Effect of Methylparaben Disinfection By-products on the Chlorine Tolerance of Biofilms

Chlorination is widely used as a tertiary treatment in water treatment plants to ensure the microbiological safety of drinking water (DW). However, this process inevitably leads to the formation of disinfection by-products (DBPs). In addition to microbial challenges, DW quality is also affected by emerging contaminants like parabens, which can react with chlorine to form parabens transformation products (PTPs). This study is the first to investigate the impact of PTPs derived from methylparaben (MP): mono-chlorinated MP (3-Cl-MP) and di-chlorinated MP (3,5-diCl-MP), on bacterial characteristics and tolerance to disinfection in 7-day-old dual-species biofilms of Acinetobacter calcoaceticus and Stenotrophomonas maltophilia. Exposure to 3-Cl-MP and 3,5-diCl-MP increased the number of viable but not culturable cells (VBNC) of A. calcoaceticus presented in dual-species biofilms by 3-fold and 5-fold, respectively. Exposure to 3-Cl-MP also resulted in a significant increase in the VBNC levels of the overall dual-species biofilms and a10 µm increase in biofilm thickness. The polysaccharide content was higher for both 3-Cl-MP and 3,5-diCl-MP exposed biofilms in comparison to non-exposed ones. Interestingly, biofilms exposed to 3-Cl-MP showed increased susceptibility to chlorination, with higher log CFU reductions for A. calcoaceticus (1.26 ± 0.07), S. maltophilia (1.21 ± 0.14), and overall dual-species biofilms (1.17 ± 0.11). Similarly, increased reductions in total cells were obtained for 3-Cl-MP- exposed A. calcoaceticus, and for the overall dual-species biofilms after 3,5-diCl-MP exposure. A greater % of bacterial cells with damaged membranes was also obtained for both 3-Cl-MP and 3,5-diCl-MP-exposed biofilms. Although MP-derived PTPs did not increase bacterial tolerance to disinfection, 3,5-diCl-MP exposure increased the antibiotic tolerance of S. maltophilia to minocycline (MINO) and trimethoprim-sulfamethoxazole (TMP-SMX). These findings underscore the complex interactions between PTPs and biofilms, highlighting the potential implications for water treatment strategies, antimicrobial resistance and public health.