Electrochemical Disinfection Promotes Community-Wide Permissiveness toward the Conjugative Antibiotic Resistance Plasmid pKJK5

Conjugation is widely recognized as the primary mechanism responsible for the dissemination of antibiotic resistance genes (ARGs). Exposure to sublethal current intensities has been observed to stimulate plasmid transfer in pure cultures of model bacteria. However, it remains unknown whether this process also facilitates conjugation at the microbial community level. To address this question, we investigated whether pre-exposing a mixed recipient community derived from the supernatant of activated sludge, as representative of the effluent of the activated sludge basin, to various current intensities affected its permissiveness (ability to take up plasmids). The permissiveness was evaluated as the frequency of plasmid transfer and the composition of the resultant transconjugant pool. This was achieved by using Pseudomonas putida carrying the fluorescently marked IncP-1ε plasmid pKJK5 as a donor. The results show that current exposure promoted the conjugative transfer of pKJK5 up to 2-fold across diverse members of the community. The overproduction of reactive oxygen species (maximum 1.45-fold) and enhanced cell membrane permeability (maximum 3- fold) induced by current exposure may be related to the enhanced transfer frequency. Cell sorting and 16S rRNA gene amplicon sequencing demonstrated that the diversity of the transconjugant pool increased with the current intensity and their composition significantly differed from that of the recipient community. The dominant transconjugant phyla shifted from Proteobacteria to Bacteroidota after current exposure. Additionally, several putative opportunistic pathogens (e.g., Klebsiella) were enriched among the transconjugants. These findings underscore the potential risks of accelerated plasmid transfer through poorly controlled electrochemical disinfection.