Targeted Probe Capture Metagenomics-Enabled Surveillance of Multidrug-Resistant Organisms and Antimicrobial Resistance Genes in Post-Handwashing Areas of Public Washrooms

Public washrooms (toilets) are potential hubs for pathogen transmission, yet the risk of microbial re-contamination via post-handwashing surfaces remains understudied. We characterized the presence and prevalence of multidrug-resistant organisms (MDROs) and antimicrobial resistance genes (ARGs) in the post-handwashing area by collecting surface samples of four high-contact sites (n = 232), including faucets, paper dispensers, hand dryers, and exit door handles, in public washrooms across healthcare, commercial, and recreational facilities. Using culture-dependent methods, we isolated 17 MDROs, including extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E, n = 10), carbapenem-resistant Pseudomonas aeruginosa (CRPA, n = 5), and methicillin-resistant Staphylococcus aureus (MRSA, n = 2). We novelly employed targeted probe capture metagenomics (TCM) to enrich and sequence low-abundance pathogen and ARG targets, revealing a core microbiome dominated by opportunistic pathogens, including Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. TCM detected clinically critical ARGs (blaCTX-M, blaNDM, blaSHV, blaVIM, mecA, vanC), with mecA, the hallmark of MRSA resistance, ubiquitously prevalent across all sites. Notably, the co-enrichment of S. aureus and mecA on exit door handles suggested their roles as critical hotspots for MRSA dissemination. Our findings highlight post-handwashing areas as underrecognized reservoirs for MDROs, particularly MRSA. Furthermore, this study demonstrates the utility of TCM in public health surveillance by enabling sensitive detection of rare but high-risk resistance determinants in low-biomass environmental samples. Integrating culture-based and metagenomic approaches, this study offers a comprehensive and nuanced assessment of the microbial and resistome landscape of washroom environments, offering a revolutionizing approach for future environmental surveillance.