Free-living lifestyle preferences drive the antibiotic resistance promotion during drinking water chlorination

The risk associated with antibiotic resistance genes (ARGs) in size-fractionated bacterial community during drinking water chlorination remains unclear, and is of paramount importance for risk mitigation through process selection and optimization. This study employed metagenomic approaches to reveal the alterations of ARGs, their potential functions and hosts within the free-living and particle-associated fractions. The total relative abundance of ARGs, mobile genetic elements (MGEs), and virulence factor genes (VFGs) significantly increased in the free-living fraction after chlorination. The contribution of the free-living fraction to the ARG relative abundance rose from 16.40 ± 1.31 % to 93.62 ± 0.47 % after chlorination. Multidrug resistance genes (e.g. mexF and mexW) were major contributors, and their co-occurrence with MGEs in the free-living fraction was enhanced after chlorination. Considering multiple perspectives, including presence, mobility, and pathogenicity, chlorination led to a significant risk of the antibiotic resistome in the free-living fraction. Moreover, potential functions of ARGs, such as cell wall/membrane/envelope biogenesis, defense mechanisms, and transcription in the free-living fraction, were intensified following chlorination. Potential pathogens, including Pseudomonas aeruginosa, Pseudomonas alcaligenes, and Acinetobacter junii, were identified as the predominant hosts of multidrug resistance genes, with their increased abundances primarily contributing to the rise of the corresponding ARGs. Overall, alterations of hosts as well as enhancing mobility and biological functions could collectively aid the proliferation and spread of ARGs in the free-living fraction after chlorination. This study provides novel insights into antibiotic resistance evolution in size-fractionated bacteria community and offers a management strategy for microbiological safety in drinking water.