Targeted probe capture metagenomics-enabled surveillance of multidrug-resistant organisms and antimicrobial resistance genes in post-handwashing areas of public washrooms.

BACKGROUND

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 prevalence and distribution of multidrug-resistant organisms (MDROs) and antimicrobial resistance genes (ARGs) in post-handwashing areas by sampling four high-contact sites, including faucets, paper dispensers, hand dryers, and exit door handles, in public washrooms across healthcare, commercial, and recreational facilities.

RESULTS

From the 232 post-handwashing surface samples collected, we isolated 17 MDROs (7.33% prevalence) from cultures, including extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (ESBL-E, n = 10), carbapenem-resistant Pseudomonas aeruginosa (CRPA, n = 5), and methicillin-resistant Staphylococcus aureus (MRSA, n = 2). Additionally, we novelly employed targeted probe capture metagenomics (TCM), which utilizes oligonucleotide probes to enrich and detect low-abundance microbial species and ARG sequences. TCM revealed the detection of human pathogenic taxa in 65.2% of samples, including P. aeruginosa (78.4%), Acinetobacter baumannii (77.9%), and S. aureus (71.1%). Clinically critical ARGs, such as blaCTX-M (2.0%), blaNDM (2.9%), blaSHV (3.4%), and mecA (62.3%), were detected in 63.7% of samples, indicating a potential transmission within the post-handwashing area.

CONCLUSIONS

Our findings highlight the role of post-handwashing areas as underrecognized reservoirs for MDROs, particularly MRSA. Furthermore, this study demonstrates the utility of TCM in public health surveillance by enabling a sensitive detection of rare but high-risk microbial species and drug resistance determinants in low-biomass environmental samples. This study offers a comprehensive and nuanced view of the microbial and resistome landscape of washroom environments, offering a revolutionary approach for future environmental surveillance.