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Background: The prevalence of healthcare-acquired infections (HAIs) and rising levels of antimicrobial resistance place a significant burden on modern healthcare systems. Cultures are typically used to track HAIs; however, culture methods provide limited information and are not applicable to all pathogens. Next-generation sequencing (NGS) can detect and characterize pathogens present within a sample, but few research studies have explored how NGS could be used to detect pathogen transmission events under HAI-relevant scenarios. The objective of this CDC-funded project was to evaluate and correlate sequencing approaches for pathogen transmission with standard culture-based analysis. Methods: We modeled pathogen transfer via hand contact using synthetic skin. These skin coupons were seeded with a community of commensal organisms to mimic the human skin microbiome. Pathogens were added at physiologically relevant high or low levels prior to skin-to-skin contact. The ESKAPE pathogens: E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp plus C. difficile were employed because they are the most common antibiotic resistant HAIs. Pathogen transfer between skin coupons was measured following direct skin contact and fomite surface transmission. The effects of handwashing or fomite decontamination were also evaluated. Transferred pathogens were enumerated via culture to establish a robust data set against which DNA and RNA sequence analyses of the same samples could be compared. These data also provide a quantitative assessment of individual ESKAPE+C pathogen transfer rates in skin contact scenarios. Results: Metagenomic and metatranscriptomic analysis using custom analysis pipelines and reference databases successfully identified the commensal and pathogenic organisms present in each sample at the species level. This analysis also identified antibiotic resistance genes and plasmids. Metatranscriptomic analysis permitted not only gene identification but also confirmation of gene expression, a critical factor in the evaluation of antibiotic resistance. DNA analysis does not require cell viability, a key differentiator between sequencing and culturing reflected in simulated handwashing data. Sensitivity remains a key limitation of metagenomic analysis, as shown by the poor species identification and gene content characterization of pathogens present at low abundance within the simulated microbial community. Species level identification typically failed as ratios fell below 1:1,000 pathogen CFU:total community CFU. Conclusions: These findings demonstrate the strengths and weaknesses of NGS for molecular epidemiology. The data sets produced for this study are publicly available so they can be employed for future metagenomic benchmarking studies.
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