

“…Quantifying metagenomics-based abundances using internal standards has substantial benefits. Theoretically, addition of internal standards could compensate for errors resulting from nonquantitative steps 13 . E. coli is one of the most used spike-in strains, in part because it is well-studied and easy to recover in sequencing 23 .…”

“…Untargeted methods are advantageous in identifying novel or rapidly emerging pathogens 12 . Metagenomics-based techniques are the most promising option to achieve these goals but are currently challenged by several limitations: 1) they are not powerful enough to extract valid signals out of the background noise for low biomass samples; 2) they do not distinguish between viable and non-viable organisms; and 3) they do not reveal the microbial load quantitatively 13,14 .…”

“…A semi-quantitative systematic evaluation concluded that PMA treatment coupled with 16S rRNA gene amplicon sequencing (PMA-Seq) is reliable when the microbial community is not very complex, while uncertainties increase dramatically with complexity 23 . The uncertainties come from both heterogeneity of microorganisms (e.g., cell envelope structure differences, spore formation), and complexity of the background matrix (e.g., turbidity, salt concentration, dead cell density) 13,24,25 . While the microbial communities to be surveilled have their inherent advantage of being low complexity, little is known about the effectiveness of incorporating PMA with multi-species internal standards.…”