Chlorination (but Not UV Disinfection) Generates Cell Debris that Increases Extracellular Antibiotic Resistance Gene Transfer via Proximal Adsorption to Recipients and Upregulated Transformation Genes

To advance the understanding of antibiotic resistance propagation from wastewater treatment plants, it is important to elucidate how different effluent disinfection processes affect the dissemination of predominantly extracellular antibiotic resistance genes (eARGs). Here, we show that, by facilitating proximal adsorption to recipient cells, bacterial debris generated by chlorination (but not by UV irradiation) increases the natural transformation frequency of their adsorbed eARG by 2.9 to 7.2-fold relative to free eARGs. This is because chlorination increases the bacterial surface roughness by 1.1 to 6.7-fold and the affinity toward eARGs by 1.6 to 5.8-fold, and 98% of the total eARGs released after chlorination were adsorbed to cell debris. In contrast, UV irradiation released predominantly free eARGs with 18% to 56% lower transformation frequency. The collision theory indicates that the ARG donor–recipient collision frequency increased by 35.1-fold for eARGs adsorbed onto chlorination-generated bacterial debris, and the xDLVO model infers a 29% lower donor–recipient contact energy barrier for these ARGs. Exposure to chlorination-generated bacterial debris also upregulated genes associated with natural transformation in Vibrio vulnificus (e.g., tfoX encoding the major activator of natural transformation) by 2.6 to 5.2-fold, likely due to the generation of chlorinated molecules (5.1-fold higher Cl content after chlorination) and persistent reactive species (e.g., carbon-centered radicals) on bacterial debris. Increased proximal eARG adsorption to bacterial debris was also observed in the secondary effluent after chlorination; this decreased eARG decay by 64% and increased the relative abundance of ARGs by 7.2-fold. Overall, this study highlights that different disinfection approaches can result in different physical states of eARGs that affect their resulting dissemination potential via transformation.