Development of reproducible, quantitative methods based on shotgun metagenome sequencing for assessment of risk of microbial transmission

The CDC has identified several limitations to the application of shotgun metagenome sequencing for environmental studies in healthcare facilities, particularly regarding sensitivity and specificity. These challenges include the “low number of the target microbes” relative to other microbes, cell types, or potential inhibitors in samples collected from surfaces in the healthcare environment. Moreover, there is a lack of validation quantitatively connecting DNA sequencing to infectious agents and thus allowing proper interpretation of metagenomics results and downstream assessment of transmission risk. We will address these concerns by determining best practices for sample processing and sequencing, specifically regarding sample pre-treatment to remove relic DNA (propidium monoazide treatment) and large particles (filtration), as well as DNA extraction methods (e.g., bead-beating, chemical lysis, enzymatic lysis, sonication). We will further recommend standardized sequencing depth-to-biomass ratios and determine limits of detection for microbes of interest, depending on the methods used. To do so, we will collect and pool surface samples from the Medical Intensive Care Unit at Rush University Medical Center, where we are currently performing ongoing analyses to link environmental microbiomes to healthcare outcomes, to generate one large aggregate representative of the surface-associated microbiome in healthcare settings and evaluate the effect of the aforementioned parameters on how informative shotgun sequencing is in identifying viable pathogens that pose a potential risk of transmission to patients.