Metagenomic Profiling of Hospital Microbiomes for Surveillance of Multidrug-Resistant Pathogens Using High-Throughput Sequencing Techniques

Abstract

Antimicrobial resistance (AMR) poses an escalating global health emergency, with hospitals acting as primary reservoirs for multidrug-resistant (MDR) pathogens. Traditional culture-based surveillance methods face significant limitations, such as culture biases and a restricted diagnostic scope. To address these gaps, high-throughput whole metagenome shotgun sequencing has emerged as a powerful, culture-independent approach for population-level environmental and clinical monitoring. This review systematically evaluates the technical capabilities, error profiles, and performance metrics of second-generation (Illumina) and third-generation (Oxford Nanopore Technologies and Pacific Biosciences) sequencing modalities in resistome tracking. While short-read platforms excel in single-nucleotide accuracy, long-read technologies enable the resolution of complex repetitive structures and mobile genetic elements (MGEs) essential for tracking horizontal gene transfer. Furthermore, this paper benchmarks sample preparation protocols designed to mitigate the bottleneck of overwhelming host DNA contamination in low-biomass environments using selective cell lysis and enzymatic depletion. It also examines the computational trade-offs between read-based mapping and assembly-based bioinformatic architectures using specialized profiling tools. Finally, we analyze the structural organization and curation strategies of prominent antimicrobial resistance databases including CARD, ResFinder, ResFinderFG, and MEGARes which facilitate ecological population modeling and the identification of novel or divergent resistance genes.