Phthalate Esters Released from Plastics Promote Biofilm Formation and Chlorine Resistance

Abstract

Phthalate esters (PAEs) are commonly released from plastic pipes in some water distribution systems. Here, we show that exposure to a low concentration (1–10 μg/L) of three PAEs (dimethyl phthalate (DMP), di-n-hexyl phthalate (DnHP), and di-(2-ethylhexyl) phthalate (DEHP)) promotes Pseudomonas biofilm formation and resistance to free chlorine. At PAE concentrations ranging from 1 to 5 μg/L, genes coding for quorum sensing, extracellular polymeric substances excretion, and oxidative stress resistance were upregulated by 2.7- to 16.8-fold, 2.1- to 18.9-fold, and 1.6- to 9.9-fold, respectively. Accordingly, more biofilm matrix was produced and the polysaccharide and eDNA contents increased by 30.3–82.3 and 10.3–39.3%, respectively, relative to the unexposed controls. Confocal laser scanning microscopy showed that PAE exposure stimulated biofilm densification (volumetric fraction increased from 27.1 to 38.0–50.6%), which would hinder disinfectant diffusion. Biofilm densification was verified by atomic force microscopy, which measured an increase of elastic modulus by 2.0- to 3.2-fold. PAE exposure also stimulated the antioxidative system, with cell-normalized superoxide dismutase, catalase, and glutathione activities increasing by 1.8- to 3.0-fold, 1.0- to 2.0-fold, and 1.2- to 1.6-fold, respectively. This likely protected cells against oxidative damage by chlorine. Overall, we demonstrate that biofilm exposure to environmentally relevant levels of PAEs can upregulate molecular processes and physiologic changes that promote biofilm densification and antioxidative system expression, which enhance biofilm resistance to disinfectants.

Effect of Phthalates and Their Substitutes on the Physiology of Pseudomonas aeruginosa

“…Indeed, we observed vesicle-like structures that are common components of biofilm matrixes [70,71], and/or ramifications between cells, which could be related to the matrixial exopolysaccharides [72]. In agreement with our data, some phthalates, including DMP, di-n-hexyl phthalate (DnHP), and DEHP, were previously shown to promote P. aeruginosa biofilm formation and resistance to the disinfectant chlorine [73]. Exposure to indoor dust including phthalate esters enhanced biofilm formation by almost two-fold in Escherichia coli, Enterococcus faecalis, and P. aeruginosa, without impacting the bacterial growth rates [74], which is in line with our data.…”

Phthalates Promote Dissemination of Antibiotic Resistance Genes: An Overlooked Environmental Risk

“…Wastewater indigenous microbiota cultures (10 μL) were inoculated in 190 μL of artificial sewage medium in 96-well microtiter plates (Monroe, NC) containing a chambered coverslip to promote the biofilm formation . After incubation at 37 °C for 48 h, each well was cleaned with PBS to remove the unattached cells.…”

“…Wastewater indigenous microbiota cultures (10 μL) were inoculated in 190 μL of artificial sewage medium in 96-well microtiter plates (Monroe, NC) containing a chambered coverslip to promote the biofilm formation. 15 After incubation at 37 °C for 48 h, each well was cleaned with PBS to remove the unattached cells. In order to resemble the natural environment and reduce the number of donor cells in the system, the donor P. putida KT2442 was suspended with PBS to 10 6 CFU/mL.…”

“…Wastewater indigenous microbiota cultures (10 μL) were inoculated in 190 μL of artificial sewage medium in 96-well microtiter plates (Monroe, NC) containing a chambered coverslip to promote the biofilm formation. 15 resemble the natural environment and reduce the number of donor cells in the system, the donor P. putida KT2442 was suspended with PBS to 10 6 CFU/mL. 30 Donor culture (200 μL) was added into the 96-well microtiter plate and incubated at 37 °C for 24 h. The DMP concentrations in 96-well microtiter plate were 0, 2, 10, 20, and 50 μg/L, respectively.…”

Zwitterionic poly(sulfobetaine methacrylate)-based hydrogel coating for drinking water distribution systems to inhibit adhesion of waterborne bacteria

“…For instance, ethylene propylene diene monomer (EPDM) rubber, an elastomer commonly used for rubber-coated valves, has repeatedly shown to support excessive biofilm development compared to other commonly applied materials ( Neu and Hammes, 2020 ). DWDS biofilm research addresses release of compounds, that either promote or inhibit microbial growth ( Wang et al, 2022 ) or material characteristics as surface roughness ( Cowle et al, 2019 ) that have an impact on biofilm development. To our knowledge, studies investigating the influence of other essential surface properties, i.e., hydrophilicity, charge or chemistry, playing an important role in early stages of biofilm formation, have been very limited in drinking water environment ( Simões et al, 2007 ; Simões et al, 2010 ).…”