P(SBMA) is a zwitterionic polymer known for its high hydrophilicity and ultralow fouling properties, which has been extensively studied in the biomedical and membrane technology fields (Zhang et al., 2018; Cascone and Lamberti, 2020). Moreover, P(SBMA) has been investigated in the context of oral drug delivery systems (Biosca et al., 2021), as it is considered non-toxic, which makes it an interesting anti-adhesive approach for DWDS. The P(SBMA)-based hydrogel coating was photografted from PDMS surface with benzophenone as a type II photoinitiator, using different monomer (SBMA) and cross-linker (BIS) concentrations (Table 1). As presented in Figure 1, UV light-induced radical formation by benzophenone that was infused into the PDMS surface, created surface-bound methyl radicals that further reacted with the monomers present in the surrounding solution initiating polymerization (Karaca Balta et al., 2015). The SEM analysis of the PDMS cross-sections showed that without the addition of the BIS cross-linker (Figure 2), the synthesis was unsuccessful, as the polymer dissolved in water in the washing steps following the polymerization, leaving the surface without any coating (Figures 2B, C). With incorporating BIS in the synthesis, the coating was present, however depending on SBMA concentration and SBMA:BIS ratio, the level of stability in terms of brittleness varied. The coatings synthetized with 20% SBMA and 10:1 SBMA:BIS ratio (Figure 2E) and 40% SBMA and 20:1 SBMA:BIS ratio (Figure 2F), were easily damaged and peeled off from a PDMS surface after drying. The most optimal outcome was achieved with 20% SBMA and 20:1 ratio of SBMA to BIS, giving a uniform and stable P(SBMA) hydrogel coating on PDMS (Figure 2D). The addition of cross-linkers in the polymerization process allows forming a covalent network that improves mechanical stability of the polymer. Selecting the right cross-linking density is essential for properties of hydrogel coatings, as it modulates not only mechanical (i.e., swelling behavior, resistance to stress and strain), but also anti-adhesive properties (i.e., stiffness) (He et al., 2019). Higher cross-linking density increases mechanical stability, however, when the cross-linking is too high, the coating becomes brittle and prone to damage (Jensen et al., 2021). The most optimal formulation of 20% SBMA with 20:1 SBMA:BIS ratio was selected for further characterization and bacterial adhesion tests.

The presence of the P(SBMA) coating was further confirmed using SEM-EDX analysis by creating an elemental map of sulfur and silica as the elements representative for SBMA and PDMS, respectively, for the SEM cross-section image (Figure 3). SEM-EDX maps for other detected elements are presented in Supplementary Figure S2. The coating resulted in a homogenous layer of surface bound hydrogel of a thickness of 19 ± 4 μm in a dry state, which significantly (p < 0.005) increased the surface hydrophilicity by reducing the water contact angle of PDMS from 106 ± 1 to 18 ± 5°, as measured with a captive bubble method (Figure 4). When the sessile drop technique for water contact angle measurements was applied on the hydrated P(SBMA)-coated PDMS, immediate spreading occurred, and an obvious hydration layer could be observed visually (data not shown), confirming the highly hydrophilic nature and high water-absorbing capacity of P(SBMA)-based hydrogel. Surface hydrophilicity is an important parameter in predicting bacterial adhesion, as it is directly connected to surface free energy of a substratum. A more hydrophilic surface responds to higher surface free energy, which in terms of bacterial adhesion, increases the energetical demand for a bacterium to replace surface-bound water molecules (Carniello et al., 2018). A hydrogel-based coating strengthens this effect, as it absorbs and immobilizes water, creating a hydration layer that weakens the interactions between bacteria and a substratum (Chen et al., 2010). While no surface is completely resistant to biofilm development, applications of hydrophilic anti-adhesive coatings can still 1) hinder the biofilm formation, 2) promote the biofilm detachment, and/or 3) affect microbial composition of the biofilm. The presence of the hydration layer highly reduces the adhesion forces exerted by the surface, resulting in bacteria being “unaware” of their presence on the surface, and thereby, remaining in the planktonic state which does not promote biofilm phenotypic changes, such as increased EPS production (Busscher and Van der Mei, 2012; Carniello et al., 2018). However, once the initial adhesion and biofilm formation do occur, weakened interaction forces between the coated surface and those initially adhering bacteria, that also become a link between the surface and the biofilm layer formed on top of them, can lead to considerable biofilm detachment under high shear (Busscher et al., 1995; Nejadnik et al., 2008; Shave et al., 2022). In DWDS circumstances, combination of high velocity flushing, commonly used as a pipe cleaning practice (Liu et al., 2017b), and a hydrogel coating could improve the effectiveness of biofilm removal from biofilm-prone materials. Moreover, it has been recently reported, that the presence of different polymer coatings can affect biofilm bacterial composition when exposed to mixed bacterial populations (Al-Ahmad et al., 2021).

So far, P(SBMA)-based hydrogel coatings have been tested against adhesion of proteins and mammalian cells (Jensen et al., 2021; Lee et al., 2018; Leigh et al., 2019), and clinically relevant bacterial strains, such as Staphylococcus aureus, Staphylococcus epidermis and Escherichia coli (Shen et al., 2021; Zhang et al., 2022), proving their excellent universal anti-adhesive properties. However, the conditions in DWDS significantly vary from these in the medical field (i.e., oligotrophic environment, high water flows, spatio-temporal variations in environmental and engineering factors) (Potgieter et al., 2018). These settings select for organisms that are equipped in specific mechanisms and behaviors in terms of their survival and biofilm forming abilities, potentially different from those in clinical circumstances (Du et al., 2020). The objective of this work was to evaluate the performance of a P(SBMA)-based hydrogel coating against attachment of environmentally relevant bacteria commonly identified in DWDS biofilm communities. The four selected bacterial strains belong to the genera Sphingomonas and Pseudomonas. Sphingomonas spp. can persist in oligotrophic conditions (Ohta et al., 2004) and are recognized as primary surface colonizers and a dominating species in various fouling phenomena, including those on spiral wound membranes (De Vries et al., 2019), and in membrane bioreactors (Huang et al., 2008). Pseudomonas spp. represent a ubiquitous group of environmental bacteria, occupying several niches, including DWDS, and the presence of P. aeruginosa, as an opportunistic pathogen, is of hygienic relevance in DWDS biofilms (Moritz et al., 2010). Bacterial suspensions were flown for 4 h over non-coated and P(SBMA)-coated PDMS surfaces, after which the images of adhered bacteria were taken and evaluated (Figure 5). Selected bacteria exhibited varying attachment behaviors, even within the same genus. While Sphingomonas Sph5, P. extremorientalis and P. aeruginosa adhered as single cells, even though still varying in density, Sphingomonas Sph10 auto-aggregated in the liquid phase and adhered as a mix of single cells and multicellular clusters. This corresponds to initial Sph10 characterization reported by De Vries and colleagues (2019), where after 24 h, up to 100% of the bacteria in the liquid culture formed aggregates. P. aeruginosa adhered in the highest numbers to PDMS. The presence of the P(SBMA) coating significantly (p < 0.05) reduced the number of adhering bacteria compared to non-coated PDMS by 97% ± 2%, 94% ± 3%, 98% ± 1%, and 99% ± 1% for Sph5, Sph10, P. extremorientalis and P. aeruginosa, respectively (Figure 5B), and: Lower anti-adhesive efficiency of the coating against Sph10 compared to other strains, could be caused by the fact that it adheres in the form of aggregates. Bright field microscopy allows the visualization of bacteria within a two-dimensional plane. While the other three strains that adhered as single cells could be accurately quantified on the collected images, for Sph10, the three-dimensional nature of the aggregates might have led to underestimation of adhered bacteria number on the non-coated PDMS, and thereby, the anti-adhesive efficiency of the coating. The versatility of the coating’s anti-adhesive mechanism is especially important since the microbial community in DWDS is a dynamic and environment-dependent system, that represents a variety of microbial species (Potgieter et al., 2018), likely exhibiting a wide range of adhesion and biofilm forming behaviors. Moreover, the fact that the P(SBMA) coating can significantly reduce the adhesion of bacterial aggregates is of value, as in DWDS, microbial concentration in bulk water is low and detached biofilm clusters, that are also more resistant to disinfectants than planktonic bacteria, could potentially be the main initiators of downstream biofilm development (Wang et al., 2021).

Biofilm formation and detachment in DWDS lead to several operational and maintenance issues for water companies, whose aim is to deliver microbiologically safe drinking water to consumers (Simões and Simões, 2013). Biofilm development is driven by a variety of complex processes along the distribution network and so far, no single control practice (i.e., periodical flushing, disinfectants dosing) proved to be sufficiently effective against biofilm formation and persistence (Liu et al., 2017a). Elements utilized in DWDS, such as pipes, appendages, and valves, are manufactured from multiple materials of whose the properties affect biofilm formation to a varying degree. With this study, we successfully introduced the concept of surface properties modification with the hydrogel-based anti-adhesive coating against drinking water bacteria as a localized biofilm control strategy.

P(SBMA)-based hydrogel coating is a safe, effective, and universal anti-adhesive solution. In terms of application in DWDS, it provides an interesting alternative that could target biofilm hotspots, characterized either by local higher temperatures, long stagnation periods and dead-ends (Simunič et al., 2020), and/or sections with elements manufactured from materials especially prone to biofilm development, such as EPDM on rubber-coated valves (Neu and Hammes, 2020). Rubber-based materials such as EPDM (M-class) rubber are generally used for production of sealing elements in fittings and other products in contact with drinking water, which organic additives, however, can migrate in water, thus are highly susceptible to biofilm formation (Szczotko et al., 2016). In particular, EPDM has showed to promote microbial growth of many different microorganisms, including protozoa (Waines et al., 2011), and to stimulate also the attachment of pathogens such as P. aeruginosa and Legionella, which can then cause secondary contamination of water (Moritz et al., 2010; Szczotko et al., 2016). In this study, the P(SBMA) coating was synthetized on a transparent PDMS surface for the sake of real-time monitoring of adhesion and non-destructive quantification of adhering bacteria, but generally the UV-mediated free radical polymerization with benzophenone infusion step can be applied to other elastomeric materials of various dimensions, including EPDM rubber (Yagci et al., 2010).

Concluding, PDMS was successfully coated with P(SBMA)-based hydrogel using photografting. The P(SBMA) coating showed excellent anti-adhesive properties against Sphingomonas and Pseudomonas after 4 h, limiting their attachment by up to 99%. These results bring a lot of promise for the potential applicability of the coating in DWDS biofilm hotspots, and on elements utilizing materials that are known to promote excessive biofilm growth. However, further investigation is needed to evaluate the coating performance and stability in more representative conditions (i.e., extended testing period, exposure to DWDS hydraulic conditions and mixed microbial community), and to optimize the coating process and coating’s mechanical properties to ensure a long-lasting durability on representative materials, so essential in the proposed field of application.