Detection of Human Gut Carriage: a Comparison of Culture, QPCR, and Whole Metagenomic Sequencing Methods

is an important opportunistic healthcare-associated pathogen and major contributor to the global spread of antimicrobial resistance. Gastrointestinal colonization with is a major predisposing risk factor for infection and forms an important hub for the dispersal of resistance. Current culture-based detection methods are time consuming, give limited intra-sample abundance and strain diversity information, and have uncertain sensitivity. Here we investigated the presence and abundance of at the species and strain level within fecal samples from 103 community-based adults by qPCR and whole metagenomic sequencing (WMS) compared to culture-based detection. qPCR demonstrated the highest sensitivity, detecting in 61.2% and 75.8% of direct-fecal and culture-enriched sweep samples, respectively, including 52/52 culture-positive samples. WMS displayed lower sensitivity, detecting in 71.2% of culture-positive fecal samples at a 0.01% abundance cutoff, and was inclined to false positives in proportion to the relative abundance of other Enterobacterales present. qPCR accurately quantified to 16 genome copies/reaction while WMS could estimate relative abundance to at least 0.01%. Quantification by both methods correlated strongly with each other (Spearman's rho = 0.91). WMS also supported accurate intra-sample sequence type (ST)-level diversity detection from fecal microbiomes to 0.1% relative abundance, agreeing with the culture-based detected ST in 16/19 samples. Our results show that qPCR and WMS are sensitive and reliable tools for detection, quantification, and strain analysis of from fecal samples with potential to support infection control and enhance insights in gastrointestinal ecology.