Although disinfection is key to infection control, the colonization patterns and resistomes of hospital-environment microbes remain underexplored. We report the first extensive genomic characterization of microbiomes, pathogens and antibiotic resistance cassettes in a tertiary-care hospital, from repeated sampling (up to 1.5 years apart) of 179 sites associated with 45 beds. Deep shotgun metagenomics unveiled distinct ecological niches of microbes and antibiotic resistance genes characterized by biofilm-forming and human-microbiome-influenced environments with corresponding patterns of spatiotemporal divergence. Quasi-metagenomics with nanopore sequencing provided thousands of high-contiguity genomes, phage and plasmid sequences (>60% novel), enabling characterization of resistome and mobilome diversity and dynamic architectures in hospital environments. Phylogenetics identified multidrug-resistant strains as being widely distributed and stably colonizing across sites. Comparisons with clinical isolates indicated that such microbes can persist in hospitals for extended periods (>8 years), to opportunistically infect patients. These findings highlight the importance of characterizing antibiotic resistance reservoirs in hospitals and establish the feasibility of systematic surveys to target resources for preventing infections.

尽管消毒是控制感染的关键，但医院环境微生物的定植模式和耐药性仍未得到充分探索。我们报告了三级医院中微生物组、病原体和抗生素耐药性盒的首次广泛基因组特征，来自与 45 张病床相关的 179 个地点的重复采样（最多相隔 1.5 年）。深鸟枪宏基因组学揭示了微生物和抗生素抗性基因的独特生态位，其特征是生物膜形成和受人类微生物组影响的环境以及相应的时空差异模式。具有纳米孔测序的准宏基因组学提供了数千个高连续性基因组、噬菌体和质粒序列（>60% 新的），能够表征医院环境中的抗性组和移动组多样性和动态架构。系统发育学鉴定出多重耐药菌株广泛分布并稳定地在不同地点定殖。与临床分离物的比较表明，这些微生物可以在医院中持续存在较长时间（> 8 年），从而机会性地感染患者。这些发现强调了描述医院抗生素耐药性储库特征的重要性，并确立了系统调查的可行性，以针对预防感染的资源。机会性地感染患者。这些发现强调了描述医院抗生素耐药性储库特征的重要性，并确立了系统调查的可行性，以针对预防感染的资源。机会性地感染患者。这些发现强调了描述医院抗生素耐药性储库特征的重要性，并确立了系统调查的可行性，以针对预防感染的资源。