A metagenomics-based approach to understanding the transmission of healthcare-associated antimicrobial resistance in Pakistan.

Hospital environments are critical yet underexamined reservoirs for hazardous antimicrobial resistance (AMR), particularly in lower-middle-income countries (LMICs) where resource constraints often hinder comprehensive surveillance. In this study, we employed 16S rRNA gene sequencing and shotgun metagenomics to characterize the microbiome, resistome, and potential transmission routes across five clinical environments within a hospital in Pakistan: the intensive care unit (ICU), surgical ward (SW), cardiac surgery ward (CSW), cardiac ward (CW), and operating theater (OT). Microbial community analysis revealed compositional similarities among the ICU, SW, and OT, with the ICU emerging as a primary source of microbial dissemination. Species-level profiling identified hospital-associated pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae, and Enterobacter cloacae, and metagenome-assembled genome (MAG) analysis enabled the linkage of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) to specific bacterial hosts. ARGs and MGEs displayed setting-specific patterns, with the SW harboring the highest abundance of ARGs, particularly those conferring resistance to β-lactams and biocides. Insertion sequences were the most prevalent MGEs and were commonly linked to ARGs, indicating potential horizontal gene transfer. Co-occurrence network analysis identified Staphylococcus, Enterococcus, and Escherichia as central hub taxa within the microbial communities of the ICU, SW, and OT, indicating their critical roles in potential ARG transmission. These findings provide critical insights into the environmental transmission dynamics of AMR in LMIC healthcare settings and underscore the urgent need for metagenomics-informed infection control strategies.