Transfer dynamics of intracellular and extracellular last-resort antibiotic resistome in hospital wastewater.

The increasing prevalence of last-resort antibiotic resistance genes (LARGs) has posed severe public health hazards. Previous studies focused primarily on the profiles of intracellular LARGs (iLARGs) in hospital wastewater (HWW), while largely neglecting the expression patterns of iLARGs and the presence of extracellular LARGs (eLARGs). Currently, wastewater resistomes and transfer dynamics of LARGs are still poorly characterized. This study integrates Nanopore-metagenomic and metatranscriptomic sequencing to conduct the comprehensive longitudinal analysis of both iLARGs and eLARGs in HWW. Our study firstly revealed the distinct seasonal patterns of iLARGs and eLARGs. Specifically, iLARGs showed higher abundance during colder seasons, whereas eLARGs showed higher abundance in warm seasons. Both clinical pathogens and functional bacteria of wastewater treatments were identified as important hosts of LARGs, while clinical pathogens played predominant roles in the high expression levels of LARGs. Acinetobacter spp. was identified as major host of bla in HWW, which is unrestricted by plasmid host range compatibility. However, HWW treatments could not remove LARGs effectively and instead facilitated their transmission by enhancing the expression and horizontal transfer of mobile genetic element (MGE)-derived LARGs. Our study provides comprehensive insights for the atlas and transfer dynamics of LARGs in HWW for the development of control strategies under worldwide spread of antibiotic resistance.