The spread of antibiotic resistance is a global health concern linked to 4.95 million deaths in 2019. (1) Therefore, it is important to advance our understanding of the fate of antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs), which are terminal collection points for antibiotics and ARGs and can be breeding grounds and point sources for discharging ARGs to the environment. (2,3) Notably, extracellular ARGs (eARGs) are commonly present during and after wastewater treatment processes, particularly effluent disinfection. Up to 1017–1018 copies/day (or 106–108 copies/L) of eARGs could be released from WWTPs, making eARGs the dominant form of ARGs discharged to the environment. (4,5) These eARGs may transform bacteria in receiving aquatic systems (e.g., river sediments (6)), which would augment the environmental resistome and associated public health risks. (7)

Chlorination and ultraviolet (UV) irradiation are the most common secondary effluent disinfection processes in most countries. (8) Both approaches are generally efficient for killing bacteria but not for removing ARGs since intracellular ARGs (iARGs) are mainly released as eARGs into the environment rather than destroyed after cell lysis. (3) Furthermore, disinfection generates bacterial debris from cell lysis (4,9) that free eARGs may adsorb to, forming adsorbed eARGs. (4) Previous studies showed that microplastic-adsorbed eARGs could have a higher transmission potential than free eARGs by facilitating proximal eARG–recipient adsorption. (10) However, little is known about the influence of different disinfection strategies and associated byproducts, specifically bacterial debris, on eARG physical states (e.g., free or adsorbed ARG) and their dissemination potential.

Chlorination can generate oxidized bacterial debris with altered affinities for eARGs due to changes in the surface charge and hydrophobicity (11) caused by the chlorinated lipids and amino acids. (12) In contrast, UV irradiation kills bacteria mainly by destroying DNA (13) without substantially affecting the cell integrity. Disinfection by chlorination can also generate environmentally persistent reactive oxidative species (e.g., chlorinated radicals) (14) or chlorinated organics (15) that could influence the susceptibility of some bacteria to transformation by eARGs. (16) Thus, different disinfection methods produce bacterial debris with distinct properties that may interact with eARGs differently and have consequences on eARG persistence and transmissivity.

This study compares the effects of chlorination versus UV irradiation on WWTP effluent in terms of eARG abundance, transformation, and persistence. E. coli with a blaNDM-1 multi-drug resistance gene (coding metallo-β-lactamase) was used as a model multidrug-resistant enteric bacterium. The importance of eARG adsorption onto cell debris for enhanced transformation was assessed separately using the competent reference strain E. coli HB101 or the natural competent strain Vibrio vulnificus (V. vulnificus) as the recipient. Notably, V. vulnificus is an important opportunistic human pathogen frequently detected in wastewater, freshwater, and marine environments. (17−19)...

Figure 2. Chlorination and UV generate bacterial debris with distinct properties that interact with eARGs differently. (A) Changes in the roughness of bacterial debris after chlorination and UV irradiation. (B) Changes in the rupture force between eARGs and cell debris after chlorination or UV irradiation. (C) eDNA fluorescence imaging in samples before and after chlorination and UV irradiation.

Figure 3. Chlorination-produced adsorbed eARGs exhibited a higher transformation frequency than that of free eARGs. (A) Transformation frequency of f-eARG and a-eARG associated with bacterial debris produced by chlorination at various doses by reference strain E. coli HB101. (B) Transformation frequency of f-eARG after exposure to UV irradiation at various fluences by reference strain E. coli HB101. (C) Transformation frequency of f-eARG and a-eARG associated with bacterial debris produced by chlorination at various doses by natural competent strain V. vulnificus. (D) Transformation frequency of f-eARG after exposure to UV irradiation at various fluences by natural competent strain V. vulnificus.

Environmental Implications
Wastewater disinfection is an important practice to mitigate the spread of waterborne infectious diseases and control the potential release of antibiotic-resistant pathogens. However, an overlooked unintended consequence of chlorination, which is the dominant effluent disinfection process, (47) is the release eARGs that are predominantly adsorbed to the generated cell debris. This debris has high affinity to both eARGs and recipient bacteria, enhancing opportunities for their effective collision and resulting transformation. Chlorination also produces persistent free radicals and chlorinated molecules on the cell debris that contribute to upregulation of transformation-related genes and further facilitate the horizontal transfer of the adsorbed eARGs. This combination of the active surface area for proximal adsorption of transforming eDNA and recipient cells and generating transformation-enhancing substrates could lead to an enrichment of the environmental resistome even in the absence of antibiotics. In contrast, disinfection by UV irradiation releases mainly free (possibly damaged) eARGs with a significantly lower transformation potential.

Overall, eARGs adsorbed to bacterial debris generated by chlorination is likely to experience higher persistence and a higher propagation potential than free eARGs in receiving waters. However, further studies at larger temporal and spatial scales are needed to determine whether this effect contributes significantly to resistome dissemination.